首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 93 毫秒
1.
Lichenysins are surface-active lipopeptides with antibiotic properties produced nonribosomally by several strains of Bacillus licheniformis. Here, we report the cloning and sequencing of an entire 26.6-kb lichenysin biosynthesis operon from B. licheniformis ATCC 10716. Three large open reading frames coding for peptide synthetases, designated licA, licB (three modules each), and licC (one module), could be detected, followed by a gene, licTE, coding for a thioesterase-like protein. The domain structure of the seven identified modules, which resembles that of the surfactin synthetases SrfA-A to -C, showed two epimerization domains attached to the third and sixth modules. The substrate specificity of the first, fifth, and seventh recombinant adenylation domains of LicA to -C (cloned and expressed in Escherichia coli) was determined to be Gln, Asp, and Ile (with minor Val and Leu substitutions), respectively. Therefore, we suppose that the identified biosynthesis operon is responsible for the production of a lichenysin variant with the primary amino acid sequence l-Gln–l-Leu–d-Leu–l-Val–l-Asp–d-Leu–l-Ile, with minor Leu and Val substitutions at the seventh position.Many strains of Bacillus are known to produce lipopeptides with remarkable surface-active properties (11). The most prominent of these powerful lipopeptides is surfactin from Bacillus subtilis (1). Surfactin is an acylated cyclic heptapeptide that reduces the surface tension of water from 72 to 27 mN m−1 even in a concentration below 0.05% and shows some antibacterial and antifungal activities (1). Some B. subtilis strains are also known to produce other, structurally related lipoheptapeptides (Table (Table1),1), like iturin (32, 34) and bacillomycin (3, 27, 30), or the lipodecapeptides fengycin (50) and plipastatin (29).

TABLE 1

Lipoheptapeptide antibiotics of Bacillus spp.
LipopeptideOrganismStructureReference
Lichenysin AB. licheniformisFAa-L-Glu-L-Leu-D-Leu-L-Val-L-Asn-D-Leu-L-Ile51, 52
Lichenysin BFAa-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu23, 26
Lichenysin CFAa-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Ile17
Lichenysin DFAa-L-Gln-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-IleThis work
Surfactant 86B. licheniformisFAa-L-Glxd-L-Leu-D-Leu-L-Val-L-Asxd-D-Leu-L-Ilee14, 15
L-Val
SurfactinB. subtilisFAa-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu1, 7, 49
EsperinB. subtilisFAb-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leue45
L-Val 
Iturin AB. subtilisFAc-L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser32
Iturin CFAc-L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asne-L-Asne34
D-Ser-L-Thr 
Bacillomycin LB. subtilisFAc-L-Asp-D-Tyr-D-Asn-L-Ser-L-Gln-D-Proe-L-Thr3
D-Ser- 
Bacillomycin DFAc-L-Asp-D-Tyr-D-Asn-L-Pro-L-Glu-D-Ser-L-Thr30, 31
Bacillomycin FFAc-L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Thr27
Open in a separate windowaFA, β-hydroxy fatty acid. The β-hydroxy group forms an ester bond with the carboxy group of the C-terminal amino acid. bFA, β-hydroxy fatty acid. The β-hydroxy group forms an ester bond with the carboxy group of Asp5. cFA, β-amino fatty acid. The β-amino group forms a peptide bond with the carboxy group of the C-terminal amino acid. dOnly the following combinations of amino acid 1 and 5 are allowed: Gln-Asp or Glu-Asn. eWhere an alternative amino acid may be present in a structure, the alternative is also presented. In addition to B. subtilis, several strains of Bacillus licheniformis have been described as producing the lipopeptide lichenysin (14, 17, 23, 26, 51). Lichenysins can be grouped under the general sequence l-Glx–l-Leu–d-Leu–l-Val–l-Asx–d-Leu–l-Ile/Leu/Val (Table (Table1).1). The first amino acid is connected to a β-hydroxyl fatty acid, and the carboxy-terminal amino acid forms a lactone ring to the β-OH group of the lipophilic part of the molecule. In contrast to the lipopeptide surfactin, lichenysins seem to be synthesized during growth under aerobic and anaerobic conditions (16, 51). The isolation of lichenysins from cells growing on liquid mineral salt medium on glucose or sucrose basic has been studied intensively. Antimicrobial properties and the ability to reduce the surface tension of water have also been described (14, 17, 26, 51). The structural elucidation of the compounds revealed slight differences, depending on the producer strain. Various distributions of branched and linear fatty acid moieties of diverse lengths and amino acid variations in three defined positions have been identified (Table (Table11).In contrast to the well-defined methods for isolation and structural characterization of lichenysins, little is known about the biosynthetic mechanisms of lichenysin production. The structural similarity of lichenysins and surfactin suggests that the peptide moiety is produced nonribosomally by multifunctional peptide synthetases (7, 13, 25, 49, 53). Peptide synthetases from bacterial and fungal sources describe an alternative route in peptide bond formation in addition to the ubiquitous ribosomal pathway. Here, large multienzyme complexes affect the ordered recognition, activation, and linking of amino acids by utilizing the thiotemplate mechanism (19, 24, 25). According to this model, peptide synthetases activate their substrate amino acids as aminoacyl adenylates by ATP hydrolysis. These unstable intermediates are subsequently transferred to a covalently enzyme-bound 4′-phosphopantetheinyl cofactor as thioesters. The thioesterified amino acids are then integrated into the peptide product through a stepwise elongation by a series of transpeptidations directed from the amino terminals to the carboxy terminals. Peptide synthetases have not only awakened interest because of their mechanistic features; many of the nonribosomally processed peptide products also possess important biological and medical properties.In this report we describe the identification and characterization of a putative lichenysin biosynthesis operon from B. licheniformis ATCC 10716. Cloning and sequencing of the entire lic operon (26.6 kb) revealed three genes, licA, licB, and licC, with structural patterns common to peptide synthetases and a gene designated licTE, which codes for a putative thioesterase. The modular organization of the sequenced genes resembles the requirements for the biosynthesis of the heptapeptide lichenysin. Based on the arrangement of the seven identified modules and the tested substrate specificities, we propose that the identified genes are involved in the nonribosomal synthesis of the portion of the lichenysin peptide with the primary sequence l-Gln–l-Leu–d-Leu–l-Val–l-Asp–d-Leu–l-Ile (with minor Val and Leu substitutions).  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.
4.
5.
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   

6.
7.
Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.Recently, a novel bacterial phylum, termed “Poribacteria,” was discovered, and members of this phylum have been found exclusively in sponges (2). Phylogenetic analyses of 16S rRNA genes indicated that poribacteria are evolutionarily deeply branching organisms and related to a superphylum composed of Planctomycetes, Verrucomicrobia, and Chlamydia (11). Poribacterial 16S rRNA genes contain 13 of 15 planctomycete signature nucleotides, but a level of sequence divergence of more than 25% compared to any other bacterial phylum, including the Planctomycetes, justifies the status of this taxon as an independent phylum. A consistent treeing pattern is difficult to resolve in comparative phylogenetic sequence analyses, making the poribacteria an unusual line of phylogenetic descent. In addition to their divergent status as a separate phylum on the basis of the 16S rRNA sequence, poribacteria are also divergent because they may have a compartmentalized cell structure, a cell plan they share only with members of the phyla Planctomycetes and Verrucomicrobia (2). They are also of interest for understanding the potential contribution of obligate sponge-associated bacteria to the sponges harboring them and as an example of a yet-to-be-cultured group of bacteria associated with invertebrate tissue apparently exclusively but for unknown reasons. This study aimed to further explore the presence and diversity of poribacteria in different marine demosponge genera using samples from around the world.The Mediterranean sponges were collected by scuba divers offshore at Banyuls sur Mer, France (42°29′N, 03°08′E). The Caribbean sponges were collected offshore at Little San Salvador Island, Bahamas (24°32′N, 75°55′W). The eastern Pacific sponge Aplysina fistularis was collected offshore at San Diego, CA (32°51′N, 117°15′W). The western Pacific sponge Theonella swinhoei was collected offshore at Palau (07°23′N, 134°38′E). All non-Great Barrier Reef (non-GBR) sponges were collected between May and July 2000, and once individual sponge specimens were brought to the surface, they were frozen in liquid nitrogen on board ship and stored at −80°C until microbiological processing (9). The GBR marine sponges were collected off Heron Island Research Station (23°27′S, 151°5′E) in April 2002 (5). Pseudoceratina clavata was collected by scuba divers at a depth of 14 m, and Rhabdastrella globostellata was collected at a depth of ca. 0.5 m after a reef walk consisting of a few hundred meters. The samples were immediately placed in plastic bags and brought to Heron Island Research Station, where they were stored at −80°C until processing. Sponge DNA was extracted as described previously (2, 5).Total sponge-derived genomic DNA was screened by PCR for the presence of poribacteria using a 16S rRNA gene primer set. Poribacterial 16S rRNA genes were amplified by employing a pair of Poribacteria-specific primers, POR389f (5′-ACG ATG CGA CGC CGC GTG-3′) and POR1130r (5′-GGC TCG TCA CCA GCG GTC-3′) (2). The poribacterial PCR products that were ca. 740 bp long derived from one sponge individual were cloned into the pGEM-T Easy vector (Promega, Madison, WI). Clone inserts were digested with restriction endonucleases MspI and HaeIII (New England Biolabs, Inc., United States), characterized to obtain restriction profiles and unique profiles, and sequenced. The compiled partial 16S rRNA gene sequences were then analyzed using BLASTN to select the most closely related poribacterial reference sequences.The sequences exhibiting levels of similarity of less than 97% were used for further analysis. Poribacterial 16S rRNA gene sequences were aligned using the ARB software package (7). The resulting alignment was imported into PAUP (10) and analyzed by using distance, maximum parsimony, and maximum likelihood algorithms together with bootstrap resamplings (3,000, 3,000, and 200 resamplings, respectively), and the resulting bootstrap values were applied to nodes on the ARB neighbor-joining tree. Signature sequences were detected using the ARB software package. A signature sequence is defined here as a short sequence that is present in a group of poribacterial sequences in a phylogenetic clade but is not found in any other clade in the poribacterial tree.Analysis of the 16S rRNA gene clone library sequences generated from sponge tissues revealed the presence of poribacteria in sponge individuals belonging to the orders Verongida, Astrophorida, Dictyoceratida, Haplosclerida, Lithistida, and Homosclerophorida, while poribacteria could not be detected in sponges belonging to the orders Hadromerida and Agelasida. In the order Halichondrida, poribacteria were detected in Xestospongia muta but not in Haliclona sp. Altogether, nine sponge species were added to the list of Poribacteria-containing sponges (Table (Table1).1). Three distinct clades were observed that were clearly supported by bootstrap values greater than 75 with every tree-building algorithm applied (Fig. (Fig.1),1), and one clade (clade I) was supported by bootstrap values of 64, 98, and 71 in distance, maximum parsimony, and maximum likelihood trees, respectively. Similarity calculations using approximately 740-bp amplified poribacterial 16S rRNA gene fragments and other poribacterial sequences from the NCBI database showed that the dissimilarity between clades was consistent with their separation in phylogenetic trees. For example, the levels of dissimilarity between members of clade I and clade II were 3 to 8%, while the levels of dissimilarity between members of clades I and III and between members of clades I and IV were 10 to 14% and 11 to 15%, respectively.Open in a separate windowFIG. 1.Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing relationships of poribacterial clones from different global regions. The poribacterial clones on the right are additional clones belonging to the same clades as strains in the tree at the same level. Bootstrap confidence values of >75% for distance, maximum parsimony, and maximum likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba; C, Aplysina cavernicola; F, Aplysina fistularis; L, Aplysina lacunosa; S, Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella globostellata; T, Theonella swinhoei; X, Xestospongia muta. Scale bar = 0.1 nucleotide substitution per site.

TABLE 1.

Distribution of poribacteria in different demosponge orders
Sponge species or seawaterOrderGeographic locationaPresence of poribacteriabReference
Aplysina aerophobaVerongidaMED+2
Aplysina lacunosaVerongidaBAH+2
Aplysina fistularisVerongidaEPAC or BAH+2
Aplysina insularisVerongidaBAH+2
Verongula giganteaVerongidaBAH+2
Smenospongia aureaDictyoceratidaBAH+2
Aplysina cauliformisVerongidaBAH+This study
Aplysina archeriVerongidaBAH+This study
Aplysina cavernicolaVerongidaMED+This study
Pseudoceratina clavataVerongidaWPAC+This study
Rhabdastrella globostellataAstrophoridaWPAC+This study
Ircinia sp.DictyoceratidaBAH+This study
Xestospongia mutaHaploscleridaBAH+This study
Theonella swinhoeiLithistidaEPAC+This study
Plakortis sp.HomosclerophoridaBAH+This study
Chondrilla nuculaHadromeridaBAH2
Agelas wiedenmayeriAgelasidaBAH2
Agelas cerebrumAgelasidaBAHThis study
Axinella polypoidesHalichondridaMEDThis study
Ptilocaulis sp.HalichondridaBAH2
Dysidea avaraDictyoceratidaMEDThis study
Haliclona sp.HaploscleridaMEDThis study
Ectyoplasia feroxPoeciloscleridaBAH2
SeawaterNAcMEDThis study
Open in a separate windowaMED, Mediterranean Sea; BAH, Bahamas; WPAC, western Pacific Ocean; EPAC, eastern Pacific Ocean.bThe presence of poribacteria was evaluated by sequencing and phylogenetic analysis of amplified PCR products. +, present; −, absent.cNA, not applicable.Within each clade in the phylum Poribacteria, there were higher similarity values, including 94 to 100% among members of clade I, 94 to 99% among members of clade II, 96 to 99% among members of clade III, and 96 to 99% among members of clade IV. When members of the the phylum Poribacteria were compared to members of the Planctomycetes (Fig. (Fig.1),1), the 16S rRNA genes exhibited levels of sequence dissimilarity of up to 38%, consistent with the conclusion of Fieseler et al. concerning the separate phylum level status of poribacteria based on a similarity value of <75%. A phylogenetic correlation between sponge phylogeny and poribacterial phylogeny is not evident, since, for example, clones from A. fistularis and Aplysina aerophoba occurred in both clade I and clade II and one clone from A. aerophoba also occurred in clade III, while clones from P. clavata and R. globostellata occurred in clades I, II, and III but not in clade IV. Clades I and II included poribacterial clones derived from all sponge species occurring in all of the widely separated geographic regions examined in this study (Fig. (Fig.2).2). Clade III represented poribacterial clones derived from sponge species obtained in the eastern Pacific region, GBR, and the Bahamas but not in the Mediterranean region. The majority of poribacterial clones in clade IV were derived from sponge species obtained in the Bahamas, and one clade IV clone was obtained from a sponge species collected in the Mediterranean region.Open in a separate windowFIG. 2.Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing the internal relationships of and occurrence of clade I members in distinct sponge species representing cosmopolitan geographic regions. For an explanation of the colors, see Fig. Fig.1.1. Bootstrap confidence values of >75% for distance, maximum parsimony, and maximum likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba; C, Aplysina cavernicola; F, Aplysina fistularis; L, Aplysina lacunosa; S, Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella globostellata; T, Theonella swinhoei; X, Xestospongia muta. Scale bar = 0.1 nucleotide substitution per site. Clones PC15, L8, T6, C2, P3, S2, and X1 were removed from this analysis to allow better branch resolution.Poribacterial clones from different sponges from widely separated marine habitats belonged to at least four major clades with similarities ranging from 94 and 96%. For clade III (Fig. (Fig.1),1), we detected a signature sequence characteristic of poribacterial clones from the GBR sponges R. globostellata and P. clavata. This signature sequence (CCA GTT AGC TTG ACG GTA) (Table (Table2)2) at E. coli positions 469 to 487 targeted 10 sequences, 5 of which were from GBR marine sponges generated in this study (clones RG68, RG112, RG105, PC96, and PC8). Another five poribacterial sequences were detected in an unpublished study investigating the microbial diversity in GBR sponges. This signature sequence indicates a specific geographic presence of poribacteria belonging to clade IV in the GBR region. In addition, a sequence (GAG TGT GAA ATG GCT TGG at E. coli positions 599 to 617) characteristic of clade IV was found in 11 sequences derived from sponges from the Bahamas and one sequence (A7) from a Mediterranean sponge.

TABLE 2.

Poribacterial signature sequences for clades III and IV, including a GBR-specific signature sequence (pori_SSIII) and a signature sequence specific to 11 of 12 sequences from the Bahamas (pori_SSIV)
Signature sequenceNameFull nameaE. coli positionSequenceb
pori_SSIIIPla101PPla101P*469GGUGAUAAG-==================-CCAUAGUA
Pla131PPla131P*469GGUGAUAAG-==================-CCAUAGUA
Pla134PPla134P*469GGUGAUAAG-==================-CCAUAGUA
Pla50PPla50P*469GGUGAUAAG-==================-CCAUAGUA
Pla82PPla82P*469GGUGAUAAG-==================-CCAUAGUA
PO68Pori clone RGPo68469GGUGAUAAG-==================-GAGAAAAG
PO112Pori clone RGPo112469GGUGAUAAU-==================-CCAUAGUA
PO105Pori clone RGPo105469GGUGAUAAG-==================-CCAUAGUA
PO96Uncultured Pori clone469GGUGAUAAG-==================-CCAUAGUA
PCPO8Pori clone PCPo8469GGUGAUAAG-==================-CCAUAGUA
pori_SSIVAY485286Uncultured Pori clone599ACAUUAGUC-==================-CUCAACCA
AY485285Uncultured Pori clone599ACAUNAGUC-==================-CUCAACNA
AY485284Uncultured Pori clone599ACAUUAGUC-==================-CUCAACCA
AY485281Uncultured Pori bacterium599ACAUUAGUC-==================-CUCAACCA
A7A7599AUAUUAGUC-==================-CUCAACCA
F2F2599ACAUAAGUC-==================-CUCAACCA
L16L16599ACAUUAGUC-==================-CUCAACCA
P20P20599ACAUAAGUC-==================-CUCAACCA
P38P38599ACAUUAGUC-==================-CUCAACCA
S6S6599AUAUUAGUC-==================-CUCAACCA
S10S10599AUAUUAGUC-==================-CUCAACCA
X18X18599ACAUUAGUC-==================-CUCAACCA
Open in a separate windowaAsterisks indicate poribacterial clones derived from the GBR sponge R. globostellata in a separate study.bThe internal sequence (indicated by equals signs) of each pori_SSIII clone is CCAGUUAGCUUGACGGUA, and that of each pori_SSIV clone is GAGUGUGAAAUGGCUUGG.Based on the data presented here, Poribacteria appears to be a bacterial phylum that is specifically found in several demosponge genera of the phylum Porifera (Table (Table1).1). To our knowledge, this is the only case of a bacterial phylum specifically associated with a marine invertebrate phylum. Certain phylum members appear to be widely distributed among sponges belonging to different species and in different geographic regions, forming sponge-specific lineages (3), but these are individual species level or at most genus level clades in a subdivision of a phylum rather than in a whole phylum.PCR analyses of seawater samples collected in this study (Table (Table1)1) and searches using nucleotide sequence databases of seawater metagenomes were negative for poribacteria. This is consistent with the concept that Poribacteria is a sponge-specific phylum. Within the sponges poribacteria are distributed among members of distinct demosponge orders that occur in various geographic locations, indicating that there is wide distribution of poribacteria among marine demosponges. Very similar 16S rRNA clone sequences that cluster in clade I were found in sponges from all geographic regions sampled in this study, including locations in the Northern and Southern hemispheres (Fig. (Fig.2).2). Similarly, clade II contains poribacterial clones from the Mediterranean Aplysina species and from GBR Pseudoceratina and Rhabdastrella species. This appears to contrast, albeit at a lower level of resolution, with results suggesting that bacterial populations are endemic in different geographic regions, e.g., with the findings that marine bacterioplankton communities include few cosmopolitan operational taxonomic units (8), that fluorescent Pseudomonas genotypes from soil are endemic at different geographic sites (1), and that hyperthermophilic Sulfolobus archaea from different geothermal areas are genetically divergent (12). Judging the endemicity of populations in different geographic regions may depend on the taxonomic scale used to distinguish populations (1). In this study we provide evidence that at least some clades may be relatively characteristic of particular regions, e.g., GBR clade III (Table (Table2).2). It is remarkable that in the case of the sponge species R. globostellata and P. clavata from a single geographic region (GBR), the microbial communities include representatives of distantly related poribacterial clades II and III, whose sequences exhibit levels of dissimilarity ranging from 10 to 13%. In another case poribacteria belonging to clades I, II, and IV were found in a single host, A. aerophoba, from the Mediterranean. Thus, members of widely divergent poribacterial clades occur in the same specimen in sponges in widely separated geographic regions in the world''s oceans. Three different sponge species belonging to three different genera exhibit this phenomenon.The morphology and life strategy of sponges have remained unchanged for the past 580 million years, as judged by the dramatic similarity of the morphologies of Precambrian fossils to the morphologies of recent sponges (6). Adaptation of the poribacteria to this niche might have taken place early in evolution before the various sponge orders separated from each other. It seems likely that poribacteria diverged from other bacterial phyla long before evolution of the metazoans as part of the fan-like radiation by which all bacterial phyla appear to have arisen (4). This bacterial radiation may have resulted in the divergence of the clades that we have observed for the poribacteria, but there is no indication of cospeciation between host sponges and the poribacteria.In summary, poribacteria exhibit considerable diversity and are classified into four phylogenetic clades. Poribacteria seem to be widely distributed among many different marine demosponge genera, and further studies are needed to explain the nature of the poribacterium-sponge interaction.  相似文献   

8.
The Norway spruce genome provides key insights into the evolution of plant genomes, leading to testable new hypotheses about conifer, gymnosperm, and vascular plant evolution.In the past year a burst of plant genome sequences have been published, providing enhanced phylogenetic coverage of green plants (Figure (Figure1)1) and inclusion of new agricultural, ecological, and evolutionary models. Collectively, these sequences are revealing some extraordinary structural and evolutionary attributes in plant genomes. Perhaps most surprising is the exceptionally high frequency of whole-genome duplication (WGD): nearly every genome that has been analyzed has borne the signature of one or more WGDs, with particularly notable events having occurred in the common ancestors of seed plants, of angiosperms, and of core eudicots (the latter ''WGD'' represents two WGDs in close succession) [1,2]. Given this tendency for plant genomes to duplicate and then return to an essentially diploid genetic system (an example is the cotton genomes, which have accumulated the effects of perhaps 15 WGDs [3]), the conservation of genomes in terms of gene number, chromosomal organization, and gene content is astonishing. From the publication of the first plant genome, Arabidopsis thaliana [4], the number of inferred genes has been between 25,000 and 30,000, with many gene families shared across all land plants, although the number of members and patterns of expansion and contraction vary. Furthermore, conserved synteny has been detected across the genomes of diverse angiosperms, despite WGDs, diploidization, and millions of years of evolution.Open in a separate windowFigure 1Simplified phylogeny of land plants, showing major clades and their component lineages. Asterisks indicate species (or lineage) for which whole-genome sequence (or sequences) is (are) available. Increases and decreases in genome size are shown by arrows.Despite the proliferation of genome sequences available for angiosperms, genome-level data for both ferns (and their relatives, collectively termed monilophytes; Figure Figure1)1) and gymnosperms have been conspicuously lacking - until recently, with the publication of the genome sequence of the gymnosperm Norway spruce (Picea abies) [5]. The large genome sizes for both monilophytes and gymnosperms have discouraged attempts at genome sequencing and assembly, whereas the smaller genome size of angiosperms has resulted in more genome sequences being available (Table (Table1)1) [6]. Because of this limited phylogenetic sample, our understanding of the timing and phylogenetic positions of WGDs, the core number of plant genes, possible conserved syntenic regions, and patterns of expansion and contraction of gene families across both tracheophytes (vascular plants) and across all land plants is imperfect. This sampling problem is particularly acute in analyses of the genes and genomes of seed plants; many hundreds of genes are present in angiosperms that are not present in mosses or lycophytes, but whether these genes arose in the common ancestor of seed plants or of angiosperms cannot be determined without a gymnosperm genome sequence. The Norway spruce genome therefore offers tremendous power, not only for understanding the structure and evolution of conifer genomes, but also as a reference for interpreting gene and genome evolution in angiosperms.

Table 1

Genome sizes in land plants
LineageRange (1C; pg)Mean
Gymnosperms
  Conifers
    Pinaceae9.5-36.023.7
    Cupressaceae8.3-32.112.8
    Sciadopitys 20.8n/a
  Gnetales
    Ephedraceae8.9-15.78.9
    Gnetaceae2.3-4.02.3
    Cycadaceae12.6-14.813.4
    Ginkgo biloba11.75n/a
Monilophytes
    Ophioglossaceae10.2-65.631.05
    Equisetaceae12.9-30422.0
    Psilotum72.7n/a
  Leptosporangiate ferns
    Polypodiaceae7.5-19.77.5
    Aspleniaceae4.1-9.16.2
    Athyriaceae6.3-9.37.6
    Dryopteridaceae6.8-23.611.7
  Water ferns
    Azolla0.77n/a
Angiosperms
    Oryza sativa 0.50n/a
    Amborella trichopoda0.89n/a
    Arabidopsis thaliana0.16n/a
    Zea mays2.73n/a
Open in a separate windown/a, not applicable. Data based on [6].  相似文献   

9.
This study investigated the added value, i.e. discriminative and concurrent validity and reproducibility, of an eye-hand coordination test relevant to table tennis as part of talent identification. Forty-three table tennis players (7–12 years) from national (n = 13), regional (n = 11) and local training centres (n = 19) participated. During the eye-hand coordination test, children needed to throw a ball against a vertical positioned table tennis table with one hand and to catch the ball correctly with the other hand as frequently as possible in 30 seconds. Four different test versions were assessed varying the distance to the
TotalNationalRegionalLocal
Total43131119
Boys268810
Girls17539
Age (years)10.4±1.410.9±1.510.4±1.510.1±1.4
7 year olds1--1
8 year olds5113
9 year olds3-3-
10 year olds12327
11 year olds11515
12 year olds11443
Length (cm)149±11150±12150±12148±10
Weight (kg )38±837±737±738±9
Right-handed359917
Left-handed8422
Training (hours*week-1)6 (0–20)11 (7–20)7 (4–11)2 (0–3)
Competition (points)173 (−52–430)297 (144–430)188 (72–317)36 (−52–130)
Open in a separate windowData are frequencies, except for age, length and weight (years±SD), and training and competition (mean (range)).  相似文献   

10.
The Russian invasion of Ukraine: a humanitarian tragedy and a tragedy for science     
Halyna R Shcherbata 《EMBO reports》2022,23(5)
The Invasion of Ukraine prompts us to support our Ukranian colleagues but also to keep open communication with the Russian scientists who oppose the war.

In the eyes of the civilized world, Russia has already lost the war: politically, it is becoming ever more isolated; economically as the sanctions take an enormous toll; militarily as the losses of the Russian army mount. In contrast, the courage of Ukrainian people fighting for their independence has united the Western world that is providing enormous support for those Ukrainians who fight the Russian invasion and those who have fled their war‐torn country. Once this war is over, Ukraine will have to heal the wounds of war, reunite families, restore its economy, reestablish infrastructure, and rebuild science and education. Russia will have to restore its dignity and overcome its self‐inflicted isolation.Europe’s unity in condemning Russia’s war of aggression and showing its solidarity with Ukraine has been impressive. This includes not the least welcoming and accommodating millions of refugees. We, the scientific community in Europe, have a moral obligation to help Ukrainian students and colleagues by providing safe space to study and to continue their research. First, European research organizations and funding agencies should develop strategies to support them in the years to come. Second, efforts by EMBO, research funders, universities, and research institutions to support Ukrainian students and scientists are necessary. As a first priority, dedicated and unbureaucratic short‐term scholarship and grant programs are required to accommodate Ukrainian scientists; such programs have been already initiated by many organizations, for example, by EMBO, Volkswagen Stiftung, Max Planck Society, and the ERC among others. These help Ukrainian scientists to stay connected to research and become integrated into the European research landscape. In the long‐term and after the war, this aid should be complemented by funding for research centers of excellence in Ukraine, to which scientists could then return.Even though the priority must be to help Ukrainians, we must also think of students and colleagues in Russia who oppose the war and are affected by the sanctions. As the Iron Curtain closes again, we have to think differently about our ongoing and future collaborations. Although freezing most, if not all, research collaborations with official Russian organizations is justified, it would be a mistake to extend these sanctions to all scientists and students. There is already an exodus of Russian and Belarusian scholars, which will only accelerate in the next months and years, and accepting scientists who ask for political asylum will be beneficial for Europe.The fraction of Russian society in open opposition to the war is, unfortunately, smaller than that officially in support of it. At the beginning of the war, a number of Russian scientists published an open letter on the internet, in which they condemn this war (https://t‐invariant.org/2022/02/we‐are‐against‐war/). They clearly state that "The responsibility for unleashing a new war in Europe lies entirely with Russia. There is no rational justification for this war”, and “demand an immediate halt to all military operations directed against Ukraine". At the same time, other prominent Russian science and education officials signed the “Statement of the Russian Union of University Rectors (Provosts)”, which expressed unwavering support for Russia, its president and its Army and their goal to “to achieve demilitarization and denazification of Ukraine and thus to defend ourselves from the ever‐growing military threat” (https://www.rsr‐online.ru/news/2022‐god/obrashchenie‐rossiyskogo‐soyuza‐rektorov1/).Inevitably, Russian scientists must decide themselves how to live and continue their scientific work under the increasingly tight surveillance of the Kremlin regime. History is repeating itself. Not long ago, during the Cold War, Soviet scientists were largely isolated from the international research community and worked in government‐controlled research. In some fields, no one knew what they were working on or where. However, even in those dark times, courageous individuals such as Andrei Sakharov spoke out against the regime and tried to educate the next generation about the importance of free will. Many Soviet geneticists had been arrested under Stalin’s regime of terror and as a result of Lysenkoism and were executed or sent to the Gulag or had to emigrate, such as Nikolaj Timofeev‐Resovskij, one of the great geneticists of his time and an opponent of communism. As a result of sending dissident scientists to Siberia, great educational institutions were created in the region, which trained many famous scientists. History tells us that it is impossible to kill free will and the search for truth.The Russian invasion of Ukraine is a major humanitarian tragedy and a tragedy for science at many levels. Our hope is that the European science community, policymakers, and funders will be prepared to continue and expand support for our colleagues from Ukraine and eventually help to rebuild the bridges with Russian science that have been torn down.This commentary has been endorsed and signed by the EMBO Young Investigators and former Young Investigators listed below.

All signatories are current and former EMBO Young Investigators and endorse the statements in this article.
Igor AdameykoKarolinska Institut, Stockholm, Sweden
Bungo AkiyoshiUniversity of Oxford, United Kingdom
Leila AkkariNetherlands Cancer Institute, Amsterdam, Netherlands
Panagiotis AlexiouMasaryk University, Brno, Czech Republic
Hilary AsheFaculty of Life Sciences, University of Manchester, United Kingdom
Michalis AverofInstitut de Génomique Fonctionnelle de Lyon (IGFL), France
Katarzyna BandyraUniversity of Warsaw, Poland
Cyril BarinkaInstitute of Biotechnology AS CR, Prague, Czech Republic
Frédéric BergerGregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria
Vitezslav BryjaInstitute of Experimental Biology, Masaryk University, Brno, Czech Republic
Janusz BujnickiInternational Institute of Molecular and Cell Biology, Warsaw, Poland
Björn BurmannUniversity Gothenburg, Sweden
Andrew CarterMRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Pedro CarvalhoSir William Dunn School of Pathology University of Oxford, United Kingdom
Ayse Koca CaydasiKoç University, Istanbul, Turkey
Hsu‐Wen ChaoMedical University, Taipei, Taiwan
Jeffrey ChaoFriedrich Miescher Institute, Basel, Switzerland
Alan CheungUniversity of Bristol, United Kingdom
Tim ClausenResearch Institute for Molecular Pathology (IMP), Vienna, Austria
Maria Luisa CochellaThe Johns Hopkins University School of Medicine, USA
Francisco CubillosSantiago de Chile, University, Chile
Uri Ben‐DavidTel Aviv University, Tel Aviv, Israel
Sebastian DeindlUppsala University, Sweden
Pierre‐Marc DelauxLaboratoire de Recherche en Sciences Végétales, Castanet‐Tolosan, France
Christophe DessimozUniversity, Lausanne, Switzerland
Maria DominguezInstitute of Neuroscience, CSIC ‐ University Miguel Hernandez, Alicante, Spain
Anne DonaldsonInstitute of Medical Sciences, University of Aberdeen, United Kingdom
Peter DraberBIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic
Xiaoqi FengJohn Innes Centre, Norwich, United Kingdom
Luisa FigueiredoInstitute of Molecular Medicine, Lisbon, Portugal
Reto GassmannInstitute for Molecular and Cell Biology, Porto, Portugal
Kinga Kamieniarz‐GdulaAdam Mickiewicz University in Poznań, Poland
Roger GeigerInstitute for Research in Biomedicine, Bellinzona, Switzerland
Niko GeldnerUniversity of Lausanne, Switzerland
Holger GerhardtMax Delbrück Center for Molecular Medicine, Berlin, Germany
Daniel Wolfram GerlichInstitute of Molecular Biotechnology (IMBA), Vienna, Austria
Jesus GilMRC Clinical Sciences Centre, Imperial College London, United Kingdom
Sebastian GlattMalopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
Edgar GomesInstitute of Molecular Medicine, Lisbon, Portugal
Pierre GönczySwiss Institute for Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
Maria GornaUniversity of Warsaw, Poland
Mina GoutiMax‐Delbrück‐Centrum, Berlin, Germany
Jerome GrosInstitut Pasteur, Paris, France
Anja GrothBiotech Research and Innovation Centre (BRIC), University of Copenhagen, Denmark
Annika GuseCentre for Organismal Studies, Heidelberg, Germany
Ricardo HenriquesInstituto Gulbenkian de Ciência, Oeiras, Portugal
Eva HoffmannCenter for Chromosome Stability, University of Copenhagen, Denmark
Thorsten HoppeCECAD at the Institute for Genetics, University of Cologne, Germany
Yen‐Ping HsuehAcademia Sinica, Taipei, Taiwan
Pablo HuertasAndalusian Molecular Biology and Regenerative Medicine Centre (CABIMER), Seville, Spain
Matteo IannaconeIRCCS San Raffaele Scientific Institute, Milan, Italy
Alvaro Rada‐IglesiasInstitue of Biomedicine and Biotechnology of Cantabria (IBBTEC)
University of Cantabria, Santander, Spain
Axel InnisInstitut Européen de Chimie et Biologie (IECB), Pessac, France
Nicola IovinoMPI für Immunbiologie und Epigenetik, Freiburg, Germany
Carsten JankeInstitut Curie, France
Ralf JansenInterfaculty Institute for Biochemistry, Eberhard‐Karls‐University Tübingen, Germany
Sebastian JessbergerHiFo / Brain Research Institute, University of Zurich, Switzerland
Martin JinekUniversity of Zurich, Switzerland
Simon Bekker‐JensenUniversity, Copenhagen, Denmark
Nicole JollerUniversity of Zurich, Switzerland
Luca JovineDepartment of Biosciences and Nutrition & Center for
Biosciences, Karolinska Institutet, Stockholm, Sweden
Jan Philipp JunkerMax‐Delbrück‐Centrum, Berlin, Germany
Anna KarnkowskaUniversity, Warsaw, Poland
Zuzana KeckesovaInstitute of Organic Chemistry and Biochemistry AS CR, Prague, Czech Republic
René KettingInstitute of Molecular Biology (IMB), Mainz, Germany
Bruno KlaholzInstitute of Genetics and Molecular and Cellular Biology (IGBMC), University of Strasbourg, Illkirch, France
Jürgen KnoblichInstitute of Molecular Biotechnology (IMBA), Vienna, Austria
Taco KooijCentre for Molecular Life Sciences, Nijmegen, Netherlands
Romain KoszulInstitut Pasteur, Paris, France
Claudine KraftInstitute for Biochemistry and Molecular Biology, Universität Freiburg, Germany
Alena KrejciFaculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic
Lumir KrejciNational Centre for Biomolecular Research (NCBR), Masaryk University, Brno, Czech Republic
Arnold KristjuhanInstitute of Molecular and Cell Biology, University of Tartu, Estonia
Yogesh KulathuMRC Protein Phosphorylation & Ubiquitylation Unit, University of Dundee, United Kingdom
Edmund KunjiMRC Mitochondrial Biology Unit, Cambridge, United Kingdom
Karim LabibMRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, United Kingdom
Thomas LecuitDevelopmental Biology Institute of Marseilles ‐ Luminy (IBDML), France
Gaëlle LegubeCenter for Integrative Biology in Toulouse, Paul Sabatier University, France
Suewei LinAcademia Sinica, Taipei, Taiwan
Ming‐Jung LiuAcademia Sinica, Taipei, Taiwan
Malcolm LoganRandall Division of Cell and Molecular Biophysics, King’s College London, United Kingdom
Massimo LopesUniversity of Zurich, Switzerland
Jan LöweStructural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Martijn LuijsterburgUniversity Medical Centre, Leiden, Netherlands
Taija MakinenUppsala University, Sweden
Sandrine Etienne‐MannevilleInstitut Pasteur, Paris, France
Miguel ManzanaresSpanish National Center for Cardiovascular Research (CNIC), Madrid, Spain
Jean‐Christophe MarineCenter for Biology of Disease, Laboratory for Molecular Cancer Biology, VIB & KU Leuven, Belgium
Sascha MartensMax F. Perutz Laboratories, University of Vienna, Austria
Elvira MassUniversität Bonn, Germany
Olivier MathieuClermont Université, Aubière, France
Ivan MaticMax Planck Institute for Biology of Ageing, Cologne, Germany
Joao MatosMax Perutz Laboratories, Vienna, Austria
Nicholas McGranahanUniversity College London, United Kingdom
Hind MedyoufGeorg‐Speyer‐Haus, Frankfurt, Germany
Patrick MeraldiUniversity of Geneva, Switzerland
Marco MilánICREA & Institute for Research in Biomedicine (IRB), Barcelona, Spain
Eric MiskaWellcome Trust/Cancer Research UK Gurdon Institute,
University of Cambridge, United Kingdom
Nuria MontserratInstitut de Bioenginyeria de Catalunya (IBEC), Barcelona, Spain
Nuno Barbosa‐MoraisInstitute of Molecular Medicine, Lisbon, Portugal
Antonin MorillonInstitut Curie, Paris, France
Rafal MostowyJagiellonian University, Krakow, Poland
Patrick MüllerUniversity of Konstanz, Konstanz, Germany
Miratul MuqitUniversity of Dundee, United Kigdom
Poul NissenCentre for Structural Biology, Aarhus University, Denmark
Ellen NollenEuropean Research Institute for the Biology of Ageing, University of Groningen, Netherlands
Marcin NowotnyInternational Institute of Molecular and Cell Biology, Warsaw, Poland
John O''NeillMRC Laboratory of Molecular Biology, Cambridge, United Kigdom
Tamer ÖnderKoc University School of Medicine, Istanbul, Turkey
Elin OrgUniversity of Tartu, Estonia
Nurhan ÖzlüKoç University, Istanbul, Turkey
Bjørn Panyella PedersenAarhus University, Denmark
Vladimir PenaLondon, The Institute of Cancer Research, United Kingdom
Camilo PerezBiozentrum, University of Basel, Switzerland
Antoine PetersFriedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland
Clemens PlaschkaIMP, Vienna, Austria
Pavel PlevkaCEITEC, Masaryk University, Brno, Czech Republic
Hendrik PoeckTechnische Universität, München, , Germany
Sophie PoloUniversité Diderot (Paris 7), Paris, France
Simona PoloIFOM ‐ The FIRC Institute of Molecular Oncology, Milan, Italy
Magdalini PolymenidouUniversity of Zurich, Switzerland
Freddy RadtkeSwiss Institute for Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
Markus RalserInstitute of Biochemistry Charité, Berlin, Germany & MRC National Institute for Medical Research, London, United Kingdom
Jan RehwinkelJohn Radcliffe Hospital, Oxford, United Kingdom
Maria RescignoEuropean Institute of Oncology (IEO), Milan, Italy
Katerina RohlenovaPrague, Institute of Biotechnology, Czech Republic
Guadalupe SabioCentro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
Ana Jesus Garcia SaezUniversity of Cologne, CECAD Research Center, Germany
Iris SaleckerInstitut de Biologie de l''Ecole Normale Supérieure (IBENS), Paris, France
Peter SarkiesUniversity of Oxford, United Kingdom
Frédéric SaudouGrenoble Institute of Neuroscience, France
Timothy SaundersCentre for Mechanochemical Cell Biology, Interdisciplinary Biomedical Research Building, Warwick Medical School, Coventry, United Kingdom
Orlando D. SchärerIBS Center for Genomic Integrity, Ulsan, South Korea
Arp SchnittgerBiozentrum Klein Flottbek, University of Hamburg, Germnay
Frank SchnorrerAix Marseille University, CNRS, IBDM, Turing Centre for Living Systems, Marseille, France
Maya SchuldinerDepartment of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
Schraga SchwartzWeizmann Institute of Science, Rehovot, Israel
Martin SchwarzerInstitute of Microbiology, Academy of Sciences of the Czech Republic
Claus MariaInstituto de Medicina Molecular Faculdade de Medicina da Universidade de Lisboa, Portugal
Hayley SharpeThe Babraham Institute, United Kingdom
Halyna ShcherbataInstitute of Cell Biochemistry, Hannover Medical School, Hannover, Germany
Eric SoDepartment of Haematological Medicine, King''s College London, United Kingdom
Victor SourjikMax Planck Institute for Terrestrial Microbiology, Marburg, Germany
Anne SpangBiozentrum, University of Basel, Switzerland
Irina StanchevaInstitute of Cell Biology, University of Edinburgh, United Kingdom
Bas van SteenselDepartment of Gene Regulation, The Netherlands Cancer Institute, Amsterdam, Netherlands
Richard SteflCEITEC, Masaryk University, Brno, Czech Republic
Yonatan StelzerWeizmann Institute of Science, Rehovot, Israel
Julian StingeleLudwig‐Maximilians‐Universität, München, Germany
Katja SträßerInstitute for Biochemistry, University of Giessen, Germany
Kvido StrisovskyInstitute of Organic Chemistry and Biochemistry ASCR, Prague, Czech Republic
Joanna SulkowskaUniversity, Warsaw, Poland
Grzegorz SumaraNencki Institute of Experimental Biology, Warsaw, Poland
Karolina SzczepanowskaInternational Institute Molecular Mechanisms & Machines PAS, Warsaw, Poland
Luca TamagnoneInstitute for Cancer Research and Treatment, University of Torino Medical School, Italy
Meng How TanSingapore, Nanyang Technological University, Singapore
Nicolas TaponCancer Research UK London Research Institute, United Kingdom
Nicholas M. I. TaylorUniversity, Copenhagen, Denmark
Sven Van TeeffelenUniversité de Montréal, Canada
Maria Teresa TeixeiraLaboratory of Molecular and Cellular Biology of Eukaryotes, IBPC, Paris, France
Aurelio TelemanGerman Cancer Research Center (DKFZ), Heidelberg, Germany
Pascal TherondInstitute Valrose Biology, University of Nice‐Sophia Antipolis, France
Pavel TolarUniversity College London, United Kingdom
Isheng Jason TsaiAcademia Sinica, Taipei, Taiwan
Helle UlrichInstitute of Molecular Biology (IMB), Mainz, Germany
Stepanka VanacovaCentral European Institute of Technology, Masaryk University, Brno, Czech Republic
Henrique Veiga‐FernandesChampalimaud Center for the Unknown, Lisboa, Portugal
Marc VeldhoenInstituto de Medicina Molecular, Lisbon, Portugal
Louis VermeulenAcademic Medical Centre, Amsterdam, Netherlands
Uwe VinkemeierUniversity of Nottingham Medical School, United Kingdom
Helen WaldenMRC Protein Phosphorylation & Ubiquitylation Unit, University of Dundee, United Kingdom
Michal WandelInstitute of Biochemistry and Biophysics, PAS, Warsaw, Poland
Julie WelburnWellcome Trust Centre, Edinburgh, United Kingdom
Ervin WelkerInstitute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary
Gerhard WingenderIzmir Biomedicine and Genome Center, Dokuz Eylul University, Izmir, Turkey
Thomas WollertInstitute Pasteur, Membrane Biochemistry and Transport, Centre François Jacob, Paris, France
Hyun YoukUniversity of Massachusetts Medical School, USA
Christoph ZechnerMPI für molekulare Zellbiologie und Genetik, Dresden, Germany
Philip ZegermanWellcome Trust / Cancer Research UK Gurdon Institute, University of Cambridge, United Kingdom
Alena ZikováInstitute of Parasitology, Biology Centre AS CR, Ceske Budejovice, Czech Republic
Piotr ZiolkowskiAdam Mickiewicz University, Poznan, Poland
David ZwickerMPI für Dynamik und Selbstorganisation, Göttingen, Germany
Open in a separate window  相似文献   

11.
Destabilized eYFP variants for dynamic gene expression studies in Corynebacterium glutamicum     
Eva Hentschel  Cornelia Will  Nurije Mustafi  Andreas Burkovski  Nadine Rehm  Julia Frunzke 《Microbial biotechnology》2013,6(2):196-201
  相似文献   

12.
Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD     
Sandy Y. M. Ng  David J. VanDyke  Bonnie Chaban  John Wu  Yoshika Nosaka  Shin-Ichi Aizawa  Ken F. Jarrell 《Journal of bacteriology》2009,191(21):6732-6740
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   

13.
Economic Benefits of Investing in Women’s Health: A Systematic Review     
Kristine Hus?y Onarheim  Johanne Helene Iversen  David E. Bloom 《PloS one》2016,11(3)

Background

Globally, the status of women’s health falls short of its potential. In addition to the deleterious ethical and human rights implications of this deficit, the negative economic impact may also be consequential, but these mechanisms are poorly understood. Building on the literature that highlights health as a driver of economic growth and poverty alleviation, we aim to systematically investigate the broader economic benefits of investing in women’s health.

Methods

Using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, we systematically reviewed health, gender, and economic literature to identify studies that investigate the impact of women’s health on micro- and macroeconomic outcomes. We developed an extensive search algorithm and conducted searches using 10 unique databases spanning the timeframe 01/01/1970 to 01/04/2013. Articles were included if they reported on economic impacts stemming from changes in women’s health (table of outcome measures included in full review, Outcome measures   FertilityIntergenerational Health SpilloverEducationProductivitySavingsMicroeconomic level    Total fertility rateChild survivalEnrollment in schoolIncomeMoneyChange in fertilityChild wellbeing and behaviorYears of schoolingPurchasing powerAssetsAge at first birth/ teenage pregnanciesAnthropometryEarly drop outPerformanceBirth spacingImproved cognitive developmentPerformance in school Life expectancyHigher education  Adult health outcomesLiteracy  Nutrition   Intrauterine growth  Macroeconomic level    Open in a separate windowGross domestic product/gross national product, gross domestic product/gross national product growth, income per capita, labor force participation, per capita income.

Results

The existing literature indicates that healthier women and their children contribute to more productive and better-educated societies. This study documents an extensive literature confirming that women’s health is tied to long-term productivity: the development and economic performance of nations depends, in part, upon how each country protects and promotes the health of women. Providing opportunities for deliberate family planning; healthy mothers before, during, and after childbirth, and the health and productivity of subsequent generations can catalyze a cycle of positive societal development.

Conclusions

This review highlights the untapped potential of initiatives that aim to address women’s health. Societies that prioritize women’s health will likely have better population health overall, and will remain more productive for generations to come.  相似文献   

14.
Genome-Wide Characterization of the SloR Metalloregulome in Streptococcus mutans     
Kevin P. O'Rourke  Jeremy D. Shaw  Mitchell W. Pesesky  Brian T. Cook  Susanne M. Roberts  Jeffrey P. Bond  Grace A. Spatafora 《Journal of bacteriology》2010,192(5):1433-1443
  相似文献   

15.
Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae Due to Narrow-Spectrum β-Lactamases as Well as Omp Mutation     
Fengzhen Yang  Qi Zhao  Lipeng Wang  Jinying Wu  Lihua Jiang  Li Sheng  Leyan Zhang  Zhaoping Xue  Maoli Yi 《Polish journal of microbiology》2022,71(2):251
Cefoperazone/sulbactam (CSL) and piperacillin/tazobactam (TZP) are commonly used in clinical practice in China because of their excellent antimicrobial activity. CSL and TZP-nonsusceptible Enterobacteriaceae are typically resistant to extended-spectrum cephalosporins such as ceftriaxone (CRO). However, 11 nonrepetitive Enterobacteriaceae strains, which were resistant to CSL and TZP yet susceptible to CRO, were collected from January to December 2020. Antibiotic susceptibility tests and whole-genome sequencing were conducted to elucidate the mechanism for this rare phenotype. Antibiotic susceptibility tests showed that all isolates were amoxicillin/clavulanic-acid resistant and sensitive to ceftazidime, cefepime, cefepime/tazobactam, cefepime/zidebactam, ceftazidime/avibactam, and ceftolozane/tazobactam. Whole-genome sequencing revealed three of seven Klebsiella pneumoniae strains harbored blaSHV-1 only, and four harbored blaSHV-1 and blaTEM-1B. Two Escherichia coli strains carried blaTEM-1B only, while two Klebsiella oxytoca isolates harbored blaOXY-1-3 and blaOXY-1-1, respectively. No mutation in the β-lactamase gene and promoter sequence was found. Outer membrane protein (Omp) gene detection revealed that numerous missense mutations of OmpK36 and OmpK37 were found in all strains of K. pneumoniae. Numerous missense mutations of OmpK36 and OmpK35 and OmpK37 deficiency were found in one K. oxytoca strain, and no OmpK gene was found in the other. No Omp mutations were found in E. coli isolates. These results indicated that narrow spectrum β-lactamases, TEM-1, SHV-1, and OXY-1, alone or in combination with Omp mutation, contributed to the resistance to CSL and TZP in CRO-susceptible Enterobacteriaceae.Antibiotic susceptibility tests
AntibioticsBreakpoint, (μg/ml)Klebsiella pneumoniae
Escherichia cou
Klebriehd axyoca
E1E3E4E7E9E10E11E6E8E2E5
CRO≤1≥4≤0.5≤0.5≤0.5≤0.5 1≤0.51≤0.5≤0.511
CAZ4 ≥161214444241 1
FEP≤2 216 110.2512220.521 1
AMC≤8 ≥32≥128≥128≥128≥128≥128≥128≥128≥128≥128≥128≥128
CSL≤16 ≥6464646464≥128128≥12864128128≥128
TZP≤16 ≥128≥256≥256≥256≥25622562256≥256≥256≥256≥256≥256
FPT≤2 ≥1610.50.060.1252120.2510.1250.25
FPZ≤2 2160.250.250.060.1250.250.25 10.1250.250.1250.125
CZA≤8 216 10.50.250.2510.2510.50.50.50.25
CZT≤2 28210.5 1222 1122
Open in a separate windowCROceftriaxone, CAZceftazidime, FEPcefepime, AMC:amoxicillin clavulanic-acid, CSLcefoperazone/sulbactam, TZP:piperadllin/tazobactam, FPT:cefepime tazobactam, FPZ:cefepime/zidebactam, CZA:ceftazidime/avibactam, CZTceftolozane/tazobactam Gene sequencing results
NumberStrainSTp-Lactamase genePromoter sequence mutationOmp mutation
ElKpn45blaSHV-1, blaTEM-lBnoneOmpK36, OmpK3 7
E3Kpn45blaSHV-1, blaTEM-lBnoneOmpK36. OmpK3 7
E4Kpn2854blaSHV-1noneOmpK36, OmpK3 7
E7Kpn2358blaSHV-1 - blaTEM-lBnoneOmpK36, OmpK3 7
E9Kpn2358blaSHV-1. blaTEM-lBnoneOmpK36. OmpK3 7
E10Kpn 189blaSHV-1noneOmpK36. OmpK3 7
EllKpn45blaSHV-1noneOmpK36, OmpK3 7
E6Eco88blaTEM-lBnonenone
ESEco409blaTEM-1Bnonenone
E2Kox194blaOXY-1-3noneOmpK36 mutations. OmpK35 and OmpK 37 deficiency
E5Kox 11blaOXY-1-1noneno OmpK (OmpK3 5, OmpK36 and OmpK37) gene found
Open in a separate window  相似文献   

16.
Plasmid pAMS1-Encoded,Bacteriocin-Related “Siblicide” in Enterococcus faecalis     
Christine M. Sedgley  Don B. Clewell  Susan E. Flannagan 《Journal of bacteriology》2009,191(9):3183-3188
  相似文献   

17.
Phage Display Selection of Cyclic Peptides That Inhibit Andes Virus Infection          下载免费PDF全文
Pamela R. Hall  Brian Hjelle  Hadya Njus  Chunyan Ye  Virginie Bondu-Hawkins  David C. Brown  Kathleen A. Kilpatrick  Richard S. Larson 《Journal of virology》2009,83(17):8965-8969
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   

18.
Functional Characterization of Naturally Occurring Variants of Human Hepatitis B Virus Containing the Core Internal Deletion Mutation     
Thomas Ta-Tung Yuan  Min-Hui Lin  Sui Min Qiu  Chiaho Shih 《Journal of virology》1998,72(3):2168-2176
  相似文献   

19.
Analysis of the Clonal Relationship of Serotype O26:H11 Enterohemorrhagic Escherichia coli Isolates from Cattle     
Lutz Geue  Sabrina Klare  Christina Schnick  Birgit Mintel  Katharina Meyer  Franz J. Conraths 《Applied and environmental microbiology》2009,75(21):6947-6953
Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (42). In humans, infections with some STEC serotypes result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by hemolytic-uremic syndrome (HUS) (49). These STEC strains are also designated “enterohemorrhagic E. coli” (EHEC). Consequently, EHEC strains represent a subgroup of STEC with a high pathogenic potential for humans. Strains of the E. coli serogroup O26 were originally classified as enteropathogenic E. coli due to their association with outbreaks of infantile diarrhea in the 1940s. In 1977, Konowalchuk et al. (37) recognized that these bacteria produced Stx, and 10 years later, the Stx-producing E. coli O26:H11/H− strains were classified as EHEC. EHEC O26 strains constitute the most common non-O157 EHEC group associated with diarrhea and HUS in Europe (12, 21, 23, 24, 26, 27, 55, 60). Reports on an association between EHEC O26 and HUS or diarrhea from North America including the United States (15, 30, 33), South America (51, 57), Australia (22), and Asia (31, 32) provide further evidence for the worldwide spread of these organisms. Studies in Germany and Austria (26, 27) on sporadic HUS cases between 1996 and 2003 found that EHEC O26 accounted for 14% of all EHEC strains and for ∼40% of non-O157 EHEC strains obtained from these patients. A proportion of 11% EHEC O26 strains was detected in a case-control study in Germany (59) between 2001 and 2003. In the age group <3 years, the number of EHEC O26 cases was nearly equal to that of EHEC O157 cases, although the incidence of EHEC O26-associated disease is probably underestimated because of diagnostic limitations in comparison to the diagnosis of O157:H7/H− (18, 34). Moreover, EHEC O26 has spread globally (35). Beutin (6) described EHEC O26:H11/H−, among O103:H2, O111:H, O145:H28/H−, and O157:H7/H−, as the well-known pathogenic “gang of five,” and Bettelheim (5) warned that we ignore the non-O157 STEC strains at our peril.EHEC O26 strains produce Stx1, Stx2, or both (15, 63). Moreover, these strains contain the intimin-encoding eae gene (11, 63), a characteristic feature of EHEC (44). In addition, EHEC strains possess other markers associated with virulence, such as a large plasmid that carries further potential virulence genes, e.g., genes coding for EHEC hemolysin (EHEC-hlyA), a catalase-peroxidase (katP), and an extracellular serine protease (espP) (17, 52). The efa1 (E. coli factor for adherence 1) gene was identified as an intestinal colonization factor in EHEC (43). EHEC O26 represents a highly dynamic group of organisms that rapidly generate new pathogenic clones (7, 8, 63).Ruminants, especially cattle, are considered the primary reservoir for human infections with EHEC. Therefore, the aim of this study was the molecular characterization of bovine E. coli field isolates of serogroup O26 using a panel of typical virulence markers. The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O26 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.In our study, 56 bovine E. coli O26:H11 isolates and one bovine O26:H32 isolate were analyzed for EHEC virulence-associated factors. The isolates had been obtained from three different beef farms during a long-term study. They were detected in eight different cattle in farm A over a period of 15 months (detected on 10 sampling days), in 3 different animals in farm C over a period of 8 months (detected on 3 sampling days), and in one cow on one sampling day in farm D (Table (Table1)1) (28).

TABLE 1.

Typing of E. coli O26 isolates
Sampling day, source, and isolateSerotypeVirulence profile by:
fliC PCR-RFLPstx1 genestx2 geneStx1 (toxin)Stx2 (toxin)Subtype(s)
efa1 genebEHEC-hlyA genekatP geneespP genePlasmid size(s) in kbCluster
stx1/stx2eaetirespAespB
Day 15
    Animal 6 (farm A)
        WH-01/06/002-1O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/06/002-2O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/06/002-3O26:H11H11++stx1ββββ+/++++110, 127
    Animal 8 (farm A)
        WH-01/08/002-2O26:H11H11++stx1ββββ+/++++110, 127
    Animal 26 (farm A)
        WH-01/26/001-2O26:H11H11++stx1ββββ+/++++130, 127
        WH-01/26/001-5O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/26/001-6O26:H11H11++stx1ββββ+/++++110, 127
        WH-01/26/001-7O26:H11H11++stx1ββββ+/−+++110, 127
Day 29
    Animal 2 (farm A)
        WH-01/02/003-1O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-2O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-5O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-6O26:H11H11++stx1ββββ+/+++110, 126
        WH-01/02/003-7O26:H11H11++stx1ββββ+/++++110, 126
        WH-01/02/003-8O26:H11H11++stx1ββββ−/++++110, 126
        WH-01/02/003-9O26:H11H11++stx1ββββ+/++++1106
        WH-01/02/003-10O26:H11H11++stx1ββββ+/++++1106
    Animal 26 (farm A)
        WH-01/26/002-2O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-5O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-8O26:H11H11++stx1ββββ+/++++130, 125
        WH-01/26/002-9O26:H11H11++stx1ββββ+/++110, 125
        WH-01/26/002-10O26:H11H11++stx1ββββ+/++++130, 125
Day 64
    Animal 20 (farm A)
        WH-01/20/005-3O26:H11H11++stx1ββββ+/+130, 2.52
Day 78
    Animal 29 (farm A)
        WH-01/29/002-1O26:H11H11++stx1ββββ+/−+130, 12, 2.54
        WH-01/29/002-2O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-3O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-4O26:H11H11++stx1ββββ+/++++130, 12, 2.54
        WH-01/29/002-5O26:H11H11++stx1ββββ+/++130, 12, 2.54
Day 106
    Animal 27 (farm A)
        WH-01/27/005-2O26:H11H11++stx1ββββ+/−+++145, 110, 123
        WH-01/27/005-5O26:H11H11++stx1ββββ+/++++130, 12, 2.55
        WH-01/27/005-6O26:H11H11++stx1ββββ+/+130, 12, 2.55
Day 113
    Animal 7 (farm C)
        WH-04/07/001-2O26:H11H11++++stx1/stx2ββββ+/+++55, 35, 2.511
        WH-04/07/001-4O26:H11H11++++stx1/stx2ββββ+/++++5512
        WH-04/07/001-6O26:H11H11++++stx1/stx2ββββ+/++++5512
Day 170
    Animal 22 (farm C)
        WH-04/22/001-1O26:H11H11++stx1ββββ+/++++110, 12, 6.312
        WH-04/22/001-4O26:H11H11++stx1ββββ+/++++110, 12, 6.312
        WH-04/22/001-5O26:H11H11++stx1ββββ+/++++110, 12, 6.312
Day 176
    Animal 14 (farm D)
        WH-03/14/004-8O26:H11H11++stx1ββββ+/+++11010
Day 218
    Animal 27 (farm A)
        WH-01/27/009-1O26:H11H11++++stx1/stx2ββββ+/++++110, 129
        WH-01/27/009-2O26:H11H11++++stx1/stx2ββββ+/++++110, 129
        WH-01/27/009-3O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/009-8O26:H11H11++++stx1/stx2ββββ+/++110, 128
        WH-01/27/009-9O26:H11H11++++stx1/stx2ββββ+/++++110, 129
Day 309
    Animal 29 (farm A)
        WH-01/29/010-1O26:H11H11++stx1ββββ+/++++110, 35, 124
        WH-01/29/010-2O26:H11H11++stx1ββββ+/++130, 55, 358
        WH-01/29/010-3O26:H11H11++stx1ββββ+/++++130, 35, 128
Day 365
    Animal 8 (farm C)
        WH-04/08/008-6O26:H11H11++stx1ββββ+/++++110, 5512
Day 379
    Animal 9 (farm A)
        WH-01/09/016-2O26:H32H32++stx1/stx2−/−145, 130, 1.81
    Animal 27 (farm A)
        WH-01/27/014-3O26:H11H11++stx1ββββ+/++++110, 129
        WH-01/27/014-4O26:H11H11++stx1ββββ+/++++110, 129
        WH-01/27/014-5O26:H11H11++stx1ββββ+/++++110, 128
Day 407
    Animal 29 (farm A)
        WH-01/29/013-4O26:H11H11++stx1ββββ+/++++110, 12, 2.58
        WH-01/29/013-7O26:H11H11++stx1ββββ+/++++110, 12, 2.58
Day 478
    Animal 27 (farm A)
        WH-01/27/017-1O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/017-5O26:H11H11++++stx1/stx2ββββ+/++++110, 128
        WH-01/27/017-6O26:H11H11++++stx1/stx2ββββ+/++++1108
        WH-01/27/017-7O26:H11H11++++stx1/stx2ββββ+/++++1108
        WH-01/27/017-10O26:H11H11+++stx1ββββ+/++++130, 12, 2.58
Open in a separate windowastx1/stx2, gene stx1 or stx2.befa1 was detected by two hybridizations (with lifA1-lifA2 and lifA3-lifA4 probes). +/+, complete gene; +/− or −/+, incomplete gene; −/−, efa1 negative.The serotyping of the O26 isolates was confirmed by the results of the fliC PCR-restriction fragment length polymorphism (RFLP) analysis performed according to Fields et al. (25), with slight modifications described by Zhang et al. (62). All O26:H11 isolates showed the H11 pattern described by Zhang et al. (62). In contrast, the O26:H32 isolate demonstrated a different fliC RFLP pattern that was identical to the H32 pattern described by the same authors. It has been demonstrated that EHEC O26:H11 strains belong to at least four different sequence types (STs) in the common clone complex 29 (39). In the multilocus sequence typing analysis for E. coli (61), the tested five EHEC O26:H11 isolates (WH-01/02/003-1, WH-01/20/005-3, WH-01/27/009-9, WH-03/14/004-8, and WH-04/22/001-1) of different farms and clusters were characterized as two sequence types (ST 21 and ST 396). The isolates from farms A and C belong to ST 21, the most frequent ST of EHEC O26:H11 isolates found in humans and animals (39), but the single isolate from farm D was characterized as ST 396.Typing and subtyping of genes (stx1 and/or stx2, eae, tir, espA, espB, EHEC-hlyA, katP, and espP) associated with EHEC were performed with LightCycler fluorescence PCR (48) and different block-cycler PCRs. To identify the subtypes of the stx2 genes and of the locus of enterocyte effacement-encoding genes eae, tir, espA, and espB, the PCR products were digested by different restriction endonucleases (19, 26, 46). The complete pattern of virulence markers was detected in most bovine isolates examined in our study. An stx1 gene was present in all O26 isolates. In addition, an stx2 gene was found in nine O26:H11 isolates in farm A and in three isolates of the same type in farm C, as well as in the O26:H32 isolate. Both Stx1 and Stx2 were closely related to families of Stx1 and Stx2 variants or alleles. EHEC isolates with stx2 genes are significantly more often associated with HUS and other severe disease manifestations than isolates with an stx1 gene, which are more frequently associated with uncomplicated diarrhea and healthy individuals (13). In contrast to STEC strains harboring stx2 gene variants, however, STEC strains of the stx2 genotype were statistically significantly associated with HUS (26). The stx2 genotype was found in all O26 isolates with an stx2 gene, while the GK3/GK4 amplification products after digestion with HaeIII and FokI restriction enzymes showed the typical pattern for this genotype described by Friedrich et al. (26). The nucleotide sequences of the A and B subunits of the stx2 gene of the selected bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. EU700491) were identical to the stx2 genes of different sorbitol-fermenting EHEC O157:H− strains associated with human HUS cases and other EHEC infections in Germany (10) and 99.3% identical in their DNA sequences to the stx2 gene of the EHEC type strain EDL933, a typical O157:H7 isolate from an HUS patient. A characteristic stx1 genotype was present in all O26 isolates. The nucleotide sequences of the A and B subunits of the stx1 gene of the tested bovine O26:H11 isolate WH-01/27/017-1 (GenBank accession no. EU700490) were nearly identical to those of the stx1 genes of the EHEC O26:H11 reference type strains H19 and DEC10B, which had been associated with human disease outbreaks in Canada and Australia. Nucleotide exchanges typical for stx1c and stx1d subtypes as described by Kuczius et al. (38) were not found. All bovine O26:H11 strains produced an Stx1 with high cytotoxicity for Vero cells tested by Stx enzyme-linked immunosorbent assay and Vero cell neutralization assay (53). The Stx2 cytotoxicity for Vero cells was also very high in the O26:H11 isolates.Not only factors influencing the basic and inducible Stx production are important in STEC pathogenesis. It has been suggested that the eae and EHEC-hlyA genes are likely contributors to STEC pathogenicity (2, 3, 13, 50). Ritchie et al. (50) found both genes in all analyzed HUS-associated STEC isolates. In all O26:H11 isolates we obtained, stx genes were present in combination with eae genes. Only the O26:H32 isolate lacked an eae gene. To date, 10 distinct variants of eae have been described (1, 19, 36, 45, 47). Some serotypes were closely associated with a particular intimin variant: the O157 serogroup was linked to γ-eae, the O26 serogroup to β-eae, and the O103 serogroup to ɛ-eae (4, 19, 20, 58). Our study confirms these associations. All bovine O26:H11 isolates were also typed as members of the β-eae subgroup. A translocated intimin receptor gene (tir gene) and the type III secreted proteins encoded by the espA and espB genes were found in all 56 O26:H11 isolates but not in the O26:H32 isolate. These other tested locus of enterocyte effacement-associated genes belonged to the β-subgroups. These results are in accord with the results of China et al. (19), who detected the pathotypes β-eae, β-tir, β-espA, and β-espB in all investigated human O26 strains. Like the eae gene, the EHEC-hlyA gene was found in association with severe clinical disease in humans (52). Aldick et al. (2) showed that EHEC hemolysin is toxic (cytolytic) to human microvascular endothelial cells and may thus contribute to the pathogenesis of HUS. In our study, the EHEC-hlyA gene was detected in 50 of the 56 bovine E. coli O26:H11 isolates which harbored virulence-associated plasmids of different sizes (Table (Table1).1). The presence of virulence-associated plasmids corresponded to the occurrence of additional virulence markers such as the espP and katP genes (17). The katP gene and the espP gene were detected in 49 and 50 of the 56 O26:H11 isolates, respectively. The espP gene was missing in six of the seven bovine O26:H11 isolates in which the katP genes were also absent. Both genes were not found in the O26:H32 isolate (Table (Table1).1). Although we found large plasmids of the same size in O26:H11 isolates, they lacked one or more of the plasmid-associated virulence factors (Table (Table1).1). Two DNA probes were used to detect the efa1 genes by colony hybridization. (DNA probes were labeled with digoxigenin [DIG] with lifA1-lifA2 and lifA3-lifA4 primers [14] using the PCR DIG probe synthesis kit [Roche Diagnostics, Mannheim, Germany]; DIG Easy Hyb solution [Roche] was used for prehybridization and hybridization.) Positive results with both DNA probes were obtained for 52 of 56 E. coli O26:H11 isolates. A positive signal was only found in three isolates with the lifA1-lifA2 DNA probe and in one isolate with the lifA3-lifA4 probe. An efa1 gene was not detected in the O26:H32 isolate (Table (Table11).We also analyzed the spatial and temporal behavior of the O26:H11/H32 isolates in the beef herds by cluster analysis (conducted in PAUP* for Windows version 4.0, 2008 [http://paup.csit.fsu.edu/about.html]). This was performed with distance matrices using the neighbor-joining algorithm, an agglomerative cluster method which generates a phylogenetic tree. The distance matrices were calculated by pairwise comparisons of the fragmentation patterns produced by genomic typing through pulsed-field gel electrophoresis analysis with four restriction endonucleases (XbaI, NotI, BlnI, and SpeI) and the presence or absence of potential virulence markers (Fig. (Fig.11 and Table Table1).1). To this end, the total character difference was used, which counts the pairwise differences between two given patterns. During a monitoring program of 3 years in four cattle farms (29), different O26:H11 cluster groups and one O26:H32 isolate were detected in three different farms. The genetic distance of the O26:H32 isolate was very high relative to the O26:H11 isolates. Therefore, the O26:H32 isolate was outgrouped. The O26:H11 isolates of each farm represented independent cluster groups. The single isolate from farm D fitted better to the isolates from farm C than to those from farm A. This finding is in accord with the geographical distance between the farms. The fact that the farms were located in neighboring villages may suggest that direct or indirect connections between the farms were possible (e.g., by person contacts or animal trade). However, the isolates from farm C and farm D belonged to different sequence types (ST 21 and ST 396), which may argue against a direct connection. Interestingly, O26:H11 isolates with and without stx2 genes were detected in the same clusters. This phenomenon was observed in both farm A and farm C. In farm A, the isolates with additional stx2 genes were found in animal 27 and were grouped in clusters 8 and 9 (day 218). An stx2 gene was repeatedly found (four isolates) in the same animal (animal 27). The isolates grouped in cluster 8 on a later day of sampling (day 478). All other O26:H11 isolates grouped in the same clusters and obtained from the same animals (27 and 29) on different sampling days lacked an stx2 gene. Also, the isolates obtained from animal 27 on previous sampling days, which grouped in clusters 3 and 5, exhibited no stx2 genes. In farm C, the three isolates with additional stx2 genes obtained from animal 7 grouped in clusters 11 and 12. An stx2 gene was absent from all other O26:H11 isolates grouped in the same cluster 12 on later sampling days, and no other isolates of cluster 11 were found later on. However, we detected members of many clusters over relatively long periods (clusters 5, 8, and 9 in farm A and cluster 12 in farm C), but members of other clusters were only found on single occasions. This patchy temporal pattern is apparently not a unique property of O26:H11, as we found similar results for cluster groups of other EHEC serotypes of bovine origin (28). The isolates grouped in the dominant cluster 8 were found on 5 of 9 sampling days over a period of 10 months. In contrast, we found the members of clusters 4, 5, 9, and 12 only on two nonconsecutive sampling days. The period during which isolates of these groups were not detected was particularly long for cluster 4 (231 days). We also observed the coexistence of different clusters over long periods in the same farm and in the same cattle (clusters 8 and 9), while one of the clusters dominated. Transmission of clusters between cattle was also observed. These results suggest that some of the EHEC O26:H11 strains had the potential for a longer persistence in the host population, while others had not. The reasons for this difference are not yet clear. Perhaps the incomplete efa1 gene found in isolates of clusters which were only detected once might explain why some strains disappeared rapidly. Efa1 has been discussed as a potential E. coli colonization factor for the bovine intestine used by non-O157 STEC, including O26 (54, 56). The O165:H25 cluster detected during a longer period in farm B may have disappeared after it had lost its efa1 gene (28). The precise biological activity of Efa1 in EHEC O26 is not yet known, but it has been demonstrated that the molecule is a non-Stx virulence determinant which can increase the virulence of EHEC O26 in humans (8).Open in a separate windowFIG. 1.Neighbor-joining tree of bovine E. coli O26:H11/H32 strains based on the restriction pattern obtained after digestion with XbaI, NotI, BlnI, and SpeI.We distinguished 12 different clusters, but complete genetic identity was only found in two isolates. The variations in the O26:H11 clusters may be due to increasing competition between the bacterial populations of the various subtypes in the bovine intestine or to potential interactions between EHEC O26:H11 and the host.The ephemeral occurrence of additional stx2 genes in different clusters and farms may be the result of recombination events due to horizontal gene transfer (16). The loss of stx genes may occur rapidly in the course of an infection, but the reincorporation by induction of an stx-carrying bacteriophage into the O26:H11 strains is possible at any time (9, 40). Nevertheless, an additional stx2 gene may increase the dangerousness of the respective EHEC O26:H11 strains. While all patients involved in an outbreak caused by an EHEC O26:H11 strain harboring the gene encoding Stx2 developed HUS (41), the persons affected by another outbreak caused by an EHEC O26:H11 strain that produced exclusively Stx1 had only uncomplicated diarrhea (60).In conclusion, our results showed that bovine O26:H11 isolates can carry virulence factors of EHEC that are strongly associated with EHEC-related disease in humans, particularly with severe clinical manifestations such as hemorrhagic colitis and HUS. Therefore, strains of bovine origin may represent a considerable risk for human infection. Moreover, some clusters of EHEC O26:H11 persisted in cattle and farms over longer periods, which may increase the risk of transmission to other animals and humans even further.  相似文献   

20.
The Chlamydomonas reinhardtii ODA3 Gene Encodes a Protein of the Outer Dynein Arm Docking Complex     
Anthony Koutoulis  Gregory J. Pazour  Curtis G. Wilkerson  Kazuo Inaba  Hong Sheng  Saeko Takada  George B. Witman 《The Journal of cell biology》1997,137(5):1069-1080
  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号