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1.
HLA-B*57-mediated selection pressure leads to a typical escape pathway in human immunodeficiency virus type 1 (HIV-1) CD8 epitopes such as TW10. Whether this T242N pathway is shared by all clades remains unknown. We therefore assessed the nature of HLA-B*57 selection in a large, observational Kenyan cohort where clades A1 and D predominate. While T242N was ubiquitous in clade D HLA-B*57+ subjects, this mutation was rare (15%) in clade A1. Instead, P243T and I247L were selected by clade A1-infected HLA-B*57 subjects but not by HLA-B*5801+ subjects. Our data suggest that clade A1 consensus proline at Gag residue 243 might represent an inherent block to T242N escape in clade A1. We confirmed immunologically that P243T and I247L likely represent escape mutations. HLA-B*57 evolution also differed between clades in the KF11 and IW9 epitopes. A better understanding of clade-specific evolution is important for the development of HIV vaccines in regions with multiple clades.Human immunodeficiency virus type 1 (HIV-1) displays extreme genetic diversity, with nine clades (subtypes) described in group M, and frequent genomic recombination among and within the clades (7, 44). HIV is also capable of rapid evolution, which can lead to mutational escape from immune control (43). Escape from CD8+ T-cell responses occurs frequently in HIV-1 infection through mutations that affect epitope processing, HLA class I binding, and/or T-cell receptor recognition (23). In early HIV-1 infection, the majority of amino acid substitutions are associated HLA class I alleles (1). The timing and consequences of mutational escape from CD8+ T-cell responses vary considerably (8, 22).HLA-B*57, and to a lesser extent HLA-B*5801, has been associated with slower progression to AIDS in several studies (18, 27, 39), and HLA-B*5701 was associated with a lower viremia set point in a genome-wide association study (16). Several attributes of HLA-B*57-restricted CD8+ T-cell responses may contribute to their protectiveness, including dominant responses in acute infection (2), recognition of protective epitopes in HIV-1 p24 (33), better recognition of epitope variation (45), and retention of proliferative capability in chronic infection (24).HLA-B*57/5801 also exert powerful selection pressure on HIV to avoid CD8+ T-cell recognition. This was first demonstrated in the HLA-B*57-restricted TW10 epitope (TSTLQEQIGW [Gag240-249]), which accounts for >30% of overall HIV-specific CD8+ T-cell responses in acutely infected HLA-B*57+ subjects (3). Escape in this epitope usually occurs early in infection, which coincidently is when HLA-B*57 is most protective (18). In clade B and C infections, >75 to 100% of HLA-B*57/5801+ subjects develop the T242N escape mutation, while HLA-B*57/5701-negative subjects rarely display polymorphism at this residue (5, 9, 10, 15, 32, 35, 38, 41). When T242N is transmitted to HLA-B*57/5801-negative subjects, it rapidly reverts to the consensus, suggesting that T242N is associated with a fitness defect (32, 35).While CD8+ T-cell cross-clade recognition has been tested extensively (6, 11, 19, 36, 48), few studies have addressed the possibility of clade-specific escape from CD8+ T-cell responses. This may be especially relevant where clade consensus sequences differ in immunologically relevant epitopes. Here we demonstrate in a large Kenyan cohort substantial differences in HLA-B*57/B*5801-mediated selection among HIV clades.Participants were enrolled from a Nairobi, Kenya-based cohort, and the relevant ethical review boards approved the study. HLA typing was performed as described previously (34). CD4 counts were measured longitudinally at biannual visits. Multiple and other clade infections were excluded. The HIV-1 p24 gene was amplified from proviral HIV DNA or RNA using a nested PCR approach and sequenced, and viral subtyping was carried out as described previously (42). Previously described HLA-B*57 epitopes IW9 (ISPRTLNAW), KF11 (KAFSPEVIPMF), and TW10 (TSTLQEQIGW) and selected variants were tested in immunological assays and described where relevant. Gamma interferon enzyme-linked immunospot (ELISPOT) assays were performed as described previously (37) using blood samples from HLA-B*57+ and -B*5801+ subjects. All peptides were tested at concentrations of 10 μg, 1 μg, 0.1 μg, and 0.01 μg/ml. Responses were considered positive if they were more than two times higher than that of the negative control and were measured at ≥100 spot-forming units ml−1. Fisher''s exact test and chi-square analyses were used to determine differences among groups in categorical analyses. Mann-Whitney U tests were used to compare response magnitudes and disease progression between groups.We confirmed the protective effects of HLA-B*57 in clade A1 infection (mean of 9.9 years versus 7.8 years until CD4 counts were <200, P = 0.041) (Fig. (Fig.1).1). Slow progressors were overrepresented in HLA-B*57+ clade A1+ subjects (52.2%) compared to both HLA-B*5801+ clade A1+ (13.3%, P = 0.02) and HLA-B*57/5801-negative clade A1+ (27.8%, P = 0.028) subjects (Fig. (Fig.1b).1b). In contrast to what has been shown for other clades (2, 27), protection was not observed for clade A1-infected HLA-B*5801+ subjects (mean of 6.5 years versus 7.8 years until CD4 counts were <200, P > 0.3) (Fig. (Fig.11).Open in a separate windowFIG. 1.HLA-B*57, but not HLA-B*5801, is associated with a lower rate of disease progression in clade A1-infected subjects than that of the overall cohort. (a) Number of years from cohort entry until sequential CD4 counts fell below 200/μl. (b) Slow progressors (>10 years with CD4 counts of >200) were also more common in HLA-B*57+ clade A1-infected subjects than in those expressing HLA-B*5801 or neither.Stratification of TW10 (Gag240-249) proviral sequences on the basis of HLA allele and clade revealed several differences in selection between clades A1 and D (Fig. (Fig.2a).2a). We observed the expected T242N substitution in 100% of HLA-B*57+ clade D-infected subjects (7/7), compared to only 14.7% variability at Gag residue 242 in HLA-B*57/5801-negative subjects (13/88, P = 3.26 × 10−9) (Fig. (Fig.2b).2b). In contrast, T242N was found infrequently in clade A1-infected HLA-B*57+ subjects (15%, 5/33, P = 0.0004). Instead, variants containing the mutations P243T and I247L were more frequently observed (both observed in 11/33 subjects). Overall, variation at residues 243 and 247 was more common in HLA-B*57+ subjects (51% and 15%, respectively; P = 2.92 × 10−6) than in HLA-B*57/5801-negative clade A1+ subjects (33% and 9%, respectively; P = 0.0008). Selection at both residues 243 and 247 was observed only in 2/33 HLA-B*57+ subjects, suggesting that these mutations are independent. Selection at residue 248, observed in clade B infection (32), was not evident in either clade A1 or D. While I247X selection has been described in other clades at low frequencies and in elite controllers (21, 40), HLA-B*57-mediated selection at Gag residue 243 has not yet been described. In summary, the T242N mutation, which is typical of other clades, does not appear to be the primary escape mutant in clade A1.Open in a separate windowFIG. 2.HLA-B*57-mediated selection in TW10 differs between clade A1 and clade D. (a) TW10 sequences were stratified by HLA-B*57, HLA-B*5801, or other alleles (HLA-B*57/5801) and compared between clades A1 and D, based on the clade B consensus TW10 sequence. Each subject is represented by one sequence, and the numbers of subjects with a given sequence are shown in parentheses. A summary of variation from the TW10 consensus at Gag residues 242, 243, 247, and 248 is shown for HLA-B*57+ (b) and -B*5801+ (c) subjects. (b and c) Clade D is shown at the top, and clade A1 is shown at the bottom. Variation is shown in dark gray, and consensus is shown in light gray. (d) The proportions of clade A1-infected subjects with selection at Gag residue 242 only, and those with selection at residues 242 and 243 in combination, are shown. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.Previous studies have suggested that HLA-B*5801 places selection pressure on TW10, similar to that of HLA-B*57 (35). Similar to clades B and C, selection of T242N was evident in HLA-B*5801+ clade D-infected subjects (TW10 variation in 8/11 HLA-B*5801+ subjects versus 13/88 HLA-B*57/5801-negative subjects; P = 0.0069) (Fig. (Fig.2c).2c). Limited T242N selection was observed in clade A1-infected HLA-B*5801+ subjects, and in contrast to HLA-B*57, there were no HLA-B*5801-associated substitutions at residues 243 and 247 in clade A1 (P values of 0.75 and 0.29, respectively) (Fig. (Fig.2c).2c). In summary, these data suggest that in addition to HLA-B*5801 not being associated with protection in clade A1 (Fig. (Fig.1),1), HLA-B*5801 does not select the HLA-B*57-associated clade A1 TW10 escape mutations.Inclusion of all clade A1 sequences with T242X substitutions (regardless of the HLA allele) reveals that in every case (10/10), there is an accompanying residue 243 mutation. Polymorphisms at these sites correlate very strongly (P = 3.71 × 10−8) (Fig. (Fig.2d).2d). Together, these data suggest that residue 242 polymorphism in clade A1 is incompatible with proline at residue 243, which is the clade A1 consensus.We next assessed the immunological implications of novel clade A1 variants in HLA-B*57+ (n = 12) and -B*5801+ (n = 6) subjects infected primarily by clade A1. Clade A1-infected subjects commonly made anamnestic, low-avidity responses to TW10. The majority of HLA-B*57+ subjects who recognized clade A1 TW10 did not respond to P243T or I247L in ELISPOT assays (Fig. (Fig.3),3), supporting the hypothesis that these represent escape mutations. Those who did recognize P243T and I247L had lower magnitude responses than those who recognized clade A1 TW10 at the 10-μg/ml peptide concentration (P of 0.0005 for both) (Fig. (Fig.3a).3a). Similarly, these variants were not well recognized by CD8+ T cells from HLA-B*5801+ subjects (Fig. (Fig.3b).3b). For two HLA-B*57+ subjects, P243T and I247L responses had lower avidity than clade A1 TW10 responses (Fig. (Fig.3c).3c). These data support the hypothesis that P243T and I247L likely represent escape mutations.Open in a separate windowFIG. 3.Peptides with novel TW10 clade A1-selected mutations are poorly recognized in ex vivo gamma interferon ELISPOT avidity assays, suggestive of escape mutations. ELISPOT responses to TW10 and variants at 10 μg/ml peptide by HLA-B*57+ (a) and HLA-B*5801+ (b) subjects are shown. (c) The functional avidity of TW10 and variants for two HLA-B*57+ subjects is shown, suggesting that P243T and I247L are less recognized than TW10, particularly at lower peptide concentrations. Sequence names are described in the text. (d) Elispot responses to A1 TW10 and T242N correlated at 10 μg/ml. SFU/m, spot-forming units/million PBMCs.Recognition of the clade B/D consensus (TSTLQEQIGW) was diminished compared to that of clade A1 TW10. However, despite the presumed absence of this variant in these subjects'' autologous sequences, the clade B/D escape variant (TSNLQEQIGW [T242N]) was recognized at a magnitude similar to that of the consensus clade A1 TW10 (r = 0.71, P = 0.0099) (Fig. (Fig.3d).3d). No responses to T242N/G248A were observed (not shown), as described previously (32). These data suggest that clade A1 and B TW10, and their escape variants, are immunologically distinct from one another.We next assessed whether clade-specific selection was evident in other immunodominant HLA-B*57 p24 epitopes that are commonly targeted in chronic clade B infection (2). In clade D IW9 (ISPRTLNAW [Gag147-155]), variants containing the escape variant I147L (14) were more common in HLA-B*57+ subjects than in HLA-B*57/5801-negative subjects (86% and 30%, respectively; P = 0.0055) (Table (Table1).1). However, this variant was not selected in clade A1 (variation in 30% versus 21% subjects; P value was not significant), where leucine is the consensus. Interestingly, ELISPOT data indicated substantial cross-reactivity between 147L and 147I in clade A1-infected subjects (10 μg/ml, r = 0.987, P < 0.0001) (data not shown), suggesting that infection with an escape variant from one clade (clade D) does not necessarily preclude recognition of this epitope in another one (clade A1). Other amino acids (F, M, and P) were common in HLA-B*57+ subjects at residue 147 (>30% versus 3% in HLA-B*57/5801-negative subjects, P = 3.85 × 10−6). Although the immunological consequences are unknown, these HLA-associated substitutions could represent novel escape variants.

TABLE 1.

p24 sequences in HLA-B*57+ subjects infected by clades A1 and D
CladeHLA-B alleleSubject no.No. of years infected prior to samplebPolymorphism at residue S146Epitope sequencea
IW9KF11TW10
LSPRTLNAWKAFSPEVIPMFTSTPQEQIGW
A157011665>130NF-----------------------------
570213921N----------------------NI------
59NDP----------------------NT---LA-
41>122T----------------------NV------
1419>41N------------------------------
616>102T---------------------------LA-
164718-P----------------------S---LQ-
613>33P-----------------------T------
718>71PM----------------------T---L--
1315>58P----------G------------TS---L--
57032125>0A------------N---------NL------
561>78P------------N-----------------
1926>39P------------c----------T------
1778>13-------------------------------
16090T---------------------------LQ-
525>135-----------G-n--------------L--
1368>57-M---------G-n----------T------
509>150TF---------G-N-----------------
260>163TF---------G-N--------------L--
111>133-----------G-N--------------LQ-
1111>34-----------G-N--------------LQ-
1638>27-----------G-N----------T------
2101>0P----------G-----------NT------
532>28-----------G-----------------A-
1669>23PP---------G-----------------A-
703>71TF---------G-------------------
1741>11TF---------G-------------------
1452>37P----------G-------------------
30>121P---------R------------------A-
1122>39P---------RG-Q-----------------
995>80PF--------RG-Q----------T------
1564>27P---------RG------------T------
5707330>164T----------G-N----------T------
D57011859P----------------------NL------
1852P----------------------NL---VA-
57031756P----------------------NL------
1423P-T--------------------NL------
1188PI---------G-N---------NL----A-
1894P----------N-----------NL----R-
199P-T--------S-----------NL---V--
Open in a separate windowaThe first row of epitope sequences shows the consensus sequences.bND, not done.In addition, a substitution at Gag residue 146 (A146P) represents an IW9 processing escape mutation in clades B and C (14), and this mutation was also selected by HLA-B*57 in both clades A1 and D (Table (Table1).1). In clade A1, substitutions at Gag residue 146 (primarily P and T) were more frequent in HLA-B*57+ subjects than in HLA-B*57/5801-negative subjects (13/33 and 10/221, respectively; P = 1.42 × 10−7) (Table (Table1).1). Therefore, although the consensus at residue 146 differs among clades, here escape at residue 146 occurs in HLA-B*57+ subjects infected by clades A1, B, C, and D.For KF11 (KAFSPEVIPMF [Gag162-172]), HLA-B*57-associated variation from the consensus was more common in clade A1 (67% versus 21%, P = 2.44 × 10−7) than in clade D (43% versus 17%, P = 0.012). Previously described A163G and A163G/S165N variants (13, 20) were most common in clade A1 (Table (Table1).1). In addition, the novel K162X substitution was present in clade A1. HLA-B*5703 and -B*5701 have previously been shown to display differences in KF11 selection (20, 47), and our data indicate that HLA-B*5702 also differs from HLA-B*5703 in terms of KF11 selection. While the KF11 consensus is present in the majority of HLA-B*5702+ subjects, it is rare in HLA-B*5703+ clade A1 infection (8/9 versus 2/22, P = 4.74 × 10−5).Mounting evidence suggests that HLA alleles are a major force in viral evolution (26). We show that in clade A1 p24, HLA-B*57 selection in three epitopes differs from earlier clade B and C data in several important aspects, while clade D selection resembles what has previously been shown. This included a low frequency of T242N in clade A1 TW10, with selection being more common at Gag residues 243 and 247, more extensive KF11 escape, and selection of different amino acids in IW9. Overall, selection was evident in the majority of HLA-B*57+ subjects (>90% of clade A1-infected subjects had selection in more than one epitope, and >75% of them had selection in more than two) (Table (Table1).1). Parallel escape in multiple epitopes demonstrates the need to avoid the pressure of CD8+ T-cell responses.One possible mechanism underlying the differences in TW10 selection is that TSNPQEQIGW (never observed) (Fig. (Fig.2d)2d) is not feasible virologically, such that T242N is possible only in conjunction with a preexisting residue 243 mutation (TSNXQEQIGW, observed in 15% of HLA-B*57+ subjects) (underlining shows mutation). One would expect to observe T242N at a higher frequency, given its dominance in HLA-B*57+ subjects infected by other clades. Therefore, while T242N has been implicated in HLA-B*57-mediated protection, this mutation is rare in clade A1. Because HLA-B*57 remains protective in clade A1, that protection may be mediated by novel mechanisms.Because TW10 is commonly recognized by 86% of clade A1 subjects, it is evident that clade A1 TW10 can bind HLA-B*57. We therefore hypothesize that TSTPQEQIGW may affect the interaction between epitope and cognate T-cell receptors, which in turn influences which escape mutations are optimal. This hypothesis is supported by our immunological data showing cross-reactivity between clade A1 TW10 and TSNLQEQIGW (underlining shows mutation), which imply that mutation at residue 242 may not lead to effective escape in clade A1 (Fig. (Fig.3d3d).In contrast to other clades (including clade D), HLA-B*5801 does not appear to place selection pressure on clade A1 TW10. A previous study in Rwanda similarly showed that in clade A1, HLA-B*5703 but not HLA-B*5801 was associated with lower HIV viral loads (30). Therefore, HLA-B*5801 was associated with neither protection nor selection in clade A1 TW10. Our data also show that HLA-B*5702- and -B*5703-mediated KF11 selection differs, despite these alleles differing at only one codon. Similar findings have been published for HLA-B7 supertype alleles (31). These data highlight the differences in immunological pressure within HLA supertype alleles, even though these alleles often present the same epitopes to the immune system.Previous reports have suggested that HIV evolution can differ among clades for a variety of reasons. HLA-B*1503 differed in its protectiveness in clade B- and clade C-infected cohorts, and the apparent mechanism is broader recognition of subdominant epitopes, which remain intact due to limited selection where HLA-B*1503 is less common (17). Similarly, Yu et al. showed that differences in KF11 evolution between clades B and C were largely the result of differences in immunological features of HLA-B*5701- and -B*5703-restricted responses, with the latter allele being more frequent in clade C-infected populations (47). The temporality of selection can also differ between clades; while TW10 and IW9 selection is similar between clade B and C, the order in which they are selected is opposite (12). Our data show that virological factors (i.e., sequence differences) can also lead to clade-specific escape. Other reports have found few differences in evolution among clades, including no differences in HLA-A2 Gag SL9 escape among clades A, B, and D (25), so the presence of clade-specific evolution will depend on the epitope and allele under study.Recent reports have suggested that Gag-specific CD8+ T-cell responses are protective in HIV infection (28), possibly because escape in Gag comes at a fitness cost. In support of this, infection by strains containing multiple Gag escape mutations was associated with lower set point viremia independent of HLA alleles in the recipients (21). One of the first demonstrations of Gag escape with fitness cost was T242N selection and reversion (32), and this substitution dominates in clade B- and clade C-infected HLA-B*57+ subjects in numerous cohorts (5, 9, 10, 15, 32, 35, 38, 41). Our data show that while clade D follows clades B and C, HLA-B*57-mediated evolution in clade A1 differs not only in TW10 but also in other p24 epitopes. Knowledge of clade-specific escape pathways will be important for vaccines that aim to cover multiple clades, particularly where clades differ in immunologically critical epitopes.  相似文献   

2.
Hepatitis C virus (HCV)-specific CD8+ T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127+ T cells is driven by viral variation.Hepatitis C virus (HCV) persists in the majority of acutely infected individuals, potentially leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The cellular immune response has been shown to play a significant role in viral control and protection from liver disease. Phenotypic and functional studies of virus-specific T cells have attempted to define the determinants of a successful versus an unsuccessful T-cell response in viral infections (10). So far these studies have failed to identify consistent distinguishing features between a T-cell response that results in self-limiting versus chronic HCV infection; similarly, the impact of viral persistence on HCV-specific memory T-cell formation is poorly understood.Interleukin-7 (IL-7) receptor alpha chain (CD127) is a key molecule associated with the maintenance of memory T-cell populations. Expression of CD127 on CD8 T cells is typically only observed when the respective antigen is controlled and in the presence of significant CD4+ T-cell help (9). Accordingly, cells specific for persistent viruses (e.g., HIV, cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) have been shown to express low levels of CD127 (6, 12, 14) and to be dependent on antigen restimulation for their maintenance. In contrast, T cells specific for acute resolving virus infections, such as influenza virus, respiratory syncytial virus (RSV), hepatitis B virus (HBV), and vaccinia virus typically acquire expression of CD127 rapidly with the control of viremia (5, 12, 14). Results for HCV have been inconclusive. The expected increase in CD127 levels in acute resolving but not acute persisting infection has been found, while a substantial proportion of cells with high CD127 expression have been observed in long-established chronic infection (2). We tried to reconcile these observations by studying both subjects with acute and chronic HCV infection and identified the presence of antigen as the determinant of CD127 expression.Using HLA-peptide multimers we analyzed CD8+ HCV-specific T-cell responses and CD127 expression levels in acute and chronic HCV infection. We assessed a cohort of 18 chronically infected subjects as well as 9 individuals with previously resolved infection. In addition, we longitudinally studied 9 acutely infected subjects (5 individuals who resolved infection spontaneously and 4 individuals who remain chronically infected) (Tables (Tables11 and and2).2). Informed consent in writing was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval from the local institutional review boards. HLA-multimeric complexes were obtained commercially from Proimmune (Oxford, United Kingdom) and Beckman Coulter (CA). The staining and analysis procedure was as described previously (10). Peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies: CD3 from Caltag; CD8, CD27, CCR7, CD127, and CD38 from BD Pharmingen; and PD-1 (kindly provided by Gordon Freeman). Primer sets were designed for different genotypes based on alignments of all available sequences from the public HCV database (http://hcvpub.ibcp.fr). Sequence analysis was performed as previously described (8).

TABLE 1.

Patient information and autologous sequence analysis for patients with chronic and resolved HCV infection
CodeGenotypeStatusEpitope(s) targetedSequencea
02-031bChronicA1 NS3 1436-1444P: ATDALMTGY
A: no sequence
00-261bChronicA1 NS3 1436-1444P: ATDALMTGY
A: no sequence
99-242aChronicA2 NS3 1073-1083P: CINGVCWTV
No recognitionA: S-S--L---
A2 NS3 1406-1415P: KLVALGINAV
No recognitionA: A-RGM-L---
A2 NS5B 2594-2602P: ALYDVVTKL
A: no sequence
1111aChronicA2 NS3 1073-1083P: CINGVCWTV
A: ---------
A2 NS5 2594-2602P: ALYDVVTKL
A: ---------
00X3aChronicA2 NS5 2594-2602P: ALYDVVTKL
No recognitionA: -----IQ--
O3Qb1aChronicA1 NS3 1436-1444P: ATDALMTGY
DiminishedA: --------F
03Sb1aChronicA1 NS3 1436-1444P: ATDALMTGY
DiminishedA: --------F
02A1aChronicA1 NS3 1436-1444P: ATDALMTGY
A: no sequence
01N1aChronicA1 NS3 1436-1444P: ATDALMTGY
DiminishedA: --------F
03H1aChronicA2 NS3 1073-1083P: CINGVCWTV
Full recognitionA: ----A----
01-391aChronicA1 NS3 1436-1444P: ATDALMTGY
DiminishedA: --------F
03-45b1aChronicA1 NS3 1436-1444P: ATDALMTGY
DiminishedA: --------F
06P3aChronicA1 NS3 1436-1444P: ATDALMTGY
DiminishedA: --------F
GS127-11aChronicA2 NS3 1073-1083P: CINGVCWTV
A: ---------
GS127-61aChronicA2 NS3 1073-1083P: CINGVCWTV
A: ---------
GS127-81bChronicA2 NS3 1073-1083P: CINGVCWTV
A: ---------
GS127-161aChronicA2 NS3 1073-1083P: CINGVCWTV
A: ---------
GS127-201aChronicA2 NS3 1073-1083P: CINGVCWTV
A: ---------
04D4ResolvedA2 NS5 1987-1996P: VLSDFKTWKL
01-49b1ResolvedA2 NS5 1987-1996P: VLSDFKTWKL
A2 NS3 1406-1415P: KLVALGINAV
01-311ResolvedA1 NS3 1436-1444P: ATDALMTGY
B57 NS5 2629-2637P: KSKKTPMGF
04N1ResolvedA1 NS3 1436-1444P: ATDALMTGY
01E4ResolvedA2 NS5 1987-1996P: VLSDFKTWKL
98A1ResolvedA2 NS3 1073-1083P: CINGVCWTV
00-10c1ResolvedA24 NS4 1745-1754P: VIAPAVQTNW
O2Z1ResolvedA1 NS3 1436-1444P: ATDALMTGY
99-211ResolvedB7 CORE 41-49P: GPRLGVRAT
OOR1ResolvedB35 NS3 1359-1367P: HPNIEEVAL
Open in a separate windowaP, prototype; A, autologous. Identical residues are shown by dashes.bHIV coinfection.cHBV coinfection.

TABLE 2.

Patient information and autologous sequence analysis for patients with acute HCV infection
CodeGenotypeOutcomeEpitope targeted and time analyzedSequencea
5541aPersistingA2 NS3 1073-1083P: CINGVCWTV
wk 8A: ---------
wk 30A: ---------
03-321aPersistingB35 NS3 1359-1367P: HPNIEEVAL
wk 8A: ---------
No recognition (wk 36)A: S--------
04-111a (1st)Persisting (1st) Resolving (2nd)A2 NS5 2594-2602P: ALYDVVTKL
1b (2nd)A: no sequence
00231bPersistingA1 NS3 1436-1444P: ATDALMTGY
Diminished (wk 7)A: --------F
Diminished (wk 38)A: --------F
A2 NS3 1073-1083P: CINGVCWTV
wk 7A: ---------
wk 38A: ---------
A2 NS3 1406-1415P: KLVALGINAV
Full recognition (wk 7)A: --S-------
Full recognition (wk 38)A: --S-------
3201ResolvingA2 NS3 1273-1282P: GIDPNIRTGV
5991ResolvingA2 NS3 1073-1083P: CINGVCWTV
11441ResolvingA2 NS3 1073-1083P: CINGVCWTV
B35 NS3 1359-1367P: HPNIEEVAL
06L3aResolvingB7 CORE 41-49P: GPRLGVRAT
05Y1ResolvingA2 NS3 1073-1083P: CINGVCWTV
Open in a separate windowaP, prototype; A, autologous. Identical residues are shown by dashes.In established persistent infection, CD8+ T-cell responses against HCV are infrequently detected in blood using major histocompatibility complex (MHC) class I tetramers and are only observed in a small fraction of those sampled (10). We were able to examine the expression of CD127 on antigen-specific T cells in such a group of 18 individuals. We observed mostly high levels of CD127 expression (median, 66%) on these populations (Fig. (Fig.1a),1a), although expression was higher on HCV-specific T-cell populations from individuals with resolved infection (median, 97%; P = 0.0003) (Fig. 1a and c). Importantly, chronically infected individuals displayed CD127 expression levels over a much broader range than resolved individuals (9.5% to 100% versus 92 to 100%) (Fig. (Fig.1a1a).Open in a separate windowFIG. 1.Chronically infected individuals express a range of CD127 levels on HCV-specific T cells. (a) CD127 expression levels on HCV-specific T-cell populations in individuals with established chronic or resolved infection. While individuals with resolved infection (11 tetramer stains in 9 subjects) uniformly express high levels of CD127, chronically infected individuals (21 tetramer stains in 18 subjects) express a wide range of CD127 expression levels. (b) CD127 expression levels are seen to be highly dependent on sequence match with the autologous virus, based on analysis of 9 responses with diminished recognition of the autologous virus and 8 responses with intact epitopes. (c) CD127 expression levels on HCV-specific T-cell B7 CORE 41-49-specific T cells from individual 01-49 with resolved HCV infection (left-hand panel). Lower CD127 expression levels are observed on an EBV-specific T-cell population from the same individual (right-hand panel). APC-A, allophycocyanin-conjugated antibody. (d) Low CD127 levels are observed on A2 NS3 1073-1083 HCV-specific T cells from individual 111 with chronic HCV infection in whom sequencing revealed an intact autologous sequence.Given the relationship between CD127 expression and antigenic stimulation as well as the potential of HCV to escape the CD8 T-cell response through viral mutation, we sequenced the autologous circulating virus in subjects with chronic infection (Table (Table1).1). A perfect match between the optimal epitope sequence and the autologous virus was found for only 8 responses. These were the only T-cell populations with lower levels of CD127 expression (Fig. (Fig.1a,1a, b, and d). In contrast, HCV T-cell responses with CD127 expression levels comparable to those observed in resolved infection (>85%) were typically mismatched with the viral sequence, with some variants compatible with viral escape and others suggesting infection with a non-genotype 1 strain (10) (Fig. (Fig.1).1). Enzyme-linked immunospot (ELISPOT) assays using T-cell lines confirmed the complete abrogation of T-cell recognition and thus antigenic stimulation in cases of cross-genotype mismatch (10). Responses targeting the epitope A1-143D expressed somewhat lower levels of CD127 (between 70% and 85%). Viral escape (Y to F at position 9) in this epitope has been shown to be associated with significantly diminished but not fully abolished recognition (11a), and was found in all chronically infected subjects whose T cells targeted this epitope. Thus, expression of CD127 in the presence of viremia is closely associated with the capacity of the T cell to recognize the circulating virus.That a decrease in antigenic stimulation is indeed associated with the emergence of CD127-expressing CD8 T cells is further demonstrated in subject 111. This subject with chronic infection targeted fully conserved epitopes with T cells with low CD127 expression; with clearance of viremia under antiviral therapy, CD127-negative HCV-specific CD8 T cells were no longer detectable and were replaced by populations expressing CD127 (data not shown). Overall these data support the notion that CD127 expression on HCV-specific CD8+ T-cell populations is dependent on an absence of ongoing antigenic stimulation.To further evaluate the dynamic relationship between antigenic stimulation and CD127 expression, we also analyzed HCV-specific T-cell responses longitudinally during acute HCV infection (Fig. (Fig.2a).2a). CD127 expression was generally low or absent during the earliest time points. After resolution of infection, we see a contraction of the HCV-specific T-cell response together with a continuous increase in CD127 expression, until virtually all tetramer-positive cells express CD127 approximately 6 months after the onset of disease (Fig. (Fig.2a).2a). A similar increase in CD127 expression was not seen in one subject (no. 554) with untreated persisting infection that maintained a significant tetramer-positive T-cell population for an extended period of time (Fig. (Fig.2a).2a). Importantly, sequence analysis of the autologous virus demonstrated the conservation of this epitope throughout persistent infection (8). In contrast, subject 03-32 (with untreated persisting infection) developed a CD8 T-cell response targeting a B35-restricted epitope in NS3 from which the virus escaped (8). The T cells specific for this epitope acquired CD127 expression in a comparable manner to those controlling infection (Fig. (Fig.2a).2a). In other subjects with persisting infection, HCV-specific T-cells usually disappeared from blood before the time frame in which CD127 upregulation was observed in the other subjects.Open in a separate windowFIG. 2.CD127 expression levels during acute HCV infection. (a) CD127 expression levels on HCV-specific T cells during the acute phase of HCV infection (data shown for 5 individuals who resolve and two individuals who remain chronically infected). (b) HCV RNA viral load and CD127 expression levels on HCV-specific T cells (A2 NS3 1073-1083 and A1 NS3 1436-1444) for chronically infected individual 00-23. PEG-IFN-α, pegylated alpha interferon. (c) Fluorescence-activated cell sorter (FACS) plots showing longitudinal CD127 expression levels on HCV-specific T cells (A2 NS3 1073-1083 and A1 NS3 1436-1444) from individual 00-23.We also characterized the levels of CD127 expression on HCV-specific CD4+ T-cell populations with similar results: low levels were observed during the acute phase of infection and increased levels in individuals after infection was cleared (data not shown). CD127 expression on CD4 T cells could not be assessed in viral persistence since we failed to detect significant numbers of HCV-specific CD4+ T cells, in agreement with other reports.In our cohort of subjects with acute HCV infection, we had the opportunity to study the effect of reencounter with antigen on T cells with high CD127 expression in 3 subjects in whom HCV viremia returned after a period of viral control. Subject 00-23 experienced viral relapse after interferon treatment (11), while subjects 05-13 and 04-11 were reinfected with distinct viral isolates. In all subjects, reappearance of HCV antigen that corresponded to the HCV-specific T-cell population was associated with massive expansion of HCV-specific T-cell populations and a decrease in CD127 expression on these T cells (Fig. (Fig.22 and and3)3) (data not shown). In contrast, T-cell responses that did not recognize the current viral isolate did not respond with an expansion of the population or the downregulation of CD127. This was observed in 00-23, where the sequence of the A1-restricted epitope 143D was identical to the frequent escape mutation described above in chronically infected subjects associated with diminished T-cell recognition (Fig. (Fig.2b2b and and3a).3a). In 05-13, the viral isolate during the second episode of viremia contained a variant in one of the anchor residues of the epitope A2-61 (Fig. (Fig.2d).2d). These results show that CD127 expression on HCV-specific T cells follows the established principles observed in other viral infections.Open in a separate windowFIG. 3.Longitudinal phenotypic changes on HCV-specific T cells. (a) HCV RNA viral load and CD127 expression (%) levels on A2 NS5B 2594-2602 HCV-specific T cells for individual 04-11. This individual was administered antiviral therapy, which resulted in a sustained virological response. Following reinfection, the individual spontaneously cleared the virus. (b) Longitudinal frequency of A2 NS5B 2594-2602 HCV-specific T cells and PD-1 expression levels (mean fluorescent intensity [MFI]) for individual 04-11. (c) Longitudinal analysis of 04-11 reveals the progressive differentiation of HCV-specific A2 259F CD8+ T cells following repetitive antigenic stimulation. FACS plots show longitudinal CD127, CD27, CD57, and CCR7 expression levels on A2 NS5B 2594-2602 tetramer-positive cells from individual 04-11. PE-A, phycoerthrin-conjugated antibody.In addition to the changes in CD127 expression for T cells during reencounter with antigen, we detected comparable changes in other phenotypic markers shortly after exposure to viremia. First, we detected an increase in PD-1 and CD38 expression—both associated with recent T-cell activation. Additionally, we observed a loss of CD27 expression, a feature of repetitive antigenic stimulation (Fig. (Fig.3).3). The correlation of CD127 and CD27 expression further supports the notion that CD127 downregulation is a marker of continuous antigenic stimulation (1, 7).In conclusion we confirm that high CD127 expression levels are common for detectable HCV-specific CD8+ T-cell populations in chronic infection and find that this phenotype is based on the existence of viral sequence variants rather than on unique properties of HCV-specific T cells. This is further demonstrated by our data from acute HCV infection showing that viral escape as well as viral resolution is driving the upregulation of CD127. We also show that some, but not all, markers typically used to phenotypically describe virus-specific T cells show a similar dependence on cognate HCV antigen. Our data further highlight that sequencing of autologous virus is vital when interpreting data obtained in chronic HCV infection and raise the possibility that previous studies, focused on individuals with established chronic infection, may have been confounded by antigenic variation within epitopes or superinfection with different non-cross-reactive genotypes. Interestingly, it should be pointed out that this finding is supported by previous data from both the chimpanzee model of HCV and from human HBV infection (3, 13).Overall our data clearly demonstrate that the phenotype of HCV-specific CD8+ T cells is determined by the level of antigen-specific stimulation. The high number of CD127 positive virus-specific CD8+ T cells that is associated with the presence of viral escape mutations is a hallmark of chronic HCV infection that clearly separates HCV from other chronic viral infections (4, 14).  相似文献   

3.
The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated (n = 67) or not vaccinated (n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6Pcy243. In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID50]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load (P = 0.0280) and CD4 loss (P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID50 (P < 0.0001), and contained acute CD4 loss at 15 MID50 (P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility (P = 0.0199) and resistance (P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion (P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.Advances in typing of the major histocompatibility complex (MHC) of Mauritian cynomolgus macaques (14, 20, 26) have provided the opportunity to address the influence of host factors on vaccine studies (13). Retrospective analysis of 22 macaques vaccinated with Tat or a Tat-expressing adenoviral vector revealed that monkeys with the H6 or H3 MHC class IB haplotype were overrepresented among aviremic or controller animals, whereas macaques with the H2 or H5 haplotype clustered in the noncontrollers (12). More recently, the H6 haplotype was reported to correlate with control of chronic infection with simian immunodeficiency virus (SIV) mac251, regardless of vaccination (18).Here, we performed a retrospective analysis of 112 Mauritian cynomolgus macaques, which included the 22 animals studied previously (12), to evaluate the impact of the challenge dose and class IB haplotype on the acquisition and severity of simian/human immunodeficiency virus (SHIV) 89.6Pcy243 infection in 45 control monkeys and 67 monkeys vaccinated with Tat from different protocols (Table (Table11).

TABLE 1.

Summary of treatment, challenge dose, and outcome of infection in cynomolgus monkeys
Protocol codeNo. of monkeysImmunogen (dose)aAdjuvantbSchedule of immunization (wk)RoutecChallenged (MID50)Virological outcomee
Reference(s) or source
ACV
ISS-ST6Tat (10)Alum or RIBI0, 2, 6, 12, 15, 21, 28, 32, 36s.c., i.m.104114, 17
ISS-ST1Tat (6)None0, 5, 12, 17, 22, 27, 32, 38, 42, 48i.d.101004, 17
ISS-PCV3pCV-tat (1 mg)Bupivacaine + methylparaben0, 2, 6, 11, 15, 21, 28, 32, 36i.m.103006
ISS-ID3Tat (6)none0, 4, 8, 12, 16, 20, 24, 28, 39, 43, 60i.d.10111B. Ensoli, unpublished data
ISS-TR6Tat (10)Alum-Iscom0, 2, 6, 11, 16, 21, 28, 32, 36s.c., i.d., i.m.10420Ensoli, unpublished
ISS-TGf3Tat (10)Alum0, 4, 12, 22s.c.1503Ensoli, unpublished
ISS-TG3Tatcys22 (10)Alum1503Ensoli, unpublished
ISS-TG4Tatcys22 (10) + Gag (60)Alum1504Ensoli, unpublished
ISS-TG4Tat (10) + Gag (60)Alum1504Ensoli, unpublished
ISS-MP3Tat (10)H1D-Alum0, 4, 12, 18, 21, 38s.c., i.m.15021Ensoli, unpublished
ISS-MP3Tat (10)Alums.c.15003Ensoli, unpublished
ISS-GS6Tat (10)H1D-Alum0, 4, 12, 18, 21, 36s.c., i.m.15132Ensoli, unpublished
NCI-Ad-tat/Tat7Ad-tat (5 × 108 PFU), Tat (10)Alum0, 12, 24, 36i.n., i.t., s.c.15232Ensoli, unpublished
NCI-Tat9Tat (6 and 10)Alum/Iscom0, 2, 6, 11, 15, 21, 28, 32, 36s.c., i.d., i.m.1524312
ISS-NPT3pCV-tat (1 mg)Bupivacaine + methylparaben-Iscom0, 2, 8, 13, 17, 22, 28, 46, 71i.m.20003Ensoli, unpublished
ISS-NPT3pCV-tatcys22 (1 mg)Bupivacaine + methylparaben-Iscom0, 2, 8, 13, 17, 22, 28, 46, 71i.m.20111
    Total vaccinated67191731
        Naive11NoneNoneNAgNA10 or 15137
        Control34None, Ad, or pCV-0Alum, RIBI, H1D, Iscom or bupivacaine + methylparaben-Iscoms.c., i.d., i.n., i.t., i.m.10, 15, or 2051316
    Total controls4561623
    Total112253354
Open in a separate windowaAll animals were inoculated with the indicated dose of Tat plasmid DNA (pCV-tat [8], adenovirus-tat [Ad-tat] [27]) or protein, Gag protein, or empty vectors (pCV-0, adenovirus [Ad]) by the indicated route. Doses are in micrograms unless indicated otherwise.bAlum, aluminum phosphate (4); RIBI oil-in-water emulsions containing squalene, bacterial monophosphoryl lipid A, and refined mycobacterial products (4); Iscom, immune-stimulating complex (4); H1D are biocompatible anionic polymeric microparticles used for vaccine delivery (10, 12, 25a).cs.c., subcutaneous; i.m., intramuscular; i.d., intradermal; i.n., intranasal; i.t., intratracheal.dAll animals were inoculated intravenously with the indicated dose of the same SHIV89.6.Pcy243 stock.eAccording to the virological outcome upon challenge, monkeys were grouped as aviremic (A), controllers (C), or viremic (V).fBecause of the short follow-up, controller status could not be determined and all infected monkeys of the ISS-TG protocol were therefore considered viremic.gNA, not applicable.  相似文献   

4.
5.
6.
7.
The transfer range of phage genes was investigated at the single-cell level by using an in situ DNA amplification technique. After absorption of phages, a phage T4 gene was maintained in the genomes of non-plaque-forming bacteria at frequencies of 10−2 gene copies per cell. The gene transfer decreased the mutation frequencies in nonhost recipients.Recently, whole-genome analyses have revealed that many bacterial genomes contain foreign genes, especially phage genes (9). The phage genes in bacterial genomes include genes for virulence or fitness factors such as extracellular toxins, superantigens, lipopolysaccharide-modifying enzymes, and proteins conferring serum resistance, etc. (1). These findings suggest that the horizontal transfer of phage genes has contributed significantly to the acquisition of new genetic traits and to the genetic diversity of bacteria (1, 9, 10). To truly appreciate the mechanisms behind phage-associated evolution, it is important to understand the frequency and range of transfer of phage genes.Most phage genomes consist of many genes derived from different origins (5, 8). Some genes are similar to those of other phages with phylogenetically different hosts or are found in the genomes of bacteria that are not the phage hosts. The mosaic nature of phage genomes has been known for some time, and a body of molecular genetic studies of phages to explain the mechanisms that drive this feature have been attempted previously (1, 5). More importantly, the horizontal transfer of phage genes has emerged as a major factor in the evolution of the phage genome. Since recombination between phage and phage/prophage can occur when these elements coexist in the same cell, coinfection with multiple phage species may result in the production of hybrid phage genomes (5). The pathways by which phages exchange genetic material vary dramatically in concert with host ranges. However, conventional plaque assays have shown that the host ranges of the phages studied are narrow. We hypothesized that phage genes can be transferred to more diverse species than previously thought.In order to accurately quantify DNA movement, gene targeting that does not require cultivation or gene expression is necessary (7). In situ DNA amplification methods allow the visualization of specific DNA sequences inside bacterial cells. In this study, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) to examine the possible range and frequency of the transfer of phage genes. CPRINS uses one primer and results in linear amplification of the target DNA inside cells, and multiply labeled fluorescent probe sets are applied for detection of the amplicons to improve the specificity and sensitivity of CPRINS (3). Previously, CPRINS-FISH did clarify the movement of DNA of a specific gene among Escherichia coli cells at the single-cell level (4).Enterobacterial phages P1 and T4 infect E. coli and have been well studied. P1 can exist as circular DNA within the bacterial cell as if it were a plasmid. Phage T4 is capable of undergoing only a lytic life cycle and not the lysogenic life cycle. Conventional methods using plaque assays have shown that the host of P1 and T4 is E. coli, but orthologous phage genes have been found in bacteria other than E. coli (6, 8). In the present study, strains of Enterobacteriaceae were allowed to grow on agar medium after the phage was adsorbed, and the maintenance of the transferred phage gene in the bacterial genomes was examined at the community level by quantitative real-time PCR and at the single-cell level by CPRINS-FISH.The following bacterial strains were used for maintenance experiments: Citrobacter freundii IFO 12681, Enterobacter aerogenes BM 2688, E. coli NBRC 12713, a Proteus mirabilis clinical isolate, Salmonella enterica serovar Enteritidis IID 640, and Yersinia enterocolitica IID 981. The bacterial strains were grown in Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl; Nacalai Tesque Inc., Kyoto, Japan) at 37°C overnight.Stationary-phase cultures of 500 μl were incubated with 500 μl of SM buffer (50 mmol liter−1 Tris-HCl [pH 7.5], 100 mmol liter−1 NaCl, 8 mmol liter−1 MgSO4, 0.01% gelatin) containing the phage P1kc NBRC 20008 (2) or T4GT7 (11) at 37°C for 10 min at a multiplicity of infection of 1:1 (ratio of PFU of the phage to CFU of the recipient bacterium). The concentration of bacterial cells was adjusted to 109 cells ml−1. After 10 min of incubation, the diluted cell suspension (105 cells) was filtered through a polycarbonate filter (Advantec, Tokyo, Japan) with a pore size of 0.2 μm and a diameter of 25 mm. Cells trapped on the filter were cultured on LB agar medium at 37°C for 24 h. The filter was transferred into a microtube, and cells on the filter were suspended in 1 ml of sterile deionized water. The numbers of cells in the suspension and cells remaining on the filter were determined by using an epifluorescence microscope (see below) after staining of the samples with 1 μg ml−1 of 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Japan, Tokyo). The level of recovery of cells from the filter into sterile deionized water was about 99%. The cultured cells were subjected to real-time PCR and CPRINS-FISH.For real-time PCR, bacterial DNA was extracted using a QIAamp DNA isolation kit (Qiagen, Tokyo, Japan). The cell suspension was mixed with 10 mg ml−1 of lysozyme solution and incubated at 37°C for 1 h. DNA extraction was then performed according to the kit manufacturer''s instructions. Table Table11 lists the oligonucleotide primers for PCR and CPRINS and the polynucleotide probes used in the present study. Tail fiber genes from phages P1kc and T4GT7 were quantified by real-time PCR with a LightCycler system (Roche Diagnostics, Tokyo, Japan). LightCycler FastStart DNA master SYBR green I (Roche Diagnostics) was used with 5 mmol liter−1 Mg2+ and 0.5 μmol liter−1 (each) primers targeting the tail fiber genes of P1kc (P1-tail931f and P1-tail1148r) and T4GT7 (T4-tail2770f and T4-tail2983r). After a hot start for 10 min at 95°C, 40 cycles of PCR were run with denaturation at 94°C for 15 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, and fluorescence detection at 83°C for 5 s. The known amounts of PCR products from the phage DNA (101 to 107 copies per reaction) were used for the standard curves to quantify the target DNA. To confirm the specificity of the reaction after real-time PCR, the PCR mixture was collected in a glass capillary and subjected to agarose gel electrophoresis in addition to a melting-curve analysis with the LightCycler system. The maintenance frequencies determined by real-time PCR were recorded as the copy number of the phage tail fiber gene per bacterial genome detected by staining with PicoGreen (Invitrogen, Tokyo, Japan) after cultivation of cells on LB agar medium for 24 h as described above. The frequencies were determined in triplicate for each sample. The increase in the phage gene copy number was determined by comparing the copy numbers in cells on the filter before and after cultivation. The phage gene copy number in cells on the filter was determined by the following formula: (total number of cells determined by DAPI staining) × (phage tail fiber gene copy number determined by real-time PCR)/(bacterial genome copy number determined by PicoGreen staining).

TABLE 1.

Probes and primers designed in this study
NameTargetTypeNucleotide sequence (5′-3′)
P1-tail931fTail fiber gene of phage P1PrimerAACGACCCGAATTACAGCAC
P1-tail1148rTail fiber gene of phage P1PrimerAGTGCTGCTGCAAGCTCATA
T4-tail2770fTail fiber gene of phage T4PrimerAGCACAAATGGTGAGCACAG
T4-tail2983rTail fiber gene of phage T4PrimerTTGCTACCGTGTGGGTATGA
T4-tail2664Tail fiber gene of phage T4ProbeGGCTTCAAGTACTGACTTAGGTACTAAAACCACATCAAGCTTTGACTATGGTACG
T4-tail2720Tail fiber gene of phage T4ProbeAAGGGAACTAACAGTACGGGTGGACACACTCACTCTGGTAGTGGTTCTA
T4-tail2769Tail fiber gene of phage T4ProbeTAGCACAAATGGTGAGCACAGCCACTACATCGAGGCATGGAATGG
T4-tail2818Tail fiber gene of phage T4ProbeGGTGTAGGTGGTAATAAGATGTCATCATATGCCATATCATACAGGGCGGG
T4-tail2869Tail fiber gene of phage T4ProbeGGGAGTAACACTAATGCAGCAGGGAACCACAGTCACACTTTCTCTTTTGGG
T4-tail2922Tail fiber gene of phage T4ProbeTAGCAGTGCTGGCGACCATTCCCACTCTGTAGGTATTGGTGCTCATA
Open in a separate windowCPRINS-FISH targeting the tail fiber gene of phage T4GT7 was performed as described by Kenzaka et al. (3, 4), except for the probe/primer sequences and thermal conditions. After cell wall permeabilization by lysozyme treatment (3), the CPRINS reaction was performed under the following conditions: a hot start at 95°C for 9 min, denaturation at 94°C for 1 min, annealing at 60°C for 30 s, and extension at 72°C for 1.5 min for primer T4-tail2983r. Amplification was repeated for 30 cycles by using a thermal cycler (PTC-200; Bio-Rad Laboratories, Inc.). After amplification, filters were rinsed with 0.1% Nonidet P-40 and sterile deionized water, dehydrated in 99% ethanol, and vacuum dried. Hybridization with Alexa Fluor 546-labeled polynucleotide probes (T4-tail2664, T4-tail2720, T4-tail2769, T4-tail2818, T4-tail2869, and T4-tail2922), washing, and DAPI staining were performed as described in a previous study (4). In order to exclude the possibility of nonspecific probe binding to cell structures other than the target DNA in the target cells, FISH using laboratory strains without amplification of target DNA and CPRINS-FISH targeting the tail fiber genes in E. coli strains that did not carry the genes were performed.In order to examine the infection ranges of phages, plaque assays and direct counting of phages were performed. Plaque assays were performed with LB soft agar (0.8% agar) as described by Kenzaka et al. (4). For the direct counting, phages were stained with 5× SYBR gold (Invitrogen, Tokyo, Japan) and trapped onto an Anodisc filter (Whatman Japan, Tokyo) with a pore size of 0.02 μm and a diameter of 25 mm.The cells or phage particles on the filters were observed under an epifluorescence microscope (E-400; Nikon, Tokyo, Japan) with the Nikon filter sets UV-2A (EX300-350, DM400, and BA420) for DAPI, B-2A (EX450-490, DM505, and BA520) for SYBR gold, and HQ-CY3 (G535/50, FT565, and BP610/75) for Alexa Fluor 546. Images were acquired using a Retiga 2000R cooled charge-coupled device camera (QImaging, Surrey, BC, Canada), and at least 2,000 DAPI- or SYBR gold-stained objects per sample were counted. The maintenance frequencies determined by CPRINS-FISH were recorded as the number of CPRINS-FISH-positive cells divided by the total direct count of recipient cells after cultivation as described above. The frequencies were determined in triplicate for each sample.After cultivation on LB agar medium for 24 h, the total number of cells on the filter as determined by DAPI staining increased by 8.7 × 102- to 1.1 × 104-fold (Table (Table2).2). Real-time PCR showed that the phage P1kc gene copy number increased only in plaque-forming strains (E. coli and E. aerogenes) and not in non-plaque-forming strains (Table (Table2).2). In contrast, the phage T4GT7 gene copy number increased in both plaque-forming and non-plaque-forming strains by 7.6 × 101- to 7.0 × 104-fold. The maintenance frequencies were more than 10−2 gene copies per bacterial genome (Table (Table2).2). Direct observation via epifluorescence microscopy showed that progeny phages were not produced in the non-plaque-forming strains (Table (Table2),2), and thus, fragments of phage genes were thought to integrate into the genomes of non-plaque-forming strains and replicate with the bacterial genomes.

TABLE 2.

Frequencies of maintenance of phage P1kc and T4GT7 genes in Enterobacteriaceae strains
PhageRecipientResult for infection range indicator:
Increase in total no. of cellscIncrease in phage gene copy no. (SD)dMaintenance frequency (SD) as determined bye:
Plaque formationaProduction of progenybReal-time PCRCPRINS-FISH
P1kcC. freundii7.0 × 103None<1.5 × 10−3ND
E. aerogenes++1.7 × 1037.7 × 103 (6.5 × 103)5.0 × 100 (4.2 × 100)ND
E. coli++7.2 × 1035.5 × 103 (2.7 × 103)9.1 × 10−1 (0.5 × 10−1)ND
P. mirabilis7.4 × 103None<1.5 × 10−3ND
S. Enteritidis8.4 × 103None<1.7 × 10−4ND
Y. enterocolitica4.6 × 103None<1.8 × 10−4ND
T4GT7C. freundii1.5 × 1037.5 × 103 (4.0 × 103)8.3 × 10−1 (4.4 × 10−1)8.6 × 10−2 (3.4 × 10−2)
E. aerogenes++8.7 × 1021.2 × 103 (0.8 × 103)8.0 × 10−1 (5.0 × 10−1)4.0 × 10−1 (0.7 × 10−1)
E. coli++1.1 × 1047.0 × 104 (2.7 × 104)8.0 × 101 (3.0 × 10)2.1 × 10−1 (0.4 × 10−1)
P. mirabilis4.0 × 1035.8 × 103 (4.2 × 103)3.3 × 10−1 (2.4 × 10−1)3.4 × 10−2 (2.2 × 10−2)
S. Enteritidis1.0 × 1047.6 × 101 (5.0 × 101)1.0 × 10−2 (0.7 × 10−2)8.8 × 10−2 (2.0 × 10−2)
Y. enterocolitica3.6 × 1031.6 × 104 (0.4 × 104)6.1 × 10−1 (1.6 × 10−1)2.2 × 10−2 (2.9 × 10−2)
Open in a separate windowaPlaque formation on soft agar was tested.bThe production of progeny phage particles was observed via epifluorescence microscopy.cThe increase (n-fold) in the total number of cells during bacterial growth for 24 h was determined via epifluorescence microscopy.dThe increase (n-fold) in the copy number of the phage tail fiber gene during bacterial growth for 24 h was determined by real-time PCR. Values in parentheses indicate standard deviations of results for triplicate samples.eMaintenance frequencies were determined by real-time PCR and CPRINS-FISH analyses targeting the phage tail fiber gene and are shown as the phage tail fiber gene copy numbers per bacterial genome and the numbers of gene-positive cells divided by the total numbers of cells, respectively. Values in parentheses indicate standard deviations of results for triplicate samples. ND, not determined.Real-time PCR provided a copy number for the target phage gene in the whole population, but the location of the target phage gene and the frequency of cells carrying the target gene were unclear. In addition, bacterial genomic DNA, which was measured using PicoGreen, included phage DNA, and thus the frequencies measured by dividing by the amount of bacterial genomic DNA were probably less accurate than those measured as described below. In order to confirm that the phage gene was located inside bacterial cells and determine a more accurate maintenance frequency for total cells, CPRINS-FISH targeting the tail fiber gene of phage T4GT7 was performed. CPRINS-FISH visualized the target phage gene in individual cells under an epifluorescence microscope (Fig. (Fig.1).1). It showed that the frequencies of maintenance of the tail fiber gene, expressed as the number of gene-positive cells divided by the total number of cells, were 2.1 × 10−1 to 4.0 × 10−1 for plaque-forming strains after growth on LB medium for 24 h (Table (Table2).2). Since phage T4GT7 is capable of undergoing only a lytic life cycle, CPRINS-FISH would detect cells in which the phage gene was replicating. For non-plaque-forming strains, the maintenance frequencies were 2.2 × 10−2 to 8.8 × 10−2 (Table (Table2).2). If the gene was amplified by the CPRINS reaction outside bacterial cells, the amplicon would not accumulate inside bacterial cells and they would not exhibit bright fluorescence. Therefore, CPRINS-FISH proved that a part of the phage T4GT7 gene was located inside cells of non-plaque-forming strains. The tail fiber gene is responsible for the phage tail structure. The DNA sequences of the phage genes responsible for phage morphology have been found in many bacterial genomes (1, 5).Open in a separate windowFIG. 1.Visualization of E. coli cells carrying the tail fiber gene transferred by phage T4GT7. (A) After being mixed with phages for 10 min, E. coli NBRC 12713 cells were cultured for 24 h and subjected to CPRINS-FISH targeting the phage gene. Only cells having amplified tail fiber gene products emitted the fluorescence of the Alexa Fluor 546-labeled probe under green excitation (exposure, 0.5 s). (B) All DAPI-stained bacterial cells were visualized under UV excitation (exposure time, 0.1 s).In order to explore the effect of integration of the phage gene into the bacterial genome on bacterial heredity, we determined the mutation frequency for a C. freundii strain that acquired the phage T4GT7 gene. Two colonies which acquired the phage T4GT7 gene were screened by colony PCR with T4-tail2770f and T4-tail2983r primers and designated Cik8-1 and Cik8-4. Mutation frequencies were determined with LB medium containing 150 μg ml−1of rifampin (rifampicin) or 10 μg ml−1of nalidixic acid. The mutation frequencies associated with nalidixic acid resistance decreased by 12- to 240-fold and the frequencies associated with rifampin resistance decreased by 40- to 83-fold compared to those for the parent strains (Fig. (Fig.2).2). Mutation increases genetic variation. The decreased mutation frequency would contribute to the genetic stability of the genome in individual cells but not to genetic variation in the population. Our results show that phage T4GT7 was capable of affecting the genomic properties of C. freundii, which was thought previously not to be the host, although the mechanism by which mutation frequencies decreased remains unknown. Further experiments are required to clarify the molecular mechanism by which mutation frequencies altered after gene transfer.Open in a separate windowFIG. 2.Mutation frequencies for T4GT7-infected C. freundii strains. Mutation frequencies were determined with LB agar medium containing nalidixic acid or rifampin. Cik8-1 and Cik8-4 were strains which acquired a phage gene transferred from phage T4GT7. Cik1 and Cik2 were the parent strains.In summary, during growth on agar medium after the phage was allowed to be adsorbed by strains of Enterobacteriaceae, the phage P1kc gene was not maintained in non-plaque-forming strains but the phage T4GT7 gene was maintained in more diverse species than previously expected. The transfer of foreign DNA molecules (DNA entry) into a bacterium is an important first step in genetic diversification through horizontal gene transfer. A previous study reported that phage P1kc is capable of injecting DNA into non-plaque-forming E. coli cells (4), but the phage P1kc gene was not maintained during bacterial growth in the present study. The results showing the difference in maintenance between phage P1kc and T4GT7 genes suggest that the maintenance of transferred phage genes depends on phage gene sequences or other phage factors. When maintained, the phage gene could alter the mutation frequency for bacteria that acquired the gene, affecting the genomic variability at the population level. Conventionally, phage-bacterium interaction has been studied with certain models consisting of a phage and a bacterium in which the phage can multiply (12, 13). Our results indicate the importance of the dynamic of phage genes among diverse bacteria that were previously thought not to be hosts and the hereditary impact of phage gene transfer on such bacteria.  相似文献   

8.
9.
Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.Melioidosis is an endemic disease in Southeast Asia and northern Australia (2, 4) and also occurs sporadically in other parts of the world (3, 7). Human melioidosis was reported to occur in Brazil only in 2003, when a family outbreak afflicted four sisters in the rural part of the municipality of Tejuçuoca, Ceará state (14). After this episode, there was one reported case of melioidosis in 2004 in the rural area of Banabuiú, Ceará (14). And in 2005, a case of melioidosis associated with near drowning after a car accident was confirmed to occur in Aracoiaba, Ceará (11).The goal of this study was to investigate the Tejuçuoca and Banabuiú municipalities, where human cases of melioidosis have been confirmed to occur, and to gain a better understanding of the ecology of Burkholderia pseudomallei in this region.We chose as central points of the study the residences and surrounding areas of the melioidosis patients in the rural areas of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50′W) (Fig. (Fig.1).1). There are two well-defined seasons in each of these locations: one rainy (running from January to May) and one dry (from June to December). A total of 600 samples were collected at five sites in Tejuçuoca (T1, T2, T3, T4, and T5) and five in Banabuiú (B1, B2, B3, B4, and B5), distributed as follows (Fig. (Fig.2):2): backyards (B1 and T1), places shaded by trees (B2 and T2), water courses (B3 and T3), wet places (B4 and T4), and stock breeding areas (B5 and T5).Open in a separate windowFIG. 1.Municipalities of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50″W).Open in a separate windowFIG. 2.Soil sampling sites in Banabuiú and Tejuçuoca.Once a month for 12 months (a complete dry/rainy cycle), five samples were gathered at five different depths: at the surface and at 10, 20, 30 and 40 cm (Table (Table1).1). The samples were gathered according to the method used by Inglis et al. (9). Additionally, the sample processing and B. pseudomallei identification were carried out as previously reported (1, 8, 9).

TABLE 1.

Distribution of samples with isolates by site and soil depth
Sitesa and depth (cm)No. of B. pseudomallei isolates in samples from:
Banabuiú (n = 300)Tejuçuoca (n = 300)Total (n = 600)
B1/T13
    Surface2
    10
    201
    30
    40
B2/T21
    Surface1
    10
    20
    30
    40
B3/T315
    Surface2
    102
    204
    303
    404
B4/T45
    Surface
    101
    201
    3011
    401
B5/T52
    Surface
    10
    20
    302
    40
Total62026
Open in a separate windowaSites designated with B are in Banabuiú, and sites designated with T are in Tejuçuoca. See the text for details.The data on weather and soil composition were obtained from specialized government institutions, such as FUNCEME, IPECE, and EMBRAPA. The average annual temperature in both municipalities is between 26 and 28°C. In 2007, the annual rainfall in Tejuçuoca was 496.8 mm, and that in Banabuiú was 766.8 mm. There are a range of soil types in both Tejuçuoca and Banabuiú: noncalcic brown, sodic planossolic, red-yellow podzolic, and litholic. In Banabuiú, there are also alluvial and cambisol soils. The characteristic vegetation in both municipalities is caatinga (scrublands).There were isolates of B. pseudomallei in 26 (4.3%) of the 600 samples collected. The bacterium was isolated at a rate (3%) similar to that previously reported (9). The bacterium isolation occurred in both the dry (53.8%) and the rainy (46.2%) seasons. Tejuçuoca represented 76.9% (20/26) of the strains isolated. Four sites in Tejuçuoca (T1, T3, T4, and T5) and three in Banabuiú (B1, B2, and B4) presented isolates of the bacterium (Table (Table1).1). The isolation of the B. pseudomallei strains varied from the surface down to 40 cm. However, 17 of the 26 positive samples (65.3%) were found at depths between 20 and 40 cm (Table (Table1).1). Only two isolates were found at the surface during the dry season.A study in Vietnam (13) and one in Australia (9) reported the presence of B. pseudomallei near the houses of melioidosis patients. In our study, the same thing happened. Site T3 (15/26; 57.6%) was located 290 m from the patient''s house, as reported by the Rolim group (14).B. pseudomallei was isolated from a sheep paddock in Australia, where animals sought shelter below mango and fig trees (17). In our study, the bacterium was isolated at site T5, a goat corral alongside the house where the outbreak occurred in Tejuçuoca. Four sites in places shaded by trees yielded positive samples (30.7%) in both Tejuçuoca (palm trees) and Banabuiú (mango trees). Additionally, B. pseudomallei was isolated on three occasions from a cornfield (site 4B) located alongside the house of the melioidosis patient in Banabuiú.In the main areas of endemicity, the disease is more prevalent in the rainy season (4, 5, 16). The outbreak in Tejuçuoca was related to rainfall (14). Besides the association of cases of the disease with rainfall itself, the isolation of B. pseudomallei in soil and water was also demonstrated during the dry season (12, 15). An Australian study isolated strains from soil and water during the dry and rainy seasons (17). A Thai study also reported B. pseudomallei in the dry season (18). In our study, the isolation of B. pseudomallei took place either at the end of the wet season or in the dry months. Fourteen of the positive samples (53.8%) were collected during the dry season, albeit near a river or reservoir (sites T3 and B4).Physical, biological, and chemical soil features appear to influence the survival of B. pseudomallei (6, 10). In the present study, the soil was classified as litholic with sandy or clayey textures. It is susceptible to erosion, and when there is a lack of water, it is subject to salinization. During the dry season, the clay layer becomes dried, cracked, and very hard. During the rainy season, it becomes soggy and sticky. The isolation of B. pseudomallei in the dry season is possibly related to the capacity for adaptation of this soil, since the extreme conditions of lithosols do not prevent the bacterial growth and survival.It has been shown that B. pseudomallei is more often isolated at depths between 25 and 45 cm (17). In our study, 65.3% of the positive samples were taken at depths between 20 and 40 cm. Moreover, of these 17 samples, 10 (58.8%) were collected during the dry months. Also, unlike in other regions, two positive samples were taken from the surface in the period without rainfall.The rainfall in Tejuçuoca and Banabuiú is generally low, and temperatures do not vary significantly during the year. Therefore, the isolation of B. pseudomallei in these places occurs outside the rainfall, temperature, and moisture conditions observed in other regions of endemicity. Our data thus suggest that peculiar environmental features, such as soil composition, might favor the multiplication of B. pseudomallei in northeast Brazil.  相似文献   

10.
In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.The bacterial type IV prepilin peptidase (TFPP) is a well-characterized enzyme belonging to a family of novel aspartic acid proteases (20). It is responsible for the cleavage of N-terminal signal peptides from prepilins and pseudopilins, prior to their incorporation into the type IV pilus structure (22, 30, 31). The prepilin peptidase is also responsible for the processing of prepilin-like proteins needed for type II secretion (22). In Archaea, the existence of bacterial TFPP-like enzymes has also been reported, and they have been most extensively studied in relation to the assembly of the archaeal flagellum. In the euryarchaeotes Methanococcus maripaludis and Methanococcus voltae, the preflagellin peptidase FlaK was demonstrated to be responsible for cleaving the N-terminal signal peptide from the preflagellin prior to its incorporation into the growing flagellar filament, a step essential to flagellar assembly (6, 7, 26). In Sulfolobus solfataricus, an acidophilic crenarchaeote, the equivalent enzyme, PibD, was also shown to process preflagellins (4). Site-directed mutagenesis of FlaK and PibD demonstrated that both aspartic acid residues that aligned with aspartic acid residues essential for bacterial TFPP activity were also essential in the archaeal enzymes (6, 32), indicating that the two archaeal peptidases belong with the bacterial TFPPs in this novel family of aspartic acid proteases (20). More recently, an additional archaeal TFPP was found to be required for cleavage of the prepilin substrates (33) that are assembled into the unique pili of M. maripaludis (37).The substrate specificity of the archaeal preflagellin peptidase remains an open question. Like prepilin peptidases, FlaK in M. voltae has stringent requirements for the amino acids surrounding the cleavage site of the substrate, especially the −1 glycine, −2 and −3 lysines, and the +3 glycine (numbers given relative to the cleavage site) (35); the last position was conserved in all archaeal flagellins (25). Upon N-terminal sequence alignment of all available archaeal flagellin amino acid sequences at the predicted cleavage site, it was found that most archaeal preflagellin signal peptides are quite conserved in length, with the typical flagellin signal peptide being 11 to 12 amino acids in length (Table (Table1).1). It is speculated that while a certain amount of flexibility might exist, some optimum and minimum length probably exists that is crucial for the juxtaposition of the signal peptide and signal peptidase with respect to each other and the membrane (18). A recent study examining possible type IV pilin-like substrates in archaea using the FlaFind program indicated that such substrates may be more widespread than initially thought (33). Since in Methanococcus the pilins are processed by a second TFPP (EppA) (33), it is very possible that the preflagellins might be the only substrates of FlaK in these archaea.

TABLE 1.

N-terminal amino acid alignment of selected archaeal flagellin sequencesa
OrganismFlagellinN-terminal sequence
Archaeoglobus fulgidusFlaB1MGMRFLKNEKGFTGLEAAIVLIAFVTVAAVFSYVLL
Aeropyrum pernixFlaB1MRRRRGIVGIEAAIVLIAFVIVAAALAFVAL
Haloarcula marismortuiFlaAMFEKIANENERGQVGIGTLIVFIAMVLVAAIAAGVLI
Halobacterium salinarumFlgA1MFEFITDEDERGQVGIGTLIVFIAMVLVAAIAAGVLI
Methanocaldococcus jannaschiiFlaB1MKVFEFLKGKRGAMGIGTLIIFIAMVLVAAVAAAVLI
Methanococcoides burtoniiFlaMKANKHLMMNNDRAQAGIGTLIIFIAMVLVAAVAAAVLI
Methanococcus aeolicusFlaMNLEHFSFLKNKKGAMGIGTLIIFIAMVLVAAVAASVLI
Methanococcus maripaludisFlaB1MKIKEFLKTKKGASGIGTLIVFIAMVLVAAVAASVLI
Methanococcus vannieliiFlaB1MSVKNFMNNKKGDSGIGTLIVFIAMVLVAAVAASVLI
Methanococcus voltaeFlaB2MKIKEFMSNKKGASGIGTLIVFIAMVLVAAVAASVLI
Methanothermococcus thermolithotrophicusFlaB1MKIAQFIKDKKGASGIGTLIVFIAMVLVAAVAASVLI
Methanogenium marisnigriFlaMKRQFNDNAFTGLEAAIVLIAFIVVAAVFSYVVL
Methanospirillum hungateiFlaMNNEDGFSGLEAMIVLIAFVVVAAVFAYATL
Natrialba magadiiFlaB1MFEQNDDRDRGQVGIGTLIVFIAMVLVAAIAAGVLI
Natronomonas pharaonisFlg1MFETLTETKERGQVGIGTLIVFIALVLVAAIAAGVLI
Pyrococcus abyssiFlaB1MRRGAIGIGTLIVFIAMVLVAAVAAGVLI
Pyrococcus furiosusFlaMKKGAIGIGTLIVFIAMVLVAAVAAGVLI
Pyrococcus horikoshiiFlaB1MRRGAIGIGTLIVFIAMVLVAAVAAAVLI
Sulfolobus solfataricusFlaMNSKKMLKEYNKKVKRKGLAGLDTAIILIAFIITASVLAYVAI
Sulfolobus tokodaiiFlaMGAKNAIKKYNKIVKRKGLAGLDTAIILIAFIITASVLAYVAI
Thermococcus kodakarensisFlaB1MKTRTRKGAVGIGTLIVFIAMVLVAAVAAAVLI
Thermoplasma acidophilumFlaMRKVFSLKADNKAETGIGTLIVFIAMVLVAAVAATVLI
Thermoplasma volcaniumFlaMYIVKKMPILKLLNSIKRIFKTDDSKAESGIGVLIVFIAMILVAAVAASVLI
Open in a separate windowaIn all organisms listed, except Sulfolobus, there are multiple flagellins but only a single example is shown. The signal peptide is shown in boldface type. In some cases, analyses of the amino acid sequences of the signal peptides with unusual lengths revealed in-frame methionines or alternative start sites (underlined) that, if they represent the true translation start site, would result in signal peptides of more typical lengths. For S. solfataricus, Albers et al. (4) used the internal start site to give a signal peptide of 13 amino acids and demonstrated signal peptide processing.Studies on PibD in S. solfataricus, however, present interesting disparities. A recent genomic survey revealed a surprisingly large group of proteins possessing type IV pilin-like signal peptides in Sulfolobus compared to other archaea (2, 33). Besides the preflagellins, other substrates for PibD include pilins and proteins involved in sugar binding. Deletions of pibD appear to be nonviable (1), unlike the case for flaK, reinforcing the role of pibD in processes other than flagellum and pilus formation. Site-directed mutagenesis on the glucose-binding protein precursor (GlcS) signal peptide revealed that a wide variety of substitutions around the cleavage site still permitted processing. The allowed substitutions were consistent with the signal peptide sequences of a list of proposed PibD substrates, some of which have predicted signal peptides as short as 3 amino acids (4). Based on the observation that homologues of S. solfataricus sugar-binding proteins that contain type IV prepilin-like sequences were absent in the genome of another species of Sulfolobus, Sulfolobus tokodaii, it was speculated that S. solfataricus PibD may have undergone a specialization allowing for a broader substrate specificity (4). However, whether the extremely short signal peptides would be functional and recognizable as preflagellin peptidase substrates remains to be biochemically demonstrated.Although the typical flagellin signal peptide is 11 to 12 amino acids in length, a small number of archaeal preflagellins contain signal peptides of unusual lengths. Some are annotated to be unusually long (e.g., MJ0893 of Methanocaldococcus jannaschii and Ta1407 of Thermoplasma acidophilum) (Table (Table1).1). These sequences, however, contain in-frame alternative translational start sites that, if they correspond to true translation start sites, would result in signal peptides more typical in length. On the other hand, organisms with preflagellins predicted to possess unusually short signal peptides of 4 to 6 amino acids include Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, and Aeropyrum pernix (Table (Table1).1). These unusual signal peptides are deduced exclusively from gene sequences. Biochemical or genetic data to explain these peculiarities are still lacking. Assuming that the annotations of these genes are accurate, this would suggest that certain archaeal TFPP-like enzymes possess the capacity to process these much shorter signal peptides.In this study, for the first time, a systematic evaluation of critical signal peptide length for recognition and cleavage by two very different archaeal TFPP-like signal peptidases, M. voltae FlaK and S. solfataricus PibD, is reported.  相似文献   

11.
12.
Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.Gastroenteritis caused by Vibrio parahaemolyticus has been reported worldwide, though only sporadic cases have been reported in Europe (7, 14). The bacterium can be naturally present in seafood, but pathogenic isolates capable of inducing gastroenteritis in humans are rare in environmental samples (2 to 3%) (15) and are often not detected (10, 19, 20).The virulence of V. parahaemolyticus is based on the presence of a thermostable direct hemolysin (tdh) and/or the thermostable direct hemolysin-related gene (trh) (1, 5). Both are associated with gastrointestinal illnesses (2, 9).Spain is not only the second-largest producer in the world of live bivalve molluscs but also one of the largest consumers of bivalve molluscs, and Catalonia is the second-most important bivalve producer of the Spanish Autonomous Regions. Currently, the cultivation of bivalves in this area is concentrated in the delta region of the Ebro River. The risk of potentially pathogenic Vibrio spp. in products placed on the market is not assessed by existing legislative indices of food safety in the European Union, which emphasizes the need for a better knowledge of the prevalence of diarrheal vibrios in seafood products. The aim of this study was to investigate the distribution and pathogenic potential of V. parahaemolyticus in bivalve species exploited in the bays of the Ebro delta.Thirty animals of each species of Mytilus galloprovincialis, Crassostrea gigas, Ruditapes decussatus, and Ruditapes philippinarum were collected. They were sampled from six sites of the culture area, three in each bay of the Ebro River delta, at the beginning (40°37′112"N, 0°37′092"E [Alfacs]; 40°46′723"N, 0°43′943"E [Fangar]), middle (40°37′125"N, 0°38′570"E [Alfacs]; 40°46′666"N, 0°45′855"E [Fangar]), and end (40°37′309"N, 0°39′934"E [Alfacs]; 40°46′338"N, 0°44′941"E [Fangar]) of the culture polygon. Clams were sampled from only one site per bay as follows: in the Alfacs Bay from a natural bed of R. decussatus (40°37′44"N, 0°38′0"E) and in the Fangar Bay from an aquaculture bed of R. philippinarum (40°47′3"N, 0°43′8"E). In total, 367 samples were analyzed in 2006 (180 oysters, 127 mussels, 30 carpet shell clams, and 30 Manila clams) and 417 samples were analyzed in 2008 (178 oysters, 179 mussels, 30 carpet shell clams, and 30 Manila clams).All animals were individually processed and homogenized, and 1 ml of the homogenate was inoculated into 9 ml of alkaline peptone water (Scharlau, Spain). Following a 6-h incubation at 37°C, one loopful of the contents of each tube of alkaline peptone water was streaked onto CHROMagar vibrio plates (CHROMagar, France) and incubated for 18 h at 37°C. Mauve-purple colonies were purified, and each purified isolate was cryopreserved at −80°C (135 isolates in 2006 and 96 in 2008). From the initial homogenate portion, 100 μl was inoculated onto marine agar (Scharlau, Spain) and onto thiosulfate citrate-bile salts-sucrose agar (Scharlau, Spain) for total heterotrophic marine bacteria counts and total vibrio counts, respectively (Table (Table11).

TABLE 1.

Vibrio parahaemolyticus isolates, serotypes, and origins and total number of vibrios/heterotrophic bacteria contained in the bivalvea
IsolateDate of collectionOrganism and site of originTemp (°C)Salinity (‰)Gene(s)SerotypeBacterial count using indicated medium (CFU ml−1)
TCBS agarMarine agar
I7458 August 2006Mg-F24.537tdhND1.5 × 1041.2 × 104
I79314 August 2006Cg-A2535tdhND9.2 × 1028.5 × 103
I80514 August 2006Cg-A2535tdhO2:KUT7.2 × 1029 × 103
I80614 August 2006Cg-A2535tdh and trhO3:K331.9 × 1034.6 × 103
I80914 August 2006Cg-A2535tdhO2:K288 × 1047.3 × 102
I6784 July 2006Rd-A28.636tdhO2:K283.1 × 1052.5 × 105
I6284 July 2006Rd-A28.636tdhO4:KUT2.9 × 1048.4 × 104
I7758 August 2006Cg-A24.537tdhND4.21 × 1031.1 × 104
I6914 July 2006Rd-A28.636trhO1:K322.2 × 1052.6 × 105
I71227 July 2006Mg-A29.435.5trhO1:KUT8.6 × 1038.4 × 103
I7658 August 2006Cg-F24.537trhO4:K341 × 104Uncountable
I98022 July 2008Cg-A26.733.5tdhO1:K322.7 × 1041.3 × 104
I98122 July 2008Cg-A26.733.5trhO1:KUT1 × 1042.2 × 104
I99322 July 2008Cg-A26.733.5tdhO5:K173 × 1031.1 × 104
I99429 July 2008Mg-A27.737trhO3:KUT3.4 × 1037 × 103
I10315 August 2008Cg-F27.737tdhO5:KUT5.5 × 1043.3 × 104
I10345 August 2008Cg-F27.737tdhO3:KUT8.7 × 1044 × 104
I10405 August 2008Cg-F27.737tdhO3:KUT1.6 × 1043.2 × 104
I10425 August 2008Cg-F27.737tdh and trhND2.8 ×1043 × 104
I10505 August 2008Cg-F27.737tdhO1:KUT4.7 × 1047.3 × 104
I106320 August 2008Mg-F25.936tdhO3:KUT7.9 ×1041.4 × 104
I106520 August 2008Mg-F25.936tdhO2:KUT2.2 × 1031.2 × 104
I106820 August 2008Mg-F25.936tdhO5:KUT2.6 × 1045.2 × 104
I106920 August 2008Mg-F25.936tdhO3:KUT2.4 × 1035.3 × 104
I107320 August 2008Mg-F25.936tdhO5:KUT2.3 × 1037.5 × 103
I107420 August 2008Mg-F25.936tdhO3:KUT7.6 × 1046.9 × 104
I107720 August 2008Mg-F25.936tdhO4:KUT1.7 × 1031.6 × 103
I107920 August 2008Mg-F25.936trhO3:KUT2.5 × 1031.1 × 104
I109220 August 2008Mg-F25.936tdhND1.7 × 1031.6 × 103
I113025 August 2008Rd-A26.435tdhND1.7 × 1043.8 × 104
I114325 August 2008Rd-A26.435tdhND1.1 × 1041.9 × 104
I116525 August 2008Rd-A26.435trhO2:KUT4.4 × 1046.8 × 104
I113325 August 2008Rp-F25.536.5tdhND3.4 × 1044 × 104
I113425 August 2008Rp-F25.536.5tdhND3.9 × 1045.8 × 104
I115825 August 2008Rp-F25.536.5trhO4:KUT6.6 × 1044.7 × 104
I116125 August 2008Rp-F25.536.5trhO3:KUT2.2 × 1046.6 × 104
Open in a separate windowaMg, Mytilus galloprovincialis; Cg, Crassostrea gigas; Rd, Ruditapes decussatus; Rp, R. phillipinarum; A, Alfacs; F, Fangar; ND, not determined; TCBS, thiosulfate citrate-bile salts-sucrose.Total DNA was extracted from each purified isolate using the Wizard genomic DNA purification kit (Promega), following the instructions of the manufacturer. A one-step PCR analysis was performed to identify/confirm which isolates were tl positive (species marker for V. parahaemolyticus). Further detection of the tdh or trh gene was carried out on all positive tl strains. All PCR analyses were carried out using the primers described by Bej et al. (2) with the following amplification conditions on the thermocycler (Eppendorf Mastercycler Personal): an initial denaturation at 95°C for 8 min, followed by 40 cycles of a 1-min denaturation at 94°C, annealing at 55°C for 1 min, elongation at 72° for 1 min, and a final extension of 10 min at 72°C. Positive and negative controls were included in all reaction mixtures: two positive controls, tl and tdh CAIM 1400 and trh CAIM 1772 (Collection of Aquatic Important Microorganisms [http://www.ciad.mx/caim/CAIM.html]), and negative control DNA-free molecular grade water (Sigma-Aldrich, Spain). Expected amplicons were visualized in 2% agarose gels stained with ethidium bromide.Fifty-eight isolates contained the gene tl in 2006 and 96 in 2008, which confirmed their identity as V. parahaemolyticus. In 2006, the distribution of the 58 isolates was as follows: 7 from 127 mussels, 34 from 180 oysters, and 17 from 30 R. decussatus clams. No tl-positive isolates were found in R. philippinarum. PCR analysis of the tl-positive isolates for the presence of the tdh or trh gene indicated that eight isolates contained the tdh gene and four contained the trh gene. In 2008, the source of the confirmed V. parahaemolyticus isolates was as follows: 31 from 88 oysters, 44 from 89 mussels, 9 from 30 R. decussatus clams, and 12 from 30 R. philippinarum clams. Of these, 17 were found to contain the tdh gene and 7 contained the trh gene. Two isolates (I806 and I1042) contained both toxigenic genes, tdh and trh.Putative tdh- and trh-positive PCR products were purified using the QIAquick PCR purification kit (Qiagen) following the manufacturer''s instructions and were sequenced bidirectionally by Macrogen Inc. Sequences were aligned using BioEdit (8) and analyzed using BLAST (National Center for Biotechnology Information). None of the toxigenic isolates was found positive by PCR analysis for the presence of open reading frame 8 of the phage 237 (16), a marker for the pandemic strain O3:K6.The isolates were fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) as described previously (3), and the resulting electrophoretic band patterns were analyzed with the GelCompar II software (v4.5; Applied Maths). The similarity matrix was calculated with the Jaccard coefficient with a band position tolerance of 0.8%, and the dendrogram was constructed with the Ward algorithm. A high level of genomic diversity was found among the 32 toxigenic isolates characterized by rep-PCR. Three clonal groups were identified (those having identical rep-PCR band patterns) (Fig. 1a to c).Open in a separate windowFIG. 1.rep-PCR dendrogram of toxigenic isolates of V. parahaemolyticus isolated in the Ebro delta. Letters denote clonal groups of isolates.In vitro antibiotic susceptibility tests were performed using the diffusion disc test following a previously described protocol (18). The antibiotics used were gentamicin (10 μg), oxolinic acid (10 μg), amoxicillin (25 μg), polymyxin B (300 UI), vancomycin (30 μg), trimethoprim sulfamethoxazole (1.25/23.75 μg), nitrofurantoin (300 μg), doxycyclin (30 μg), ceftazidime (30 μg), streptomycin (10 μg), neomycin (30 UI), penicillin (6 μg), flumequine (30 μg), tetracycline (30 μg), ampicillin (10 μg), kanamycin (30 μg), ciprofloxacin (5 μg), and sulfonamide (300 μg). All tests were performed in duplicate. A Student t test for two samples with unequal variance was performed to compare the sensitivity of all 2006 isolates against the sensitivity of 2008 isolates for each antibiotic (Microsoft Office Excel 97-2003). Antibiogram results revealed a lower susceptibility in 2008 than in 2006, indicating a possible shift in overall susceptibility. Results from the t test indicated that significantly lower susceptibility in 2008 was detected (P ≤ 0.05; n = 36) for the following antibiotics: vancomycin, polymyxin B, ampicillin, amoxicillin, gentamicin, neomycin, trimethoprim sulfamethoxazole, nitrofurantoin, doxycyclin, ceftazidime, tetracycline, flumequine, and ciprofloxacin.The serological types for 27 strains were determined by the agglutination method using commercially available V. parahaemolyticus antisera (Denka Seiken Ltd.; Cosmos Biomedical Ltd, United Kingdom) following the manufacturer''s instructions. Potentially toxigenic V. parahaemolyticus isolates collected in 2006 were serologically heterogeneous (8 out of the 11 isolates) (Table (Table1).1). In isolates collected in 2008, results were more homogenous, with seven serotypes found among 19 isolates analyzed. The O3:K6 serotype was not detected in any of the strains analyzed, in agreement with the open reading frame 8 PCR results.The present study is the first to report the detection of potentially diarrheal V. parahaemolyticus strains isolated from cultured bivalves on Spanish Mediterranean coasts, providing data on the presence of both tdh- and trh-positive isolates. V. parahaemolyticus has previously been detected in several European countries (4, 13, 21, 22). A recent study carried out in Spain detected tdh-positive V. parahaemolyticus strains from patients who had consumed fresh oysters in a market in Galicia on the Atlantic coast of Spain (12) and potentially pathogenic V. parahaemolyticus strains have also been reported in France (17). These studies indicate that the risk of infections caused by V. parahaemolyticus in Europe is low compared to that in America or Asia (15). However, this risk could have been underestimated, since V. parahaemolyticus is not included in the current European surveillance programs, such as the European Network for Epidemiological Surveillance and Control of Communicable Diseases.Toxigenic V. parahaemolyticus strains detected in this study were genomically and serologically heterogeneous. The pandemic serotype O3:K6 was not detected, and although attempts to isolate O3:K6 from the environment and from seafood have not always been successful in previous studies reviewed by Nair and coauthors (15), this finding seems to be in agreement with the fact that no outbreak of diarrhea was observed in the area. Interestingly, isolates I806 and I1042 have been found positive for both tdh and trh in PCR tests. The coexistence of tdh and trh genes has already been reported in isolates from Japan, the United States, and Mexico (3, 6, 11, 19, 23). To our knowledge, no occurrence of an environmental isolate positive for both tdh and trh had previously been reported in Europe. All isolates tested were slightly different in their antibiotic resistance profiles. Typically, a high level of resistance could be determined. The detection of tdh- and/or trh-positive V. parahaemolyticus strains for the first time on the Mediterranean coast emphasizes the need to monitor for the presence of potentially diarrheal vibrios and bacterial gastroenteritis, and these data should be taken into consideration to revise the European legislation on the requirements for shellfish harvested for consumption in order to include the surveillance of these pathogens in Europe.  相似文献   

13.
Twenty-one salts were tested for their effects on the growth of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum. In liquid medium, 11 salts (0.2 M) exhibited strong inhibition of bacterial growth. The inhibitory action of salts relates to the water-ionizing capacity and the lipophilicity of their constituent ions.Different biochemical mechanisms have been put forth to explain the antimicrobial activity of organic and inorganic salts, including inhibition of several steps of the energy metabolism (benzoate, bicarbonate, propionate, sorbate, and sulfite salts) (2, 3, 11, 16, 17, 19, 25) and complexation to DNA and RNA (aluminum and sulfites) (12, 13, 15, 20, 27, 28). However, little is known about the physicochemical basis for the general antimicrobial action of salts. The objective of this work was to gain an understanding of the relationship between the inhibitory action of salts on bacterial growth and their physicochemical properties by using the bacteria Pectobacterium carotovorum subsp. carotovorum (formerly Erwinia carotovora subsp. carotovora) and Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica). These bacteria are responsible for soft rot, a disease of economic importance affecting numerous stored vegetable crops (14, 22).Pectobacterium carotovorum subsp. carotovorum (strain Ecc 1367) and P. atrosepticum (strain Eca 709), provided by the Laboratoire de Diagnostic en Phytoprotection (MAPAQ, Québec, Canada), were grown in 250-ml flasks containing 50 ml of 20% tryptic soy broth (Difco Laboratories, Becton Dickinson, Sparks, MD) amended with salts (200 mM) or unamended (control), by incubation at 24°C with agitation (150 rpm; Lab-Line Instruments Inc., Melrose Park, IL) for 24 h. The pHs of the media were not adjusted but varied with the type of salts, unless stated otherwise. Flasks were inoculated with 100 μl of each bacterial suspension (1 × 107 CFU/ml). Bacterial growth was determined by turbidimetry at 600 nm with a UV/visible spectrophotometer (Ultrospec 2000; Pharmacia Biotech Ltd, Cambridge, United Kingdom), using appropriate blanks. Results were expressed as the percentage of growth inhibition compared with the growth of the control. A completely randomized experimental design with three replicates was used, the experimental unit being a flask. Analysis of variance was carried out with the GLM (general linear model) procedure of SAS (SAS Institute, Cary, NC) software. When they were significant (P < 0.05), treatment means were compared using Fisher''s protected least-significant-difference test.Among the 21 salts tested, sodium carbonate, sodium metabisulfite, trisodium phosphate, aluminum lactate, aluminum chloride, sodium bicarbonate, sodium propionate, ammonium acetate, aluminum dihydroxy acetate, potassium sorbate, and sodium benzoate exhibited strong inhibition (≥97%) of the growth of both P. carotovorum subsp. carotovorum and P. atrosepticum (Table (Table1).1). Calcium chloride, sodium formate, sodium acetate, ammonium hydrogen phosphate, and sodium hydrogen phosphate exhibited a moderately inhibitory effect; sodium lactate and tartrate had no effect. On the other hand, ammonium chloride, potassium chloride, and sodium chloride stimulated the growth of P. atrosepticum.

TABLE 1.

Effect of salts on the growth of P. atrosepticum and P. carotovorum subsp. carotovorum
Salt (0.2 M)apHbOsmotic pressure (atm)cGrowth inhibition (%)d
P. atrosepticumP. carotovorum subsp. carotovorum
Aluminum dihydroxy acetate [Al(OH)2C2H3O2]4.99.79100 a100 a
Aluminum chloride (AlCl3·6H2O)2.519.57100 a100 a
Aluminum lactate [Al(C3H5O3)3]3.419.57100 a100 a
Ammonium acetate (NH4C2H3O2)7.29.79100 a100 a
Ammonium chloride (NH4Cl)7.09.79−18 dND
Ammonium hydrogen phosphate [(NH4)2HPO4]8.314.6843 b23 c
Calcium chloride (CaCl2·2H2O)5.814.6885 a70 b
Potassium chloride (KCl)7.39.79−27 dND
Potassium sorbate (KC6H7O2)7.79.79100 a97 a
Sodium acetate (NaC2H3O2·3H2O)7.49.7963 bND
Sodium benzoate (NaC7H5O2)7.49.79100 a100 a
Sodium bicarbonate (NaHCO3)8.19.79100 a100 a
Sodium carbonate (Na2CO3)10.614.68100 a100 a
Sodium chloride (NaCl)7.29.79−29 dND
Sodium formate (NaCHO2)7.39.7924 cND
Sodium lactate (C3H5O3Na)7.39.793 cND
Sodium metabisulfite (Na2S2O5)4.519.57100 a100 a
Sodium hydrogen phosphate (Na2HPO4)8.714.6869 b61 b
Sodium propionate (NaC3H5O2)7.49.79100 a99 a
Sodium tartrate (Na2C4H4O6·2H2O)7.314.682 cND
Trisodium phosphate (Na3PO4·12H2O)11.919.57100 a100 a
Open in a separate windowaSalts were purchased from Sigma Chemical Co. (St. Louis, MO), except for ammonium acetate (BDH Inc., Toronto, Canada), sodium chloride (BDH), sodium bicarbonate (BDH), and aluminum lactate (Aldrich Chemical, Milwaukee, WI).bpH of the medium amended with each salt.cOsmotic pressure of the salt solution was calculated using van’t Hoff''s equation, Π = iRTc, where R is the gas constant, T is the absolute temperature (K), c is the concentration of the salt (mol/liter), and i is the number of ions into which the salt dissociates in solution.dPercentage of growth inhibition compared to growth of the control. Each value represents the mean of three replicates. Values in the same column followed by the same letter are not significantly different according to Fisher''s protected least-significant-difference test (P > 0.05). ND, not determined. Negative values signify bacterial growth stimulation.Several factors in the salt solutions can contribute to bacterial growth inhibition. Elevated osmolarity due to salt addition may trigger the osmoregulatory process, causing an increased maintenance metabolism and leading to reduction in bacterial growth. Thus, we calculated the osmotic pressure (Π) of salt solutions using van''t Hoff''s equation (26). As shown in Table Table1,1, salts with comparable osmolarities displayed complete or no bacterial growth inhibition, indicating that osmotic stress or reduction in water activity alone may not have brought about the inhibition of the bacterial growth. Therefore, other factors may play a role.The acidity or alkalinity of the medium resulting from the addition of some of the salts can have profoundly adverse effects on bacterial growth. Extreme pH conditions can lead to denaturation of proteins like enzymes present on the cell surface, depolarization of transport for essential ions and nutrients, modification of cytoplasmic pH, and DNA damage (12, 18). Table Table11 shows that the addition of aluminum lactate, aluminum chloride, and sodium metabisulfite, whose ΔpHs (ΔpH = |7.5 [the optimal pH for growth] − the pH of the salt-amended medium|) are ≥3, strongly acidified the medium, whereas the addition of sodium carbonate and trisodium phosphate strongly increased the pH (ΔpH ≥ 3.1). Except for ammonium acetate, sodium acetate, sodium bicarbonate, and the preservative salts (potassium sorbate, sodium benzoate, and sodium propionate), whose ΔpHs are <1, all the other salts generally display inhibitory effects when ΔpH values are ≥1 (Fig. (Fig.1).1). Based on this result, the effect of the highly acidic or alkaline salts (which strongly affected the pH of the medium) on the growth of P. atrosepticum was evaluated at pH 7.5. Sodium carbonate and sodium metabisulfite completely inhibited bacterial growth at pH 7.5, as they did at pHs 10.6 and 4.5, respectively; trisodium phosphate (pH 11.9) exhibited a slightly lower inhibitory effect (growth inhibition of 83.2%) at pH 7.5. These observations suggest that growth inhibition by sodium carbonate, sodium metabisulfite, and trisodium phosphate cannot be attributed solely to extreme pH and passive proton transfer (extreme pH) across the bacterial membrane. Since aluminum salts precipitate at pH 7.5 (due to formation of hydrated aluminum hydroxide), it was not possible to test their inhibitory effect at pH 7.5.Open in a separate windowFIG. 1.Relationship between ΔpH (|7.5 [the optimal pH for growth] − the pH of the salt-amended medium|) and growth inhibition of Pectobacterium atrosepticum. 1, Sodium chloride; 2, potassium chloride; 3, ammonium chloride; 4, sodium tartrate; 5, sodium lactate; 6, sodium formate; 7, ammonium hydrogen phosphate; 8, sodium acetate; 9, sodium hydrogen phosphate; 10, calcium chloride; 11, ammonium acetate; 12, sodium benzoate; 13, sodium propionate; 14, potassium sorbate; 15, sodium bicarbonate; 16, aluminum dihydroxy acetate; 17, sodium metabisulfite; 18, sodium carbonate; 19, aluminum lactate; 20, aluminum chloride; 21, trisodium phosphate.The dissociation of salts in aqueous medium generates ionic species which can participate in proton exchange reactions with water molecules. The capacity of an ion to dissociate water is an intrinsic characteristic, determined by its pK value (pKa for acidic species or pKb for basic ones) (4, 21, 24). For an ionic strength of >0.1 M, pKa and pKb values of the ions are more accurate when they are defined as apparent constants (pK′a or pK′b) in terms of the activities of hydronium and hydroxyl ions, ionic species concentrations and activity coefficients (6). Thus, for the acidic ions, we have the equation ), and for the basic anions, pK′b = pKb + log(γHB/γB), where pK′a and pK′b are the apparent acidity constant and basicity constant, respectively; is the activity coefficient of the conjugate base (B); and γHB is that of the acidic (HB) species. The activity coefficient (γ) of the species i can be expressed as a function of ionic strength (μ), using the Güntelberg approximation of the Debye-Hückel equation (21), as follows: −log γi=[(0.51Zi2 μ1/2)/(1 + μ1/2)], where Zi is the charge on the species i, and μ is the ionic strength. Thus, log(/γHB) = [(0.51μ1/2)/(1 + μ1/2)] (), and log(γHB/) = −[(0.51μ1/2)/(1 + μ1/2)] ().Polytropic acid-potentiating ions (bicarbonate, carbonate, monohydrogen phosphate, phosphate, sulfite, and tartrate) in an aqueous solution can exist as (n + 1) possible species for which the parent acid is HnA. These species may coexist in equilibrium under certain pH conditions. For these ions, pK′a or pK′b were expressed as the means of the coexisting species at a specified pH. Calculated values for pK′a of acidic anions and cations and calculated values for pK′b of basic anions are presented in Table Table2.2. Figure Figure2A2A shows a sigmoidal relationship between the inhibitory effect of salts on bacterial growth and the pK′b value of the basic ions (with a common cation, sodium or potassium, in the salt) and the pK′a value of the acidic ions (with a common anion, chloride, in the salt). The plot exhibits a sharp linear relationship in the pK′ range of 8.0 to 12.0. Below the pK′ value of 8.0, inhibition is maximal, whereas above the pK′ value of 11.0, ions appear to stimulate growth (growth was maximal above the pK′ value of 12). This result demonstrates that the capacity of the constitutive ions of the salts to either donate or subtract protons to water molecules, either in the growth environment (as reflected in the modification of the medium pH) or in the developing cells, generally plays a role in their inhibitory action. The consequent transmembrane pH gradient generated leads to a passive H+ transport across the microbial membrane and to acidification (in the case of ions with low pK′a) or alkalinization (in the case of ions with low pK′b) of the cytoplasm, once the capacity for proton-coupled active transport is outstripped. In both cases, proton exchange with outer membrane proteins will destabilize these proteins, their interaction with membrane lipids, and ultimately, their function in solute transport, leading to growth inhibition. The modification of cytoplasmic pH can also alter nucleic acid structures and functions and contribute to growth inhibition (18).Open in a separate windowFIG. 2.(A) Relationship between the growth inhibition of Pectobacterium atrosepticum and the apparent basicity constant (pK′b,•) of basic anions with common Na+ (or K+) cations in the salt, the apparent acidity constant (pK′a,○) of acidic bisulfite anion (HSO3), and the cations with common Cl ions in the salt. (B) Relationship between the growth inhibition of Pectobacterium atrosepticum and the addition parameter (pK′ + pPo/w) combining the partition coefficient (Po/w) and pK′b (•) of basic anions (common cation, Na+ or K+, in the salt) or pK′a (○) of cations (common anion, Cl, in the salt) and the acidic bisulfite anion (HSO3).

TABLE 2.

Calculated apparent values for acidity, pK′a, and basicity, pK′ba
SaltBasic anion
Cation and acidic anion
pHIonic species or species in equilibriumpK′bpHIonic species or species in equilibriumpK′a
Sodium acetate7.4Acetate9.5
Sodium benzoate7.4Benzoate10.0
Sodium bicarbonate8.1H2CO3/HCO3b7.7
Sodium carbonate10.6HCO3/CO32−6.1
Sodium formate7.3Formate10.4
Sodium hydrogen phosphate8.7H2PO4/HPO42−9.8
Sodium lactate7.3Lactate11.1
Trisodium phosphate11.9HPO42−/PO43−5.3
Sodium propionate7.4Propionate9.3
Potassium sorbate7.7Sorbate9.4
Sodium tartrate7.3Tartrate2−10.6
Sodium chloride7.2Cl17.2
Sodium metabisulfite4.5SO2·H2O/HSO34.0
Aluminum chloride2.5Al3+6.2
Calcium chloride5.8Ca2+13.4
Potassium chloride7.3K+16.2
Sodium chloride7.2Na+15.0
Ammonium chloride7.0NH4+9.5
Open in a separate windowaCalculation of pK′ was performed according to Edsall and Wyman (6). pH values were measured at 0.2 M.bIncludes CO2·H2O and H2CO3.However, the water-ionizing capacity of the constituent ions of the salts and the consequent modification of the pH of the medium are not the sole factors accounting for growth inhibition, as suggested by the exceptional inhibitory actions of benzoate, propionate, and sorbate (Fig. (Fig.11 and and2A).2A). These ions provide a higher inhibition than is expected from their pK′ values (pK′b values of 10.0, 9.3, and 9.4, respectively), while the pH of their solution is optimal for bacterial growth (pHs of 7.4, 7.4, and 7.7, respectively). This suggests that they possess additional characteristics mediating their action, in addition to their water-ionization property. In fact, these preservative agents have been shown to be active either as undissociated acids (like other weak acids) or as anions (7, 8), due to their possibly hydrophobic nature which would allow them to interact with lipid constituents of the cell envelope of gram-negative bacteria such as Pectobacterium spp., and to modify their functionality (5), resulting in growth inhibition. They can also cross the cell envelope due to their lipophilicity, and their acidification inside the cell can cause additional adverse effects.Thus, we determined the octanol/water partition coefficient (Po/w), an indicator of the lipophilic character of a compound, for the effective salts with common sodium (or potassium) or chloride ions. The Po/w coefficients of the salts were determined in duplicate by using the general solvent-solvent separation procedure (9). Equal volumes (50 ml) of 1-octanol (Sigma Chemical Co., St. Louis, MO) and bidistilled water were poured into a separating flask and thoroughly shaken for 5 min. Four grams of each salt was then added, and the flask content was thoroughly mixed three times for 5 min each time, with a rest period of 5 min after each agitation. After complete separation (20 to 24 h at room temperature), the two phases were recovered separately in different flasks, and the concentration of the accompanying ion of the salt was measured in each phase by atomic absorption (model 3300 unit; Perkin-Elmer, Ueberlinger, Germany). The Po/w coefficient was calculated as the ratio of the concentration of ion in 1-octanol to the concentration of ion in the aqueous phase. Sodium benzoate was found to be the most lipophilic (Po/w = 1.41 × 10−2), followed by potassium sorbate (Po/w = 7.6 × 10−3) and sodium metabisulfite (Po/w = 2.0 × 10−4). Most other salts, sodium chloride (reference salt), sodium bicarbonate and carbonate, sodium propionate, sodium acetate, calcium chloride, and aluminum chloride mainly remained in the aqueous phase (Po/w = 2.0 × 10−5 to 5.0 × 10−5). This lipophilic characteristic of benzoate and sorbate ions would result from a reduced charge density in their molecules (due to the conjugated double bonds in their molecules). An addition parameter, pK′ + pPo/w, which combines the two properties of salts ions, i.e., the water-ionizing capacity (pK′) and the lipophilicity (pPo/w = −log Po/w), appears to provide a more general basis for the inhibitory effect of salts (Fig. (Fig.2B).2B). This suggests that while the dissociation constant of ions plays a major role in growth inhibition, as seen in Fig. Fig.2A,2A, the lipophilic character of the preservative-salt ions confers to them an added ability to penetrate the cell envelope and to inhibit bacterial growth (5, 10). The exclusion of ammonium (lower inhibition than expected from its pK′a value) and calcium (higher inhibition than expected from its pK′a value) ions from the sigmoidal pattern portrayed in Fig. Fig.2B2B would have resulted from their interactions with water and other molecules (NH4+) (1) or from cell membrane destabilization (Ca2+) (23).In conclusion, the study has shown that several salts (0.2 M concentration), including aluminum dihydroxy acetate, aluminum chloride, aluminum lactate, ammonium acetate, potassium sorbate, sodium benzoate, sodium metabisulfite, sodium bicarbonate, sodium carbonate, sodium propionate, and trisodium phosphate, strongly inhibited the growth of P. carotovorum subsp. carotovorum and P. atrosepticum. In addition, the study has established for the first time a basic sigmoidal relationship between the antimicrobial activity of the salts and the physicochemical characteristics of their constituent ions, namely their water-ionizing capacity and their lipophilicity. The constituent ions of the highly inhibiting salts generally displayed a high capacity to ionize water molecules (low pK′a or pK′b values) (Al3+, CO32−, PO43−, HCO3, and HSO3) or a high lipophilicity (benzoate and sorbate), and these two parameters in combination with known biochemical activities of salts ions would affect bacterial growth.  相似文献   

14.
Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table (Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table (Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table (Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table Table22.

TABLE 1.

E. coli strains used in this study
E. coli strain (n)Source
ECOR strains (72)Thomas Whittman
Laboratory adapted (6)
    K-12 DavisPaul Blum
    CG5C 4401Paul Blum
    K-12 StanfordPaul Blum
    W3110Paul Blum
    BTyler Kokjohn
    AB 1157Tyler Kokjohn
O157:H7 (10)
    FRIK 528Andrew Benson
    ATCC 43895Andrew Benson
    MC 1061Andrew Benson
    C536Tim Cebula
    C503Tim Cebula
    C535Tim Cebula
    ATCC 43889William Cray, Jr.
    ATCC 43890William Cray, Jr.
    ATCC 43888Willaim Cray, Jr.
    ATCC 43894William Cray, Jr.
Open in a separate window

TABLE 2.

Physiological comparison of 88 strains of Escherichia coli
Growth medium or conditionOxygencNo. of strains with type of growthb
ECOR strains (n = 72)
Laboratory strains (n = 6)
O157:H7 strains (n = 10)
GoodPoorNoneVariableGoodPoorNoneVariableGoodPoorNoneVariable
LB controlaBoth72000600010000
1% SDSAerobic6930060008002
5% SDSAerobic6840060008200
1% SDSAnaerobic53154023101702
5% SDSAnaerobic0684004200704
CTABd (all)Both00720006000100
0.05% BACAerobic31158202220091
0.2% BACAerobic01710105000100
0.05% BACAnaerobic2367001500091
0.2% BACAnaerobic00720006000100
pH 6.5Both72000600010000
pH 6Both72000600010000
pH 5Both7020060009001
pH 4.6Both70200600010000
pH 4.3Aerobic14015731203205
pH 4.3Anaerobic6930031201100
pH 4.1 or 4.2Aerobic00720NDgND
pH 4.0Both0072000600091
M63 with supplemente
    GlucoseAerobicf6912050109010
    GlucoseAnaerobicf7002050109010
    GluconateBoth6912050109010
    GlucuronateAerobic6822050109010
    GlucuronateAnaerobic6912050109010
Open in a separate windowaEight LB controls were run, two for each set of LB experiments: SDS, CTAB, benzalkonium chloride (BAC), and pH stress.bGrowth was measured as either +++, +, or 0 (good, poor, and none, respectively), with +++ being the growth achieved on the LB control plates. “Variable” means that two or three replicates did not agree. All experiments were done at 37°C.c“Anaerobic” refers to use of an Oxoid anaerobic chamber. Aerobic and anaerobic growth data are presented together when the results were identical and separately when the results were not the same or the anaerobic set had not been done. LB plates were measured after 1 (aerobic) or 2 (anaerobic) days, and the M63 plates were measured after 2 or 3 days.dCTAB used at 0.05, 0.2%, and 0.4%.eM63 defined medium (3) was supplemented with glucose, gluconate, or glucuronate, all at 0.2%.fIdentical results were obtained with and without 0.0001% thiamine.gND, not determined.  相似文献   

15.
A molecular diagnostic system using single nucleotide polymorphisms (SNPs) was developed to identify four Sclerotinia species: S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. DNAs of samples are hybridized with each of five 15-bp oligonucleotide probes containing an SNP site midsequence unique to each species. For additional verification, hybridizations were performed using diagnostic single nucleotide substitutions at a 17-bp sequence of the calmodulin locus. The accuracy of these procedures was compared to that of a restriction fragment length polymorphism (RFLP) method based on Southern hybridizations of EcoRI-digested genomic DNA probed with the ribosomal DNA-containing plasmid probe pMF2, previously shown to differentiate S. sclerotiorum, S. minor, and S. trifoliorum. The efficiency of the SNP-based assay as a diagnostic test was evaluated in a blind screening of 48 Sclerotinia isolates from agricultural and wild hosts. One isolate of Botrytis cinerea was used as a negative control. The SNP-based assay accurately identified 96% of Sclerotinia isolates and could be performed faster than RFLP profiling using pMF2. This method shows promise for accurate, high-throughput species identification.Sclerotinia is distinguished morphologically from other genera in the Sclerotiniaceae (Ascomycota, Pezizomycotina, Leotiomycetes) by the production of tuberoid sclerotia that do not incorporate host tissue, by the production of microconidia that function as spermatia but not as a disseminative asexual state, and by the development of a layer of textura globulosa composing the outer tissue of apothecia (8). Two hundred forty-six species of Sclerotinia have been reported, most distinguished morphotaxonomically (Index Fungorum [www.indexfungorum.org]). These include the four species of agricultural importance now recognized plus many that are imperfectly known, seldom collected, or apparently endemic to relatively small geographic areas (2, 5, 6, 7, 8, 9, 17).The main species of phythopathological interest in the genus Sclerotinia are S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. Sclerotinia species 1 is an important cause of disease in vegetables in Alaska (16) and has been found in association with wild Taraxacum sp., Caltha palustris, and Aconitum septentrionalis in Norway (7). It is morphologically indistinguishable from S. sclerotiorum, but it was shown to be a distinct species based on distinctive polymorphisms in sequences from internal transcribed spacer 2 (ITS2) of the nuclear ribosomal repeat (7). The other three species have been delimited using morphological, cytological, biochemical, and molecular characters (3, 8, 9, 10, 12, 15). Interestingly, given that the ITS is sufficiently polymorphic in many fungal genera to resolve species, in Sclerotinia, only species 1 and S. trifoliorum are distinguished by characteristic ITS sequence polymorphisms; S. sclerotiorum and S. minor cannot be distinguished based on ITS sequence (2, 7).Sclerotinia sclerotiorum is a necrotrophic pathogen with a broad host range (1). S. minor has a more restricted host range but causes disease in a variety of important crops such as lettuce, peanut, and sunflower crops (11). S. trifoliorum has a much narrower host range, limited to the Fabaceae (3, 8, 9). Sclerotial and ascospore characteristics also serve to differentiate among the three species. Sclerotinia minor has small sclerotia that develop throughout the colony in vitro and aggregate to form crusts on the host, while the sclerotia of S. sclerotiorum and S. trifoliorum are large and form at the colony periphery in vitro, remaining separate on the host (8, 9). The failure of an isolate to produce sclerotia or apothecia in vitro is not unusual, especially after serial cultivation (8). The presence of dimorphic, tetranucleate ascospores characterizes S. trifoliorum, while S. sclerotiorum and S. minor both have uniformly sized ascospores that are binucleate and tetranucleate, respectively (9, 14).With the apparent exception of Sclerotinia species 1, morphological characteristics are sufficient to delimit Sclerotinia species given that workers have all manifestations of the life cycle in hand. In cultures freshly isolated from infected plants, investigators usually have mycelia and sclerotia but not apothecia. Restriction fragment length polymorphisms (RFLPs) in ribosomal DNA (rDNA) are diagnostic for Sclerotinia species (3, 10), but the assay requires cloned probes (usually accessed from other laboratories) hybridized to Southern blots from vertical gels, an impractical procedure for large samples. We have analyzed sequence data from previous phylogenetic studies (2) and have identified diagnostic variation for the rapid identification of the four Sclerotinia species. The single nucleotide polymorphism (SNP) assay that we report here is amenable to a high throughput of samples and requires only PCR amplification with a standard set of primers and oligonucleotide hybridizations to Southern blots in a dot format.The SNP assay was performed using two independent sets of species-specific oligonucleotide probes, all with SNP sites shown to differentiate the four Sclerotinia species (Fig. (Fig.1).1). A panel of 49 anonymously coded isolates (Table (Table1)1) was screened using these species-specific SNP probes, as outlined in Fig. Fig.1.1. The assay was validated by comparison to Southern hybridizations of EcoRI-digested genomic DNA hybridized with pMF2, a plasmid probe containing the portion of the rDNA repeat with the 18S, 5.8S, and 26S rRNA cistrons of Neurospora crassa (4, 10).Open in a separate windowFIG. 1.Protocol for the SNP-based identification of Sclerotinia species, with diagnostic SNP sites underlined and in boldface type for each hybridization probe.

TABLE 1.

Isolates and hybridization results for all SNP-based oligonucleotide probesf
Collector''s isolateAnonymous codePrescreened presumed species identityOriginHostSpecies-specific SNP
IGS50CAL448 S.trifolCAL124CAL448 S.minorRAS148CAL446 S.sp1CAL19ACAL19BCAL448 S.sclero
LMK1849Botrytis cinereaOntario, CanadaAllium cepa
FA2-13Sclerotinia minorNorth CarolinaArachis hypogaea++
W15Sclerotinia minorNorth CarolinaCyperus esculentus++
W1030Sclerotinia minorNorth CarolinaOenothra laciniata++
PF1-138Sclerotinia minorNorth CarolinaArachis hypogaea++
PF18-49714Sclerotinia minorOklahomaArachis hypogaea++
PF17-48246Sclerotinia minorOklahomaArachis hypogaea++
PF19-51948Sclerotinia minorOklahomaArachis hypogaea++
LF-2720Sclerotinia minorUnited StatesLactuca sativa++
AR12811Sclerotinia sclerotiorumArgentinaArachis hypogaea++
AR128216Sclerotinia sclerotiorumArgentinaArachis hypogaea++
LMK2116Sclerotinia sclerotiorumCanadaBrassica napus++
LMK5725Sclerotinia sclerotiorumNorwayRanunculus ficaria++
LMK75415Sclerotinia sclerotiorumNorwayRanunculus ficaria++
UR1939Sclerotinia sclerotiorumUruguayLactuca sativa++
UR4789Sclerotinia sclerotiorumUruguayLactuca sativa++
CA90132Sclerotinia sclerotiorumCaliforniaLactuca sativa++
CA99540Sclerotinia sclerotiorumCaliforniaLactuca sativa++
CA104441Sclerotinia sclerotiorumCaliforniaLactuca sativa++
1980a34Sclerotinia sclerotiorumNebraskaPhaseolus vulgaris++
Ss00113Sclerotinia sclerotiorumNew YorkbGlycine max++
Ssp00531Sclerotinia sclerotiorumNew YorkGlycine max++
H02-V2833Sclerotinia species 1AlaskacUnknown vegetable crop++
H01-V1426Sclerotinia species 1AlaskaUnknown vegetable crop++
LMK74521Sclerotinia species 1NorwayTaraxacum sp.++
02-2611Sclerotinia trifoliorumFinlanddTrifolium pratense+
06-1429Sclerotinia trifoliorumFinlandTrifolium pratense++
2022Sclerotinia trifoliorumFinlandTrifolium pratense++
2-L945Sclerotinia trifoliorumFinlandTrifolium pratense++
3-A524Sclerotinia trifoliorumFinlandTrifolium pratense
5-L912Sclerotinia trifoliorumFinlandTrifolium pratense++
K14Sclerotinia trifoliorumFinlandTrifolium pratense++
K237Sclerotinia trifoliorumFinlandTrifolium pratense++
L-11223Sclerotinia trifoliorumFinlandTrifolium pratense++
L-11944Sclerotinia trifoliorumFinlandTrifolium pratense++
LMK3619Sclerotinia trifoliorumTasmaniaTrifolium repens++
Ssp00118Sclerotinia trifoliorumNew YorkLotus corniculatus++
Ssp00210Sclerotinia trifoliorumNew YorkLotus corniculatus++
Ssp00328Sclerotinia trifoliorumNew YorkLotus corniculatus++
Ssp00436Sclerotinia trifoliorumNew YorkLotus corniculatus++
LMK4743Sclerotinia trifoliorumVirginiaMedicago sativa++
MBRS-127UnknownAustraliaeBrassica spp.++
MBRS-27UnknownAustraliaBrassica spp.++
MBRS-342UnknownAustraliaBrassica spp.++
MBRS-522UnknownAustraliaBrassica spp.++
WW-135UnknownAustraliaBrassica spp.++
WW-28UnknownAustraliaBrassica spp.++
WW-317UnknownAustraliaBrassica spp.++
WW-447UnknownAustraliaBrassica spp.++
Open in a separate windowaThe annotated genome for S. sclerotiorum strain 1980 (ATCC 18683) is publicly available through the Broad Institute, Cambridge, MA (http://www.broad.mit.edu/annotation/genome/sclerotinia_sclerotiorum/Home.html).bAll isolates from New York were provided by Gary C. Bergstrom, Cornell University, Ithaca, NY. Isolates Ss001 and Ssp005 were submitted as S. sclerotiorum, and Ssp001 through Ssp004 were submitted as S. trifoliorum.cAll isolates from Alaska, submitted as Sclerotinia species 1, were provided by Lori Winton, USDA-ARS Subarctic Agricultural Research Unit, University of Alaska, Fairbanks.dAll isolates from Finland, submitted as S. trifoliorum, were provided by Tapani Yli-Mattila, University of Turku, Turku, Finland.eAll isolates from Australia, presumed to be S. sclerotiorum but requiring species confirmation, were provided by Martin Barbetti, DAF Plant Protection Branch, South Perth, Australia.fThe probes that are diagnostic for S. minor, S. sclerotiorum, S. trifoliorum, and Sclerotinia species 1 are listed, with a “+” indicating a positive hybridization for the probe and a “−” indicating no hybridization of the probe.  相似文献   

16.
Sulfonamide-resistant Escherichia coli and Salmonella isolates from pigs and chickens in Ontario and Québec were screened for sul1, sul2, and sul3 by PCR. Each sul gene was distributed differently across populations, with a significant difference between distribution in commensal E. coli and Salmonella isolates and sul3 restricted mainly to porcine E. coli isolates.Resistance to sulfonamides is frequent in bacteria from farm animals (7, 8, 9, 10) and is usually caused by the acquisition of the genes sul1, sul2, and sul3 (20, 22). The objectives of this study were (i) to assess the distribution of these genes in Escherichia coli and Salmonella enterica isolates in swine and chickens from two major provinces in Canada, (ii) to assess whether differences occur in the distribution of these genes among bacterial species found within two different animal host species, and (iii) to assess whether significant differences in the distribution of these genes are present between the commensal E. coli strains used as indicators for surveillance of antimicrobial resistance and the zoonotic Salmonella pathogens found in the same ecological niche. In contrast to previous studies, a multivariable logistic regression model was used to analyze the data, control for confounding factors, and assess the interaction effect between animal and bacterial species in terms of the probability of an isolate carrying a specific sul gene. The distribution of sulfonamide resistance genes among sulfonamide-resistant E. coli (393 isolates from chickens and 311 from swine) and Salmonella (13 isolates from chickens and 221 from swine) isolates was assessed. These isolates were collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) between 2003 and 2005 from ceca of apparently healthy animals at abattoirs in Ontario (n = 435) and Québec (n = 503). The methods used by CIPARS are presented in detail elsewhere (8-10). The isolates were screened with a previously published multiplex PCR for sul1, sul2, and sul3 (16). The sul1 and sul2 genes were found in E. coli and Salmonella isolates from both animal species. The sul3 gene was detected in both E. coli and Salmonella isolates from swine but only in E. coli isolates from chickens (Table (Table1).1). Three percent of the isolates had no detectable sul gene, 12.5% possessed two genes, and two isolates carried three genes (Table (Table1).1). Similar (2, 3, 14, 19) or higher (4, 11, 17) values for multiple genes have been reported by others. The overall higher prevalence of sul1 in Salmonella isolates and of sul2 and sul3 in E. coli isolates was in agreement with the results of previous studies (2-4, 11, 12, 14, 21).

TABLE 1.

Frequency of the three resistance genes sul1, sul2, and sul3 in sulfonamide-resistant E. coli and Salmonella isolates from chickens and swine in Ontario and Québec between 2003 and 2005
Bacterial speciesSource (no. of isolates)No. (%) of isolates with indicated gene(s)
sul1sul2sul3sul1 + sul2sul1+ sul3sul2 + sul3sul1 + sul2 + sul3None
E. coliSwine (311)61 (19.6)66 (21.2)132 (42.4)11 (3.5)9 (2.9)11 (3.5)2 (0.6)19 (6.1)
Chickens (393)103 (26.2)211 (53.7)9 (2.3)64 (16.3)0 (0.0)0 (0.0)0 (0.0)6 (1.5)
Total (704)164 (23.3)277 (39.3)141 (20.0)75 (10.7)9 (1.3)11 (1.6)2 (0.3)25 (3.6)
SalmonellaSwine (221)173 (78.3)20 (9.0)5 (2.3)7 (3.2)0 (0.0)14 (6.3)0 (0.0)2 (0.9)
Chickens (13)7 (53.8)5 (38.5)0 (0.0)0 (0.0)0 (0.0)0 (0.0)0 (0.0)1 (7.7)
Total (234)180 (76.9)25 (10.7)5 (2.1)7 (3.0)0 (0.0)14 (6.0)0 (0.0)3 (1.3)
Open in a separate windowThree logistic regression models (Table (Table2)2) for associations between the presence of each sul gene and the bacterial species, animal species, province of origin of the animals, and year of isolation were built using Stata 9 (StataCorp, College Station, TX). Statistical interactions between bacterial and animal species were assessed in the sul1 and sul2 models but not in the sul3 model (the sample size was insufficient). Tests were two tailed, with significance at a P value of ≤0.05. A significant interaction between bacterial and animal species was observed for sul1 (Table (Table2).2). The presence of statistical interactions indicates that the effect of each variable depends on the state of the other variable. Thus, the effects of bacterial and animal species on the distribution of sul1 cannot be interpreted independently. For instance, sul1 was more frequent in Salmonella isolates from swine than from chickens, but the reverse was true for E. coli isolates (Table (Table3).3). Genomic islands containing sul1 are present in some Salmonella serovars, including S. enterica serovar Typhimurium and S. Derby (1, 6, 18), which were the most frequent in the swine samples of this study (Table (Table4).4). This contrasts with E. coli strains, in which sul1 is usually associated with transposons and large transferable plasmids (22). Thus, the presence of significant statistical interactions in the sul1 model could be an indicator of the differential importance of horizontal gene transfer in E. coli strains versus clonal expansion of specific Salmonella serovars in the animal species investigated. No significant interaction was detected for sul2 (P = 0.66) (Table (Table2).2). This could be the consequence of the relatively small number of sul2-positive Salmonella isolates and the resultant lack of statistical power; however, it is also possible that sul2 has a different epidemiology than sul1. The sul2 gene has not been shown to be located on genomic islands and is usually plasmid borne (22). It may therefore be transferred more frequently than sul1 between E. coli and Salmonella and between bacteria from swine and chickens, leading to the absence of a significant interaction in the sul2 model. Serotyping, molecular typing, and assessment of gene transferability would be required to test these hypotheses.

TABLE 2.

Multivariable models for the distribution of the sul1, sul2, and sul3 sulfonamide resistance genes from swine and chickens at slaughter in Ontario and Québec between 2003 and 2005
Explanatory variable (referent group) or interactionOdds ratio (95% confidence interval)b
sul1sul2sul3
Salmonella (E. coli)1.54 (0.50-4.74)0.33 (0.21-0.51)0.10 (0.06-0.16)
Swine (chickens)0.49 (0.36-0.68)c0.16 (0.12-0.23)c39.57 (20.21-77.48)c
Québec (Ontario)0.80 (0.60-1.07)2.20 (1.61-2.99)c0.83 (0.56-1.23)
2004 (2003)0.77 (0.56-1.06)0.99 (0.71-1.40)1.84 (1.18-2.86)
2005 (2003)0.68 (0.45-1.04)1.36 (0.88-2.09)1.66 (0.99-2.79)
2005 (2004)0.88 (0.58-1.36)1.36 (0.88-2.11)0.90 (0.53-1.53)
Bacterial species × animal speciesa12.84 (3.79-43.46)cNINI
Open in a separate windowaStatistical interaction between data for bacterial species and animal species. The terms for this interaction are described in Table Table33.bExample of odds ratio interpretation: the odds of a porcine isolate carrying sul3 were 39.57 times higher than for an isolate obtained from chickens. NI, not included in the model. The sul2 model was not included because the P value for this interaction was equal to 0.66, and the sul3 model was not included because it did not converge when including this interaction.cP < 0.001.

TABLE 3.

Interaction terms for the association between bacterial and animal species for the presence of sul1
Contrast variablesOdds ratioa95% Confidence intervalP value
Salmonella in pigs vs Salmonella in chickens6.301.95-20.390.002
E. coli in pigs vs E. coli in chickens0.490.36-0.67<0.001
Salmonella in chickens vs E. coli in chickens1.540.50-4.740.451
Salmonella in pigs vs E. coli in pigs19.7912.28-31.87<0.001
Open in a separate windowaExample of interpretation: the interaction effect suggests that sul1-mediated sulfonamide resistance is 19.79 times more likely to occur in Salmonella in pigs than in E. coli in pigs.

TABLE 4.

Salmonella serovars and frequency of resistance genes detected in each serovar
Salmonella serovarNo. of isolatesaNo. of isolates with indicated genea
sul1sul2sul3
Agona14/014/00/00/0
Berta3/00/03/00/0
Bovismorbificans1/00/00/00/0
Brandenburg3/01/01/00/0
Derby63/059/04/02/0
Give O:15+1/01/00/00/0
Heidelberg2/40/30/02/0
4,12:−:−3/03/00/00/0
4,12:i:−2/02/00/00/0
4,5,12:−:−1/01/00/01/0
Rough-O:fg:−1/01/00/00/0
Rough-O:l,v:enz151/01/01/00/0
Infantis3/01/02/00/0
Johannesburg1/01/00/00/0
London2/01/00/01/0
Manhattan2/00/02/00/0
Mbandaka6/06/01/00/0
Ohio O:14+1/00/01/00/0
Putten1/01/00/00/0
Schwarzengrund0/60/10/50/0
Typhimurium110/3101/312/013/0
Total221/13194/727/519/0
Open in a separate windowaThe first and second numbers in each column represent swine and chicken isolates, respectively; the total number of tabulated occurrences of sulfonamide genes is larger than the number of resistant isolates investigated because some isolates carried several genes simultaneously.A significant increase in the prevalence of sul3 was detected between 2003 and 2004 (P = 0.007) but not between 2004 and 2005 (P = 0.055) nor at any time for sul1 and sul2 (Table (Table2).2). The sul3 gene has emerged recently (5), and its prevalence was probably still increasing in 2003. The odds of finding sul3 in Salmonella isolates were 10 times lower than for E. coli isolates and 40 times higher in swine than in chickens (Table (Table2).2). Other studies have also found high frequencies of sul3 in porcine E. coli isolates (5, 12, 20) and much lower frequencies in other sources (4, 11, 12, 14). Although these isolates were not typed, previous results have shown that sul3 is present in both pathogenic and commensal porcine E. coli isolates (5) of at least 13 different serotypes (P. Boerlin and R. M. Travis, unpublished data). The sul3 gene has spread extensively across porcine E. coli populations in North America and Europe but remains uncommon in other major farm animal species and in Salmonella populations (Table (Table1)1) (2, 13). It may require more time to spread to other populations. There may be biological and ecological barriers slowing its spread to bacteria of other animal species or coselection factors that favor its presence in porcine E. coli populations.Using the example of sulfonamides as a model for the application of multivariable statistical approaches to the study of antimicrobial resistance epidemiology, this study indicates that the relative frequencies of genes encoding resistance to the same antimicrobial either present in bacterial populations for decades or recently emerged can vary significantly between animal hosts and phylogenetically related bacterial species sharing the same ecological niche. Differences in the distribution of resistance determinants may remain hidden when assessing resistance phenotypes. Similar antimicrobial susceptibility results do not necessarily imply similar resistance genes. These findings highlight the need to further explore the interactions between commensals and pathogens and the ways in which commensal bacteria are interpreted as indicators of antimicrobial resistance in pathogens.  相似文献   

17.
Vertebrate genomic assemblies were analyzed for endogenous sequences related to any known viruses with single-stranded DNA genomes. Numerous high-confidence examples related to the Circoviridae and two genera in the family Parvoviridae, the parvoviruses and dependoviruses, were found and were broadly distributed among 31 of the 49 vertebrate species tested. Our analyses indicate that the ages of both virus families may exceed 40 to 50 million years. Shared features of the replication strategies of these viruses may explain the high incidence of the integrations.It has long been appreciated that retroviruses can contribute significantly to the genetic makeup of host organisms. Genes related to certain other viruses with single-stranded RNA genomes, formerly considered to be most unlikely candidates for such contribution, have recently been detected throughout the vertebrate phylogenetic tree (1, 6, 13). Here, we report that viruses with single-stranded DNA (ssDNA) genomes have also contributed to the genetic makeup of many organisms, stretching back as far as the Paleocene period and possibly the late Cretaceous period of evolution.Determining the evolutionary ages of viruses can be problematic, as their mutation rates may be high and their replication may be rapid but also sporadic. To establish a lower age limit for currently circulating ssDNA viruses, we analyzed 49 published vertebrate genomic assemblies for the presence of sequences derived from the NCBI RefSeq database of 2,382 proteins from known viruses in this category, representing a total of 23 classified genera from 7 virus families. Our survey uncovered numerous high-confidence examples of endogenous sequences related to the Circoviridae and to two genera in the family Parvoviridae: the parvoviruses and dependoviruses (Fig. (Fig.11).Open in a separate windowFIG. 1.Phylogenetic tree of vertebrate organisms and history of ssDNA virus integrations. Times of integration of ancestral dependoviruses (yellow icosahedrons), parvoviruses (blue icosahedrons), and circoviruses (triangles) are approximate.The Dependovirus and Parvovirus genomes are typically 4 to 6 kb in length, include 2 major open reading frames (encoding replicase proteins [Rep and NS1, respectively] and capsid proteins [Cap and VP1, respectively]), and have characteristic hairpin structures at both ends (Fig. (Fig.2).2). For replication, these viruses depend on host enzymes that are recruited by the viral replicase proteins to the hairpin regions, where self-primed viral DNA synthesis is initiated (2). Circovirus genomes are typically ∼2-kb circles. DNA of the type species, porcine circovirus 1 (PCV-1), contains a stem-loop structure within the origin of replication (Fig. (Fig.2),2), and the largest open reading frame includes sequences that are homologous to the Parvovirus replicase open reading frame (9, 11). The circoviruses also depend on host enzymes for replication, and DNA synthesis is self-primed from a 3′-OH end formed by endonucleolytic cleavage of the stem-loop structure (4). The frequency of Dependovirus infection is estimated to be as high as 90% within an individual''s lifetime. None of the dependoviruses have been associated with human disease, but related viruses in the family Parvoviridae (e.g., erythrovirus B19 and possibly human bocavirus) are pathogenic for humans, and members of both the Parvoviridae and the Circoviridae can cause a variety of animal diseases (2, 4).Open in a separate windowFIG. 2.Schematics illustrating the structure and organization of Parvoviridae and Circoviridae genomes and origins of several of the longest-integrated ancestral viral sequences found in vertebrates. Integrations were aligned to the Dependovirus adeno-associated virus 2 (AAV2), the Parvovirus minute virus of mice (MVM), and the Circovirus porcine circovirus 1 (PCV-1). The inverted terminal repeat (ITR) sequences in the Dependovirus and Parvovirus genomes are depicted on an expanded scale. A linear representation of the circular genome of PCV-1 is shown with the 10-bp stem-loop structure on an expanded scale. Horizontal lines beneath the maps indicate the lengths of similar sequences that could be identified by BLAST. The numbers indicate the locations of amino acids in the viral proteins where the sequence similarities in the endogenous insertions start and end. The actual ancestral virus-derived integrated sequences may extend beyond the indicated regions.With some ancestral endogenous sequences that we identified, phylogenetic comparisons can be used to estimate age. For example, as a Dependovirus-like sequence is present at the same location in the genomes of mice and rats, the ancestral virus must have existed before their divergence, more than 20 million years ago. Some Circovirus- and Dependovirus-related integrations also predate the split between dog and panda, about 42 million years ago. However, in most other cases, we rely on an indirect method for estimating age (1). As genomic sequences evolve, they accumulate new stop codons and insertion/deletion-induced frameshifts. The rates of these events can be tied directly to the rates of neutral sequence drift and, therefore, the time of evolution. To apply this method, we first performed a BLAST search of vertebrate genomes for all known ssDNA virus proteins (BLAST options, -p tblastn -M BLOSUM62 -e 1e−4). Candidate sequences were then recorded, along with 5 kb of flanking regions, and then again aligned against the database of ssDNA viruses to find the most complete alignment (BLAST options, -t blastx -F F -w 15 -t 1500 -Z 150 -G 13 -E 1 -e 1e−2). Detected alignments were then compared with a neutral model of genome evolution, as described in the supplemental material, and the numbers of stop codons and frameshifts were converted into the expected genomic drift undergone by the sequences. The age of integration was then estimated from the known phylogeny of vertebrates (7, 10). Using these methods, we discovered that as many as 110 ssDNA virus-related sequences have been integrated into the 49 vertebrate genomes considered, during a time period ranging from the present to over 40 to 60 million years ago (Table (Table1;1; see also Tables S1 to S3 in the supplemental material).

TABLE 1.

Selected endogenous sequences in vertebrate genomes related to single-stranded DNA viruses
Virus group and vertebrate speciesInitial genomic search using TBLASTN
Best sequence homology identified using BLASTX
Predicted nucleotide drift (%)Integration labelAge (million yr) or timing of integration based on sequence aging
Chromosomal or scaffold locationProteinBLAST E value/% sequence identityMost similar virusaProteinCoordinatesNo. of stop codons/frameshifts
Circoviruses
    CatScaffold_62068Rep6E−05/37Canary circovirusRep4-2833/7 in 268 aab14.2fcECLG-182
Scaffold_24038Rep6E−06/51Columbid circovirusRep44-3174/5 in 231 aac15.2fcECLG-287
    DogChr5dRep7E−16/46Raven circovirusRep16-2636/5 in 250 aa17.6cfECLG-198
Chr22Rep1E−14/43Beak and feather disease virusRep7-2642/1 in 261 aac4.5cfECLG-254
    OpossumChr3Rep4E−46/44Finch circovirusRep2-2910/2 in 282 aa2.3mdECLG12
Cap6-360/0 in 30 aa
Dependoviruses
    DogChrXRep6E−05/55AAV5Rep239-4453/4 in 200 aa14.0cfEDLG-178
    DolphinGeneScaffold1475Rep8E−39/39Avian AAV DA1Rep79-4863/4 in 379 aac6.6ttEDLG-255
Cap4E−61/47Cap1-7384/7 in 678 aac
    ElephantScaffold_4Rep0/55AAV5Rep3-5890/0 in 579 aa0.0laEDLGRecent
    HyraxGeneScaffold5020Cap3E−34/53AAV3Cap485-7350/5 in 256 aa7.0pcEDLG-129
Scaffold_19252Rep9E−72/47Bovine AAVRep2-3488/4 in 348 aa14.3pcEDLG-260
    MegabatScaffold_5601Rep2E−13/31AAV2Rep315-4791/5 in 175 aa13.1pvEDLG-376
    MicrobatGeneScaffold2026Rep1E−117/50AAV2Rep1-6172/5 in 612 aa5.8mlEDLG-127
Cap9E−33/51Cap1-7312/9 in 509 aac
Scaffold_146492Cap6E−32/42AAV2Cap479-7320/3 in 252 aa4.2mlEDLG-219
    MouseChr1Rep2E−06/34AAV2Rep4-2063/5 in 191 aa17.1mmEDLG-139
Chr3Rep2E−24/31AAV5Rep71-47812/7 in 389 aa16.5mmEDLG-237
Cap2E−22/45Cap22-72412/10 in 649aac
Chr8Rep1E−08/46AAV2Rep314-4733/3 in 147 aa13.8mmEDLG-331
Cap1-1371/2 in 114 aa
    PandaScaffold2359Rep2E−06/37Bovine AAVRep238-4262/3 in 186 aa10.4amEDLG-159
    PikaScaffold_9941Rep4E−14/28AAV5Rep126-4152/2 in 282 aa5.4opEDLG14
    PlatypusChr2Rep9E−10/35Bovine AAVRep297-4374/3 in 138 aa17.1oaEDLG-179
Cap272-4191/2 in 150 aac
Contig12430Rep2E−09/47Bovine AAVRep353-4503/1 in 123 aa12.0oaEDLG-255
Cap2E−05/32Cap253-3672/1 in 116 aa
    RabbitChr10Rep3E−97/39AAV2Rep1-6193/9 in 613 aa9.3ocEDLG43
Cap5E−50/45Cap1-72310/9 in 675 aa
    RatChr13Rep2E−09/33AAV2Rep4-1752/4 in 177 aa13.3rnEDLG-128
Chr2Rep4E−18/40AAV5Rep1-46112/12 in 454 aa22.7rnEDLG-251
Chr19Rep2E−07/33AAV5Rep329-4642/4 in 136 aa16.1rnEDLG-335
Cap31-1332/1 in 93 aa
    TarsierScaffold_178326Rep4E−14/23AAV5Rep96-4652/3 in 356 aa5.3tsEDLG23
Parvoviruses
    Guinea pigScaffold_188Rep3E−24/46Porcine parvovirusRep313-5675/3 in 250 aa12.3cpEPLG-140
Cap1E−16/36Cap10-68911/12 in 672 aa
Scaffold_27Rep1E−50/39Canine parvovirusRep11-6401/4 in 616 aa5.3cpEPLG-217
Cap1E−38/39Porcine parvovirusCap3-7192/14 in 700 aa
    TenrecScaffold_260946Rep2E−20/38LuIII virusRep406-5984/4 in 190 aa19.0etEPLG-260
Cap11-63916/15 in 595 aa
    RatChr5Rep6E−10/56Canine parvovirusRep1-2820/0 in 312 aa0.6rnEPLGRecent
Cap0/62Cap637-6670/2 in 760 aa
Rep0/631-751
    OpossumChr3Rep2E−39/33LuIII virusRep7-57011/3 in 502 aa10.9mdEPLG-256
Cap7E−8/33Cap11-72914/7 in 704 aa
Chr6Rep6E−58/44Porcine parvovirusRep16-5633/7 in 534 aac4.6mdEPLG-324
Cap6E−60/38Cap10-7152/5 in 707 aac
    WallabyScaffold_108040Rep4E−74/62Canine parvovirusRep341-6450/0 in 287 aa1.3meEPLG-37
Cap8E−37/32Cap35-7380/4 in 687 aa
Scaffold_72496Rep2E−61/42Porcine parvovirusRep23-5674/3 in 531 aa5.7meEPLG-630
Cap2E−31/38Cap10-5326/4 in 514 aa
Scaffold_88340Rep7E−37/55Mouse parvovirus 1Rep344-5660/3 in 223 aa6.7meEPLG-1636
Cap7E−22/33Cap11-7136/9 in 700 aa
Open in a separate windowaSome ambiguity in choosing the most similar virus is possible. We generally used the alignment with the lowest E value in the BLAST results. However, one or two points in the exponent of an E value were sometimes sacrificed to achieve a longer sequence alignment.baa, amino acids.cThese sequences have long insertions compared to the present-day viruses. In all cases tested, these insertions originated from short interspersed elements (SINEs). These insertions were excluded from the counts of stop codons and frameshifts and the estimation of integration age.dChr, chromosome.It is important to recognize that there is an intrinsic limit on how far back in time we can reach to identify ancient endogenous viral sequences. First, the sequences must be identified with confidence by BLAST or similar programs. This requirement places a lower limit on sequence identity at about 20 to 30% of amino acids, or about 75% of nucleotides (nucleotides evolve nearly 2.5 times slower than the amino acid sequence they encode). Second, the related, present-day virus must have evolved at a rate that is not much higher than that of the endogenous sequences. The viruses for which ancestral endogenous sequences were identified in this study exhibit sequence drift similar to that associated with mammalian genomes. Setting this rate at 0.14% per million years of evolution (8), we arrive at 90 million years as the theoretical limit for the oldest sequences that can be identified using our methods. This limit drops to less than 35 million years for endogenous viral sequences in rodents and even lower for sequences related to viruses that evolve faster than mammalian genomes.The most widespread integrations found in our survey are derived from the dependoviruses. These include nearly complete genomes related to adeno-associated virus (AAV) in microbat, wallaby, dolphin, rabbit, mouse, and baboon (Fig. (Fig.2).2). We did not detect inverted terminal repeats in several integrations tested, even though repeats are common in the present-day dependoviruses. This result could be explained by sequence decay or the absence of such structures in the ancestral viruses. However, we do see sequences that resemble degraded hairpin structures to which Dependovirus Rep proteins bind, with an example from microbat integration mlEDLG-1 shown in Fig. Fig.3.3. The second most widespread endogenous sequences are related to the parvoviruses. They are found in 6 of 49 vertebrate species considered, with nearly complete genomes in rat, opossum, wallaby, and guinea pig (Fig. (Fig.22).Open in a separate windowFIG. 3.Hairpin structure of the inverted terminal repeat of adeno-associated virus 2 (left) and a candidate degraded hairpin structure located close to the 5′ end of the mlEDLG-1 integration in microbats (right). Structures and mountain plots were generated using default parameters of the RNAfold program (5), with nucleotide coloring representing base-pairing probabilities: blue is below average, green is average, and red is above average. Mountain plots represent hairpin structures based on minimum free energy (mfe) calculations and partition function (pf) calculations, as well as the centroid structure (5). Height is expressed in numbers of nucleotides; position represents nucleotide.The Dependovirus AAV2 has strong bias for integration into human chromosome 19 during infection, driven by a host sequence that is recognized by the viral Rep protein(s). Rep mediates the formation of a synapse between viral and cellular sequences, and the cellular sequences are nicked to serve as an origin of viral replication (14). The related integrations in mice and rats, located in the same chromosomal locations, might be explained by such a mechanism. However, the extent of endogenous sequence decay and the frequency of stop codons indicate that these integrations occurred some 30 to 35 million years ago, implying that they are derived from a single event in a rodent ancestor rather than two independent integration events at the same location. Similarly, integrations EDLG-1 in dog and panda lie in chromosomal regions that can be readily aligned (based on University of California—Santa Cruz [UCSC] genome assemblies) and show sequence decay consistent with the age of the common ancestor, about 42 million years. Endogenous sequences related to the family Parvoviridae can thus be traced to over 40 million years back in time, and viral proteins related to this family have remained over 40% conserved.Sequences related to circoviruses were detected in five vertebrate species (Table (Table11 and Table S1 in the supplemental material). At least one of these sequences, the endogenous sequence in opossum, likely represents a recent integration. Several integrations in dog, cat, and panda, on the other hand, appear to date from at least 42 million years ago, which is the last time when pandas and dogs shared a common ancestor. We see evidence for this age in data from sequence degradation (Table (Table1),1), phylogenetic analyses of endogenous Circovirus-like genomes (see Fig. S2 in the supplemental material), and genomic synteny where integration ECLG-3 is surrounded by genes MTA3 and ARID5A in both dog and panda and integration ECLG-2 lies 35 to 43 kb downstream of gene UPF3A. In fact, Circovirus integrations may even precede the split between dogs and cats, about 55 million years ago, although the preliminary assembly and short genomic contigs for cats make synteny analysis impossible.The most common Circovirus-related sequences detected in vertebrate genomes are derived from the rep gene. We speculate that, like those of the Parvoviridae, the ancestral Circoviridae sequences might have been copied using a primer sequence in the host DNA that resembled the viral origin and was therefore recognized by the virus Rep protein. Higher incidence of rep gene identifications may represent higher conservation of this gene with time, or alternatively, possession of these sequences may impart some selective advantage to the host species. The largest Circovirus-related integration detected, in the opossum, comprises a short fragment of what may have been the cap gene immediately adjacent to and in the opposite orientation from the rep gene. This organization is similar to that of the present day Circovirus genome in which these genes share a promoter in the hairpin regions but are translated in opposite directions (Fig. (Fig.22).In summary, our results indicate that sequences derived from ancestral members of the families Parvoviridae and Circoviridae were integrated into their host''s genomes over the past 50 million years of evolution. Features of their replication strategies suggest mechanisms by which such integrations may have occurred. It is possible that some of the endogenous viral sequences could offer a selective advantage to the virus or the host. We note that rep open reading frame-derived proteins from some members of these families kill tumor cells selectively (3, 12). The genomic “fossils” we have discovered provide a unique glimpse into virus evolution but can give us only a lower estimate of the actual ages of these families. However, numerous recent integrations suggest that their germ line transfer has been continuing into present times.   相似文献   

18.
Feeding high levels of zinc oxide to piglets significantly increased the relative abundance of ileal Weissella spp., Leuconostoc spp., and Streptococcus spp., reduced the occurrence of Sarcina spp. and Neisseria spp., and led to numerical increases of all Gram-negative facultative anaerobic genera. High dietary zinc oxide intake has a major impact on the porcine ileal bacterial composition.Zinc oxide (ZnO) is used as a feed additive for diarrhea prophylaxis in piglets (23). However, the mode of action of ZnO is not fully understood. Besides its effects on the host (10, 30, 31), high dietary zinc levels may affect the diversity of intestinal microbial communities (2, 11, 20). The prevention of postweaning diarrhea in piglets due to high dietary ZnO intake may not be directly related to a reduction of pathogenic E. coli (8) but, rather, to the diversity of the coliform community (15). Studies on the impact of high ZnO levels on the porcine ileal bacterial community are scarce but nevertheless important, as bacterial diarrhea is initiated in the small intestine (9, 17). The small intestine is a very complex habitat with many different factors shaping the bacterial community. Studies on the ecophysiology (22) and maturation of the porcine ileal microbiota (13, 27) indicate a drastic impact directly after weaning and a gradual decline of modifications during the following 2 weeks. Thus, the time point for analysis chosen in this study (14 days postweaning) does reflect a more stable period of the ileal porcine microbiota. In this study, we used bar-coded pyrosequencing of 16S rRNA genes to gain further insight into the mode of action of pharmacological levels of ZnO in the gastrointestinal tract of young pigs.Total DNA was extracted from the ileal digesta of 40- to 42-day-old piglets using a commercial kit (Qiagen stool kit; Qiagen, Hilden, Germany) and PCR amplified with unique bar-coded primer sets targeting the V1-to-V3 and the V6-to-V8 hypervariable regions (see the supplemental material for detailed methods). The rationale behind this approach was derived from the fact that no single “universal” primer pair can completely cover a complex bacterial habitat (4, 24, 32, 33). Furthermore, these studies also show that in silico information on the coverage of selected primer sets diverges from empirical results, and hence, two hypervariable regions were chosen in this study to maximize the detection of phylogenetically diverse bacterial groups.Equimolar dilutions of all samples were combined into one master sample. Pyrosequencing was performed by Agowa (Berlin, Germany) on a Roche genome sequencer FLX system using a Titanium series PicoTiterPlate. The resulting data files were uploaded to the MG-RAST server (http://metagenomics.nmpdr.org/) (19) and processed with its SEED software tool using the RDP database (5) as the reference database. After automated sequence analysis, all sequences with less than five identical reads per sample were deleted in order to increase the confidence of sequence reads and reduce bias from possible sequencing errors (12, 16). Thus, 0.43% of all sequences were not considered (1,882 of 433,302 sequences). These sequences were assigned to a total of 238 genera, of which most only occurred in a few samples (see the supplemental material). Furthermore, all unclassified sequences were removed (8.7%; 41,467 of 474,769 sequences). Due to the use of the RDP reference database, the SEED software incorrectly assigned the majority of unclassified sequences as unclassified Deferribacterales (83%; 34,393 sequences), which were actually identified as 16S soybean or wheat chloroplasts by BLAST or as cyanobacterial chloroplasts by the RDP II seqmatch tool.The pyrosequencing results for the two primer combinations were merged by taking only sequences from the primer combination that yielded the higher number of reads for a specific sequence assignment in a sample. The remaining reads were used to calculate the relative contribution of assigned sequences to total sequence reads in a sample.The Firmicutes phylum dominated the small intestinal bacterial communities in both the control group and the group with high dietary ZnO intake, with 98.3% and 97.0% of total sequence reads, respectively. No significant influence of high dietary ZnO intake was found for the main phyla Proteobacteria (0.92% versus 1.84%), Actinobacteria (0.61% versus 0.75%), Bacteroidetes (0.15% versus 0.17%), and Fusobacteria (0.09% versus 0.12%).On the order level, a total of 20 bacterial orders were detected (data not shown). Lactobacillales dominated bacterial communities in the control and high-dietary-ZnO-intake groups, with 83.37% and 93.24% of total reads. Lactic acid bacteria are well known to dominate the bacterial community in the ileum of piglets (11, 22). No significant difference between the control group and the group with high dietary ZnO intake was observed on the order level, although high dietary ZnO intake led to a strong numerical decrease for Clostridiales (14.4 ± 24.0% [mean ± standard deviation] versus 2.8 ± 1.7%), as well as to numerical increases for Pseudomonadales (0.3 ± 0.3% versus 0.6 ± 0.6%) and Enterobacteriales (0.2 ± 0.2% versus 0.5 ± 0.6%).On the genus level, a total of 103 genera were detected. Table Table11 summarizes the main 31 genera which exceeded 0.05% of total reads (see the supplemental material for a complete list). Lactobacilli clearly dominated the bacterial communities in both trial groups, but they also were numerically lower due to high dietary ZnO intake.

TABLE 1.

Bacterial genera in the ileum of piglets fed diets supplemented with 200 or 3,000 ppm ZnO
GenusProportion (% ± SD) of ileal microbiota in groupa receiving:
200 ppm ZnO3,000 ppm ZnO
Lactobacillus59.3 ± 30.640.7 ± 19.1
Weissella11.6 ± 7.8 A24.1 ± 8.3 B
Sarcina11.4 ± 20.5 A0.84 ± 1.2 B
Leuconostoc4.7 ± 3.2 A9.4 ± 3.1 B
Streptococcus1.8 ± 1.6 A5.7 ± 5.1 B
Lactococcus1.6 ± 1.52.6 ± 3.1
Veillonella0.57 ± 0.630.34 ± 0.30
Gemella0.34 ± 0.67 A0.45 ± 0.25 B
Acinetobacter0.25 ± 0.210.44 ± 0.50
Clostridium0.25 ± 0.400.22 ± 0.21
Enterococcus0.19 ± 0.150.26 ± 0.24
Acidovorax0.14 ± 0.040.16 ± 0.19
Arcobacter0.14 ± 0.150.16 ± 0.17
Neisseria0.14b0.03 ± 0.01
Enterobacter0.13 ± 0.090.29 ± 0.34
Lachnospira0.12 ± 0.130.13 ± 0.03
Peptostreptococcus0.11 ± 0.100.07 ± 0.09
Chryseobacterium0.10 ± 0.070.15 ± 0.16
Actinomyces0.09 ± 0.040.15 ± 0.16
Anaerobacter0.07 ± 0.080.02 ± 0.01
Aerococcus0.07 ± 0.040.07 ± 0.04
Dorea0.07b0.05 ± 0.05
Fusobacterium0.06 ± 0.090.08 ± 0.11
Microbacterium0.06 ± 0.010.07 ± 0.04
Carnobacterium0.06 ± 0.020.08 ± 0.13
Granulicatella0.06 ± 0.020.09 ± 0.10
Staphylococcus0.06 ± 0.040.05 ± 0.02
Facklamia0.05 ± 0.060.03 ± 0.01
Comamonas0.05 ± 0.030.04 ± 0.02
Citrobacter0.05 ± 0.020.07 ± 0.08
Erysipelothrix0.05 ± 0.010.22 ± 0.40
Open in a separate windowan = 6 piglets per trial group. A,B, results are significantly different by Kruskal-Wallis test.bSingle sample.Significant changes due to high dietary ZnO intake were observed for other lactic acid bacteria, including Weissella spp., Leuconostoc spp., and Streptococcus spp. A significant and strong decrease was observed for Sarcina spp., which is a genus of acid-tolerant strictly anaerobic species found in the intestinal tract of piglets and other mammals (6, 28, 29). This genus thus appeared to be very sensitive to modifications induced by high dietary ZnO intake.An interesting result was observed for Gram-negative Proteobacteria, (i.e., enterobacteria and relatives). Although not statistically significant, virtually all detected proteobacteria increased numerically due to high dietary ZnO intake (Enterobacter spp., Microbacterium spp., Citrobacter spp., Neisseria spp., and Acinetobacter spp.). Apparently, enterobacteria gained colonization potential by high dietary ZnO intake. This is in good agreement with the results of studies by Hojberg et al. (11), Amezcua et al. (1), and Castillo et al. (3). Therefore, the frequently observed diarrhea-reducing effect of zinc oxide may not be directly related to a reduction of pathogenic E. coli strains. Considering a possible antagonistic activity of lactobacilli against enterobacteria (25), it can be speculated that a numerical decrease of dominant lactobacilli may lead to increased colonization with Gram-negative enterobacteria. On the other hand, specific plasmid-borne genes for resistance against heavy metals have been reported for both Gram-positive and Gram-negative bacteria present in the intestine (21, 26), and an increased resistance against Zn ions may exist for Gram-negative enterobacteria. Zinc oxide is an amphoteric molecule and shows a high solubility at acid pH. The low pH in the stomach of piglets (pH 3.5 to 4.5) transforms a considerable amount of insoluble ZnO into zinc ions (54 to 84% free Zn2+ at 150 ppm and 24 ppm ZnO, respectively) (7), and thus, high concentrations of toxic zinc ions exist in the stomach. The stomach of piglets harbors large numbers of lactic acid bacteria, especially lactobacilli. Zn ions may thus lead to a modification of the lactic acid bacterial community in the stomach, and the changes observed in the ileum could have been created in the stomach. A reduction of dominant lactobacilli may thus point to an increased adaptation potential of Gram-negative facultative anaerobes and a generally increased bacterial diversity.Additionally, the direct effects of dietary ZnO on intestinal tissues include altered expression of genes responsible for glutathione metabolism and apoptosis (30), enhanced gastric ghrelin secretion, which increases feed intake (31), and increased production of digestive enzymes (10). An analysis of the intestinal morphology was beyond the scope of this study, but although ZnO concentrations are markedly increased in intestinal tissue, the influence of ZnO on morphology is apparently not always observed (10, 14, 18). Consequently, any changes in epithelial cell turnover, feed intake, or digestive capacity may influence the composition of bacterial communities in the small intestine.In conclusion, this study has shown that high dietary zinc oxide has a major impact on ileal bacterial communities in piglets. Future studies on the impact of zinc oxide in pigs should include a detailed analysis of host responses in order to identify the cause for the observed modifications of intestinal bacterial communities.  相似文献   

19.
Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β3 (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table (Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).

TABLE 1.

Peptide-bearing phage eluted from ANDV
Phage% Inhibition (SD)aP valueb
Phage bearing the following peptides eluted with anti-Gn antibody 6B9/F5
    Group 1 (<30% inhibition)
        CDQRTTRLC8.45 (15.34)0.0002
        CPHDPNHPC9.94 (7.72)0.333
        CQSQTRNHC11.76 (13.25)0.0001
        CLQDMRQFC13.26 (9.92)0.0014
        CLPTDPIQC15.70 (14.05)0.0005
        CPDHPFLRC16.65 (15.22)0.8523
        CSTRAENQC17.56 (16.50)0.0004
        CPSHLDAFC18.98 (20.06)0.0017
        CKTGHMRIC20.84 (7.47)0.0563
        CVRTPTHHC20.89 (27.07)0.1483
        CSGVINTTC21.57 (19.61)0.0643
        CPLASTRTC21.65 (5.98)0.004
        CSQFPPRLC22.19 (8.26)0.0004
        CLLNKQNAC22.34 (7.78)0.001
        CKFPLNAAC22.89 (6.15)0.0001
        CSLTPHRSC23.63 (16.74)0.0563
        CKPWPMYSC23.71 (6.68)0.0643
        CLQHDALNC24.01 (7.60)1
        CNANKPKMC24.67 (11.67)0.0004
        CPKHVLKVC25.30 (28.36)0.0003
        CTPDKKSFC26.91 (11.15)0.399
        CHGKAALAC27.22 (32.53)0.005
        CNLMGNPHC28.08 (21.35)0.0011
        CLKNWFQPC28.64 (18.49)0.0016
        CKEYGRQMC28.76 (29.33)0.0362
        CQPSDPHLC29.44 (31.22)0.0183
        CSHLPPNRC29.70 (17.37)0.0061
    Group 2 (30-59% inhibition)
        CSPLLRTVC33.05 (20.26)0.0023
        CHKGHTWNC34.17 (12.50)0.0795
        CINASHAHC35.62 (13.03)0.3193
        CWPPSSRTC36.75 (26.95)0.0006
        CPSSPFNHC37.78 (7.11)0.0001
        CEHLSHAAC38.47 (7.60)0.0115
        CQDRKTSQC38.74 (9.12)0.1802
        CTDVYRPTC38.90 (25.03)0.006
        CGEKSAQLC39.11 (27.52)0.0013
        CSAAERLNC40.13 (6.33)0.0033
        CFRTLEHLC42.07 (5.01)0.0608
        CEKLHTASC43.60 (27.92)0.1684
        CSLHSHKGC45.11 (49.81)0.0864
        CNSHSPVHC45.40 (28.80)0.0115
        CMQSAAAHC48.88 (44.40)0.5794
        CPAASHPRC51.84 (17.09)0.1935
        CKSLGSSQC53.90 (13.34)0.0145
    Group 3 (60-79% inhibition)
        CPSNVNNIC61.11 (25.41)0.1245
Negative control0 (6.15)
6B9/F5 (5 μg/ml)26.77 (5.33)
ReoPro (80 μg/ml)79.86 (4.88)
Phage bearing the following peptides eluted with anti-Gc antibody 6C5/D12
    Group 1 (<30% inhibition)
        CHPGSSSRC1.01 (7.03)0.0557
        CSLSPLGRC10.56 (13.62)0.7895
        CTARYTQHC12.86 (3.83)0.3193
        CHGVYALHC12.91 (7.32)0.0003
        CLQHNEREC16.79 (13.72)0.0958
        CHPSTHRYC17.23 (14.53)0.0011
        CPGNWWSTC19.34(9.91)0.1483
        CGMLNWNRC19.48 (19.42)0.0777
        CPHTQFWQC20.44 (13.65)0.0008
        CTPTMHNHC20.92 (11.68)0.0001
        CDQVAGYSC21.79 (23.60)0.0063
        CIPMMTEFC24.33 (9.28)0.2999
        CERPYSRLC24.38 (9.09)0.0041
        CPSLHTREC25.06 (22.78)0.1202
        CSPLQIPYC26.30 (34.29)0.4673
        CTTMTRMTC (×2)29.27 (8.65)0.0001
    Group 2 (30-59% inhibition)
        CNKPFSLPC30.09 (5.59)0.4384
        CHNLESGTC31.63 (26.67)0.751
        CNSVPPYQC31.96 (6.51)0.0903
        CSDSWLPRC32.95 (28.54)0.259
        CSAPFTKSC33.40 (10.64)0.0052
        CEGLPNIDC35.63 (19.90)0.0853
        CTSTHTKTC36.28 (13.42)0.132
        CLSIHSSVC36.40 (16.44)0.8981
        CPWSTQYAC36.81 (32.81)0.5725
        CTGSNLPIC36.83 (31.64)0.0307
        CSLAPANTC39.73 (4.03)0.1664
        CGLKTNPAC39.75 (16.98)0.2084
        CRDTTPWWC40.08 (18.52)0.0004
        CHTNASPHC40.26 (4.77)0.5904
        CTSMAYHHC41.89 (8.61)0.259
        CSLSSPRIC42.13 (29.75)0.2463
        CVSLEHQNC45.54 (6.55)0.5065
        CRVTQTHTC46.55 (8.45)0.3676
        CPTTKSNVC49.28 (14.00)0.3898
        CSPGPHRVC49.50 (42.60)0.0115
        CKSTSNVYC51.20 (4.60)0.0611
        CTVGPTRSC57.30 (11.31)0.0176
    Group 3 (60-79% inhibition)
        CPMSQNPTC65.60 (13.49)0.014
        CPKLHPGGC71.88 (27.11)0.0059
Negative control0.26 (4.53)
6C5/D12 (5 μg/ml)22.62 (8.40)
ReoPro (80 μg/ml)80.02 (76.64)
Open in a separate windowaStandard deviations of four experiments are shown in parentheses. Peptide-bearing phage were added at 109 phage/μl.bP values for the pairwise amino acid alignment score of each peptide versus that of integrin β3 were determined using an unpaired Student''s t test. P values considered statistically significant are shown in bold.To determine whether the peptide sequences of any of the identified inhibitory phage showed homology to integrin β3, a known entry receptor for pathogenic hantaviruses (6, 7), we used the Gap program to perform a pairwise amino acid alignment of each peptide versus the extracellular portion of integrin β3 and determined P values for the alignments. Of 45 phage eluted with the anti-Gn antibody, 6B9/F5, 27 of the peptide sequences showed homology to integrin β3 (P < 0.05), and 9 were highly significant (P ≤ 0.0005) (Fig. (Fig.1A).1A). Of the latter, CKFPLNAAC and CSQFPPRLC map to the hybrid domain (Fig. (Fig.1B),1B), which is proximal to the plexin-semaphorin-integrin domain (PSI) containing residue D39, shown to be critical for viral entry in vitro (19). Five sequences (CPSSPFNH, CPKHVLKVC, CNANKPKMC, CQSQTRNHC, and CDQRTTRLC) map to the I-like (or βA) domain near the binding site of ReoPro (2). Finally, CLPTDPIQC maps to the epidermal growth factor 4 (EGF-4) domain, and CSTRAENQC aligns to a portion of β3 untraceable in the crystal structure, specifically the linker region between the hybrid domain and EGF-1. Although this represents a disordered portion of the protein (22), the location of this loop proximal to the PSI domain is worth noting, due to the role of the PSI domain in facilitating viral entry (19). Therefore, 60% of phage eluted with the anti-Gn antibody showed some homology to integrin β3, and those with highly significant P values predominantly mapped to or proximal to regions of known interest in viral entry.Open in a separate windowFIG. 1.Inhibitory peptides identified through phage panning against ANDV show homology to integrin β3. (A) Alignment of phage peptide sequences with P values for integrin β3 pairwise alignment of less than 0.05. Residues comprising the signal peptide, transmembrane, and cytoplasmic domains, which were not included during pairwise alignment, are underlined. Residues 461 to 548, which are missing in the crystal structure, are italicized. Residues involved in the ReoPro binding site are highlighted in green (2). Residue D39 of the PSI domain is highlighted in yellow (19). Peptides are shown above the sequence of integrin β3, with antibody 6C5/D12-eluted sequences shown in blue text and sequences eluted with antibody 6B9/F5 shown in red. Peptide sequences with alignment P values of ≤0.0005 are highlighted in yellow. Percent inhibition of the peptide-bearing phage is shown in parentheses. (B) View of integrin αvβ3 (PDB ID 1U8C [23]). αv is shown in blue ribbon diagram, and β3 is shown in salmon-colored surface representation, with specific domains circled. Residues corresponding to the ReoPro binding site are shown in green, as in panel A, and D39 is shown in yellow. Regions corresponding to 6C5/D12-eluted peptides with P values of ≤0.0005 for alignment with integrin β3 (highlighted in panel A) are shown in blue, and those corresponding to 6B9/F5-eluted peptides with P values of ≤0.0005 for alignment with integrin β3 are shown in red. Alignment of peptide PLASTRT (P value of 0.0040) adjacent to D39 of the PSI domain is shown in magenta. Graphics were prepared using Pymol (DeLano Scientific LLC, San Carlos, CA).Of the 41 peptide-bearing phage eluted with the anti-Gc antibody 6C5/D12, 14 showed sequence homology to integrin β3 (P < 0.05), 4 of which had P values of ≤0.0005 (Fig. (Fig.1A).1A). Of the latter, sequence CTTMTRMTC mapped to the base of the I-like domain (Fig. (Fig.1B),1B), while CHGVYALHC and CRDTTPWWC mapped to the EGF-3 domain. Finally, sequence CTPTMHNHC mapped to the linker region untraceable in the crystal structure. Therefore, in contrast to peptide sequences identified by competition with the anti-Gn antibody, sequences identified by competition with the anti-Gc antibody 6C5/D12 appear to be mostly unrelated to integrin β3.As a low level of pathogenic hantavirus infection can be seen in cells lacking integrin β3, such as CHO cells (19), we asked if any of the identified peptide sequences could represent a previously unidentified receptor. We used the Basic Local Alignment Search Tool to search a current database of human protein sequences for potential alternate receptors represented by these peptides. However, none of the alignments identified proteins that are expressed at the cell surface, eliminating them as potential candidates for alternate viral entry receptors. This suggests that the majority of the peptides identified here likely represent novel sequences for binding ANDV surface glycoproteins.To determine whether synthetic peptides would also block infection, we synthesized cyclic peptides based on the 10 most-potent peptide-bearing phage. These peptides, in the context of phage presentation, showed levels of inhibition ranging from 44 to 72% (Table (Table2).2). When tested by IFA at 1 mM, four of the synthetic peptides showed inhibition levels significantly lower than those of the same peptide presented in the context of phage. This is not surprising, as steric factors due to the size of the phage and the multivalent presentation of peptide in the context of phage may both contribute to infection inhibition (8). However, there was no significant difference in inhibition by synthetic peptide versus peptide-bearing phage for six of the sequences, implying that inhibition in the context of phage was due solely to the nature of the peptide itself and not to steric factors or valency considerations contributed by the phage, which contrasts with our previous results, determined by using phage directed against αvβ3 integrin (10).

TABLE 2.

Synthetic cyclic peptides inhibit ANDV infection
TargetSample% Inhibition bya:
Peptide-bearing phageSynthetic peptide
GnCMQSAAAHC48.88 (44.40)59.66 (11.17)
GcCTVGPTRSC57.30 (11.31)46.47 (7.61)
GnCPSNVNNIC61.11 (25.41)44.14 (10.74)
GnCEKLHTASC43.60 (27.92)34.87 (9.26)
GcCPKLHPGGC71.88 (27.11)30.95 (7.73)b
GnCSLHSHKGC45.11 (49.81)29.79 (9.34)
GcCPMSQNPTC65.60 (13.49)18.19 (8.55)b
GnCKSLGSSQC53.90 (13.34)18.10 (7.55)b
GnCNSHSPVHC45.40 (28.80)15.52 (10.48)
GnCPAASHPRC51.84 (17.09)0 (10.72)b
Integrin β3ReoPro80.10 (7.72)
Gn6B9/F5 antibody42.72 (6.75)
Gc6C5/D12 antibody31.04 (7.81)
Open in a separate windowaStandard deviations of the results of at least four experiments are shown in parentheses.bMean percent inhibition between phage and synthetic peptide differs significantly (P < 0.05).The three most-potent synthetic peptides were examined for their ability to inhibit ANDV entry in a dose-dependent manner. The concentration of each peptide that produces 50% of its maximum potential inhibitory effect was determined. As shown in Fig. Fig.2A,2A, the 50% inhibitory concentration for each of the peptides was in the range of 10 μM, which from our experience is a reasonable potency for a lead compound to take forward for optimization.Open in a separate windowFIG. 2.Activities of synthetic peptides in inhibition of ANDV infection in vitro. (A) Peptides were examined for their ability to block ANDV infection of Vero E6 cells in a dose-dependent manner by IFA. (B) Peptides were tested in parallel for the ability to block infection of Vero E6 cells by ANDV, SNV, HTNV, and PHV. (C) Peptides were tested, singly or in combination, for the ability to block ANDV infection of Vero E6 cells. For all experiments, controls included media, ReoPro at 80 μg/ml, and monoclonal antibodies 6C5/D12 and 6B9/F5 at 5 μg/ml. All peptides were used at 1 mM. Data points represent n = 2 to 6, with error bars showing the standard errors of the means. Statistical analyses were performed on replicate samples using an unpaired Student''s t test.In order to determine the specificity of the three most-potent synthetic cyclic peptides in blocking ANDV, we examined them for inhibition of ANDV infection versus two other pathogenic hantaviruses, SNV and Hantaan virus (HTNV), or the nonpathogenic hantavirus Prospect Hill virus (PHV). As shown in Fig. Fig.2B,2B, ReoPro, which binds integrin β3, showed inhibition of infection by each of the pathogenic hantavirus strains, known to enter cells via β3, but not the nonpathogenic PHV, which enters via integrin β1 (6, 7). In contrast, peptides selected for the ability to bind ANDV were highly specific inhibitors of ANDV versus SNV, HTNV, or PHV. The specificities of peptides eluted by the anti-Gn monoclonal antibody are not surprising, as they are likely due to global differences in the Gn amino acid sequence. Specifically, sequence homologies between ANDV and SNV, HTNV, and PHV are 61%, 36%, and 51%, respectively, for the region corresponding to the immunogen for antibody 6B9/F5. Although homology between the immunogen for antibody 6C5/D12 and the corresponding Gc region of these viruses is somewhat higher (82% with SNV, 63% with HTNV, and 71% with PHV), the possibility that the monoclonal antibody used here recognizes a three-dimensional epitope lends itself to the high specificity of the peptides.The current model for cellular infection by hantaviruses (14) is as follows. Viral binding of the host cell surface target integrin is followed by receptor-mediated endocytosis and endosome acidification. Lowered pH induces conformational changes in Gn and/or Gc, which facilitate membrane fusion and viral release into the cytosol. As there is currently little information available about whether one glycoprotein is dominant in mediating infection, and as neutralizing epitopes have been found on both Gn and Gc glycoproteins (1, 4, 12, 13, 20), we examined whether combining anti-Gn- and anti-Gc-targeted synthetic peptides would lead to an increased infection blockade compared to those for single treatments. As shown in Fig. Fig.2C,2C, the combination of anti-Gn and anti-Gc peptides CMQSAAAHC and CTVGPTRSC resulted in a significant increase in infection inhibition (P = 0.0207 for CMQSAAAHC, and P = 0.0308 for CTVGPTRSC) compared to that resulting from single treatments. Although the high specificity of the peptides for ANDV makes it unlikely that this combination treatment will lead to more cross-reactivity with other pathogenic hantaviruses, this can be determined only by additional testing. Regardless, these data suggest a unique role for each of these viral proteins in the infection process as well as the benefits of targeting multiple viral epitopes for preventing infection.To our knowledge, the peptides reported here are the first identified that directly target ANDV, and this work further illustrates the power of coupling phage display and selective elution techniques in the identification of novel peptide sequences capable of specific protein-protein interactions from a large, random pool of peptide sequences. These novel peptide inhibitors (R. S. Larson, P. R. Hall, H. Njus, and B. Hjelle, U.S. patent application 61/205,211) provide leads for the development of more-potent peptide or nonpeptide organics for therapeutic use against HCPS.  相似文献   

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