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Victoria Kasprowicz Yu-Hoi Kang Michaela Lucas Julian Schulze zur Wiesch Thomas Kuntzen Vicki Fleming Brian E. Nolan Steven Longworth Andrew Berical Bertram Bengsch Robert Thimme Lia Lewis-Ximenez Todd M. Allen Arthur Y. Kim Paul Klenerman Georg M. Lauer 《Journal of virology》2010,84(3):1656-1663
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).
Open in a separate windowaP, prototype; A, autologous. Identical residues are shown by dashes.bHIV coinfection.cHBV coinfection.
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
TABLE 1.
Patient information and autologous sequence analysis for patients with chronic and resolved HCV infectionCode | Genotype | Status | Epitope(s) targeted | Sequencea |
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02-03 | 1b | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
A: no sequence | ||||
00-26 | 1b | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
A: no sequence | ||||
99-24 | 2a | Chronic | A2 NS3 1073-1083 | P: CINGVCWTV |
No recognition | A: S-S--L--- | |||
A2 NS3 1406-1415 | P: KLVALGINAV | |||
No recognition | A: A-RGM-L--- | |||
A2 NS5B 2594-2602 | P: ALYDVVTKL | |||
A: no sequence | ||||
111 | 1a | Chronic | A2 NS3 1073-1083 | P: CINGVCWTV |
A: --------- | ||||
A2 NS5 2594-2602 | P: ALYDVVTKL | |||
A: --------- | ||||
00X | 3a | Chronic | A2 NS5 2594-2602 | P: ALYDVVTKL |
No recognition | A: -----IQ-- | |||
O3Qb | 1a | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
Diminished | A: --------F | |||
03Sb | 1a | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
Diminished | A: --------F | |||
02A | 1a | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
A: no sequence | ||||
01N | 1a | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
Diminished | A: --------F | |||
03H | 1a | Chronic | A2 NS3 1073-1083 | P: CINGVCWTV |
Full recognition | A: ----A---- | |||
01-39 | 1a | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
Diminished | A: --------F | |||
03-45b | 1a | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
Diminished | A: --------F | |||
06P | 3a | Chronic | A1 NS3 1436-1444 | P: ATDALMTGY |
Diminished | A: --------F | |||
GS127-1 | 1a | Chronic | A2 NS3 1073-1083 | P: CINGVCWTV |
A: --------- | ||||
GS127-6 | 1a | Chronic | A2 NS3 1073-1083 | P: CINGVCWTV |
A: --------- | ||||
GS127-8 | 1b | Chronic | A2 NS3 1073-1083 | P: CINGVCWTV |
A: --------- | ||||
GS127-16 | 1a | Chronic | A2 NS3 1073-1083 | P: CINGVCWTV |
A: --------- | ||||
GS127-20 | 1a | Chronic | A2 NS3 1073-1083 | P: CINGVCWTV |
A: --------- | ||||
04D | 4 | Resolved | A2 NS5 1987-1996 | P: VLSDFKTWKL |
01-49b | 1 | Resolved | A2 NS5 1987-1996 | P: VLSDFKTWKL |
A2 NS3 1406-1415 | P: KLVALGINAV | |||
01-31 | 1 | Resolved | A1 NS3 1436-1444 | P: ATDALMTGY |
B57 NS5 2629-2637 | P: KSKKTPMGF | |||
04N | 1 | Resolved | A1 NS3 1436-1444 | P: ATDALMTGY |
01E | 4 | Resolved | A2 NS5 1987-1996 | P: VLSDFKTWKL |
98A | 1 | Resolved | A2 NS3 1073-1083 | P: CINGVCWTV |
00-10c | 1 | Resolved | A24 NS4 1745-1754 | P: VIAPAVQTNW |
O2Z | 1 | Resolved | A1 NS3 1436-1444 | P: ATDALMTGY |
99-21 | 1 | Resolved | B7 CORE 41-49 | P: GPRLGVRAT |
OOR | 1 | Resolved | B35 NS3 1359-1367 | P: HPNIEEVAL |
TABLE 2.
Patient information and autologous sequence analysis for patients with acute HCV infectionCode | Genotype | Outcome | Epitope targeted and time analyzed | Sequencea |
---|---|---|---|---|
554 | 1a | Persisting | A2 NS3 1073-1083 | P: CINGVCWTV |
wk 8 | A: --------- | |||
wk 30 | A: --------- | |||
03-32 | 1a | Persisting | B35 NS3 1359-1367 | P: HPNIEEVAL |
wk 8 | A: --------- | |||
No recognition (wk 36) | A: S-------- | |||
04-11 | 1a (1st) | Persisting (1st) Resolving (2nd) | A2 NS5 2594-2602 | P: ALYDVVTKL |
1b (2nd) | A: no sequence | |||
0023 | 1b | Persisting | A1 NS3 1436-1444 | P: ATDALMTGY |
Diminished (wk 7) | A: --------F | |||
Diminished (wk 38) | A: --------F | |||
A2 NS3 1073-1083 | P: CINGVCWTV | |||
wk 7 | A: --------- | |||
wk 38 | A: --------- | |||
A2 NS3 1406-1415 | P: KLVALGINAV | |||
Full recognition (wk 7) | A: --S------- | |||
Full recognition (wk 38) | A: --S------- | |||
320 | 1 | Resolving | A2 NS3 1273-1282 | P: GIDPNIRTGV |
599 | 1 | Resolving | A2 NS3 1073-1083 | P: CINGVCWTV |
1144 | 1 | Resolving | A2 NS3 1073-1083 | P: CINGVCWTV |
B35 NS3 1359-1367 | P: HPNIEEVAL | |||
06L | 3a | Resolving | B7 CORE 41-49 | P: GPRLGVRAT |
05Y | 1 | Resolving | A2 NS3 1073-1083 | P: CINGVCWTV |
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Francisco J. Roig A. Llorens B. Fouz C. Amaro 《Applied and environmental microbiology》2009,75(8):2577-2580
This work demonstrates that Vibrio vulnificus biotype 2, serovar E, an eel pathogen able to infect humans, can become resistant to quinolone by specific mutations in gyrA (substitution of isoleucine for serine at position 83) and to some fluoroquinolones by additional mutations in parC (substitution of lysine for serine at position 85). Thus, to avoid the selection of resistant strains that are potentially pathogenic for humans, antibiotics other than quinolones must be used to treat vibriosis on farms.Vibrio vulnificus is an aquatic bacterium from warm and tropical ecosystems that causes vibriosis in humans and fish (http://www.cdc.gov/nczved/dfbmd/disease_listing/vibriov_gi.html) (33). The species is heterogeneous and has been subdivided into three biotypes and more than eight serovars (6, 15, 33; our unpublished results). While biotypes 1 and 3 are innocuous for fish, biotype 2 can infect nonimmune fish, mainly eels, by colonizing the gills, invading the bloodstream, and causing death by septicemia (23). The disease is rapidly transmitted through water and can result in significant economic losses to fish farmers. Surviving eels are immune to the disease and can act as carriers, transmitting vibriosis between farms. Interestingly, biotype 2 isolates belonging to serovar E have been isolated from human infections, suggesting that serovar E is zoonotic (2). This serovar is also the most virulent for fish and has been responsible for the closure of several farms due to massive losses of fish. A vaccine, named Vulnivaccine, has been developed from serovar E isolates and has been successfully tested in the field (14). Although the vaccine provides fish with long-term protection from vibriosis, at present its use is restricted to Spain. For this reason, in many fish farms around the world, vibriosis is treated with antibiotics, which are usually added to the food or water.Quinolones are considered the most effective antibiotics against human and fish vibriosis (19, 21, 31). These antibiotics can persist for a long time in the environment (20), which could favor the emergence of resistant strains under selective pressure. In fact, spontaneous resistances to quinolones by chromosomal mutations have been described for some gram-negative bacteria (10, 11, 17, 24, 25, 26). Therefore, improper antibiotic treatment of eel vibriosis or inadequate residue elimination at farms could favor the emergence of human-pathogenic serovar E strains resistant to quinolones by spontaneous mutations. Thus, the main objective of the present work was to find out if the zoonotic serovar of biotype 2 can become quinolone resistant under selective pressure and determine the molecular basis of this resistance.Very few reports on resistance to antibiotics in V. vulnificus have been published; most of them have been performed with biotype 1 isolates. For this reason, the first task of this study was to determine the antibiotic resistance patterns in a wide collection of V. vulnificus strains belonging to the three biotypes that had been isolated worldwide from different sources (see Table S1 in the supplemental material). Isolates were screened for antimicrobial susceptibility to the antibiotics listed in Table S1 in the supplemental material by the agar diffusion disk procedure of Bauer et al. (5), according to the standard guideline (9). The resistance pattern found for each isolate is shown in Table S1 in the supplemental material. Less than 14% of isolates were sensitive to all the antibiotics tested, and more than 65% were resistant to more than one antibiotic, irrespective of their biotypes or serovars. The most frequent resistances were to ampicillin-sulbactam (SAM; 65.6% of the strains) and nitrofurantoin (F; 60.8% of the strains), and the least frequent were to tetracycline (12%) and oxytetracycline (8%). In addition, 15% of the strains were resistant to nalidixic acid (NAL) and oxolinic acid (OA), and 75% of these strains came from fish farms (see Table S1 in the supplemental material). Thus, high percentages of strains of the three biotypes were shown to be resistant to one or more antibiotics, with percentages similar to those found in nonbiotyped environmental V. vulnificus isolates from Asia and North America (4, 27, 34). In those studies, resistance to antibiotics could not be related to human contamination. However, the percentage of quinolone-resistant strains found in our study is higher than that reported in other ones, probably due to the inclusion of fish farm isolates, where the majority of quinolone-resistant strains were concentrated. This fact suggests that quinolone resistance could be related to human contamination due to the improper use of these drugs in therapy against fish diseases, as has been previously suggested (18, 20). Although no specific resistance pattern was associated with particular biotypes or serovars, we found certain differences in resistance distribution, as shown in Table Table1.1. In this respect, biotype 3 displayed the narrowest spectrum of resistances and biotype 1 the widest. The latter biotype encompassed the highest number of strains with multiresistance (see Table S1 in the supplemental material). Within biotype 2, there were differences among serovars, with quinolone resistance being restricted to the zoonotic serovar (Table (Table11).
Open in a separate windowaCTX, cefotaxime; E, erythromycin; OT, oxytetracycline; SXT-TMP, sulfamethoxazole-trimethoprim; TE, tetracycline.The origin of resistance to quinolones in the zoonotic serovar was further investigated. To this end, spontaneous mutants of sensitive strains were selected from colonies growing within the inhibition halo around OA or NAL disks. Two strains (strain CG100 of biotype 1 and strain CECT 4604 of biotype 2, serovar E) developed isolated colonies within the inhibition zone. These colonies were purified, and maintenance of resistance was confirmed by serial incubations on medium without antibiotics. Using the disk diffusion method, CG100 was shown to be resistant to SAM and F and CECT 4604 to F (see Table S1 in the supplemental material). The MICs for OA, NAL, flumequine (UB), and ciprofloxacin (CIP) were determined by using the microplate assay according to the recommendations of the Clinical and Laboratory Standards Institute and the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases (8, 12) and interpreted according to the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases (13). The MICs for OA and NAL and for the fluoroquinolones UB and CIP exhibited by the mutants and their counterparts are shown in Table Table2.2. The inhibition zone diameters correlated well with MICs (data not shown). Mutants FR1, FR2, FR3, and FR4 were resistant to NAL and sensitive to the remaining quinolones, although they showed higher resistances than their parental strains (Table (Table2).2). Thus, these four mutants showed increases of 32- to 128-fold for NAL MICs, 4- to 8-fold for UB MICs, and 16-fold for CIP MICs (Table (Table2).2). The fifth mutant, FR5, was resistant to the two tested quinolones and to UB, a narrow-spectrum fluoroquinolone. This mutant, although sensitive to CIP, multiplied its MIC for this drug by 128 with respect to the parental strain (Table (Table22).
Open in a separate windowaMutations in a nucleotide (nt) that gave rise to a codon change and to a change in amino acids (aa) are indicated. NC, no change detected.bThe resistance (R) or sensitivity (S) against the antibiotic determined according to the Clinical and Laboratory Standards Institute and the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases (9, 13) is indicated in parentheses.For other gram-negative pathogens, quinolone resistance relies on spontaneous mutations in the gyrA, gyrB, parC, and parE genes that occur in a specific region of the protein known as the quinolone resistance-determining region (QRDR) (1, 11, 17, 24, 25, 26, 28). To test the hypothesis that mutations in these genes could also produce quinolone resistance in V. vulnificus, the QRDRs of these genes were sequenced in the naturally resistant strains and in the two sensitive strains that had developed resistances by selective pressure in vitro. The genomic DNA was extracted (3), and the QRDRs of gyrA, gyrB, parE, and parC were amplified using the primers shown in Table Table3,3, which were designed from the published genomes of biotype 1 strains YJ016 and CMCP6 (7, 22). PCR products of the predicted size were sequenced in an ABI 3730 sequencer (Applied Biosystems). Analysis of the QRDR sequences for gyrA, gyrB, parC, and parE of the mutants and the naturally resistant strains revealed that all naturally resistant strains, except one, shared a specific mutation at nucleotide position 248 with the laboratory-induced mutants (Table (Table2).2). This mutation gave rise to a change from serine to isoleucine at amino acid position 83. The exception was a mutation in the adjacent nucleotide that gave rise to a substitution of arginine for serine at the same amino acid position (Table (Table2).2). All the isolates that were resistant to the quinolone NAL had a unique mutation in the gyrA gene, irrespective of whether resistance was acquired naturally or in the laboratory (Table (Table2).2). This result strongly suggests that a point mutation in gyrA that gives rise to a change in nucleotide position 83 can confer resistance to NAL in V. vulnificus biotypes 1 and 2 and that this mutation could be produced by selective pressure under natural conditions. gyrA mutations consisting of a change from serine 83 to isoleucine have also been described in isolates of Aeromonas from water (17) and in diseased fish isolates of Vibrio anguillarum (26). Similarly, replacement of serine by arginine at amino acid position 83 in diseased fish isolates of Yersinia ruckeri (16) suggests that this mechanism of quinolone resistance is widespread among gram-negative pathogens. In all cases, these single mutations were also related to increased resistance to other quinolones (OA) and fluoroquinolones (UB and CIP) (Table (Table2),2), although the mutants remained sensitive according to the standards of the Clinical and Laboratory Standards Institute and the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases (9, 13). A total of 50% of the naturally resistant strains, all of them of biotype 1, showed additional mutations that affected parC (a change in amino acid position 113) or gyrB (changes in amino acids at positions 425 and 438) (Table (Table2).2). These strains exhibited higher MICs for OA and fluoroquinolones (Table (Table2),2), although they were still sensitive to these drugs (9, 13). Finally, one isolate of biotype 2, serovar E, which was naturally resistant to quinolones and UB, showed a mutation in parC that gave rise to a substitution of leucine for serine at amino acid position 85 (Table (Table2).2). This mutation was shared only with the laboratory-induced mutant, also a biotype 2, serovar E mutant, which was resistant to the fluoroquinolone UB. The same mutation in parC had been previously described in diseased fish isolates of V. anguillarum that were highly resistant to quinolones (28), but this had not been related to fluoroquinolone resistance in Vibrio spp. nor in other gram-negative bacteria. These results strongly suggest that resistance to fluoroquinolones in V. vulnificus is related to specific mutations in gyrA and parC and that mutations in different positions for parC or in gyrB could contribute to increased resistance to quinolones and fluoroquinolones. Our results also agree with previous studies confirming that the acquisition of higher quinolone resistance is more probable when arising from a gyrA parC double mutation than from a gyrA gyrB double mutation (29).
Open in a separate windowFinally, the evolutionary history for each protein was inferred from previously published DNA sequences of the whole genes from different Vibrio species after multiple sequence alignment with MEGA4 software (32) by applying the neighbor-joining method (30) with the Poisson correction (35). The distance tree for each whole protein showed a topology similar to the phylogenetic tree based on 16S rRNA analysis, with the two isolates of V. vulnificus forming a single group, closely related to Vibrio parahaemolyticus, Vibrio cholerae, V. anguillarum, and Vibrio harveyi (see Fig. S1A in the supplemental material). A second analysis was performed with the QRDR sequences of the different mutants and isolates of V. vulnificus (GenBank accession numbers ) to infer the intraspecies relationships (see Fig. S1B in the supplemental material). This analysis showed that QRDRs of gyrA, gyrB, parC, and parE were highly homogeneous within V. vulnificus.In summary, the zoonotic serovar of V. vulnificus can mutate spontaneously to gain quinolone resistance, under selective pressure in vitro, due to specific mutations in gyrA that involve a substitution of isoleucine for serine at amino acid position 83. This mutation appears in biotype 2, serovar E diseased-fish isolates and biotype 1 strains, mostly recovered from fish farms. An additional mutation in parC, resulting in a substitution of lysine for serine at amino acid position 85, seems to endow partial fluoroquinolone resistance on biotype 2, serovar E strains. This kind of double mutation is present in diseased-fish isolates of the zoonotic serovar but not in resistant biotype 1 isolates, which show different mutations in gyrB or in parC that increase their resistance levels but do not make the strains resistant to fluoroquinolones. Thus, antibiotics other than quinolones should be used at fish farms to prevent the emergence and spread of quinolone resistances, especially to CIP, a drug widely recommended for human vibriosis treatment. FJ379836 to FJ379927 相似文献
TABLE 1.
Percentage of resistant strains distributed by biotypes and serovarsV. vulnificus | No. of isolates | Resistance distribution (%) for indicated antibiotica
| ||||||||
---|---|---|---|---|---|---|---|---|---|---|
SAM | CTX | E | NAL | F | OT | OA | SXT-TMP | TE | ||
Biotype 1 | 49 | 75.5 | 24.5 | 14.3 | 30.6 | 83.7 | 8.2 | 30.6 | 28.6 | 8.2 |
Biotype 2 (whole) | 72 | 58.3 | 13.9 | 12.5 | 4.2 | 47.2 | 9.7 | 4.2 | 4.2 | 13.9 |
Biotype 2 | ||||||||||
Serovar E | 36 | 30.3 | 12.1 | 3 | 9.1 | 27.3 | 15.2 | 9.1 | 3 | 21.2 |
Serovar A | 23 | 100 | 9.1 | 18.2 | 0 | 77.3 | 0 | 0 | 9.1 | 4.6 |
Nontypeable | 8 | 29 | 14.3 | 25 | 0 | 57.1 | 14.3 | 0 | 0 | 14.3 |
Serovar I | 5 | 100 | 20 | 20 | 0 | 20 | 20 | 0 | 0 | 0 |
Biotype 3 | 5 | 100 | 0 | 20 | 0 | 80 | 0 | 0 | 0 | 20 |
TABLE 2.
MICs for quinolones and fluoroquinolones and mutations in gyrA, gyrB, and parC detected in naturally and artificially induced resistant strainsStrain(s) | MIC (μg ml−1) for indicated antibioticb
| Gene mutationa
| ||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
gyrA
| gyrB
| parC
| ||||||||||||||
Position
| Codon change | aa change | Position
| Codon change | aa change | Position
| Codon change | aa change | ||||||||
NAL | OA | UB | CIP | nt | aa | nt | aa | nt | aa | |||||||
CG100 | 0.5 (S) | 0.125 (S) | 0.0625 (S) | 0.0078 (S) | ||||||||||||
FR1 | 16 (R) | 1 (S) | 0.25 (S) | 0.125 (S) | 248 | 83 | AGT→ATT | S→I | NC | NC | NC | NC | NC | NC | NC | NC |
FR2 | 16 (R) | 1 (S) | 0.25 (S) | 0.125 (S) | 248 | 83 | AGT→ATT | S→I | NC | NC | NC | NC | NC | NC | NC | NC |
CECT 4604 | 0.25 (S) | 0.0625 (S) | 0.0625 (S) | 0.0078 (S) | ||||||||||||
FR3 | 32 (R) | 2 (S) | 0.5 (S) | 0.125 (S) | 248 | 83 | AGT→ATT | S→I | NC | NC | NC | NC | NC | NC | NC | NC |
FR4 | 32 (R) | 2 (S) | 0.5 (S) | 0.125 (S) | 248 | 83 | AGT→ATT | S→I | NC | NC | NC | NC | NC | NC | NC | NC |
FR5 | 256 (R) | 16 (R) | 16 (R) | 1 (S) | 248 | 83 | AGT→ATT | S→I | 1156 | 386 | GCA→ACA | A→T | 254 | 85 | TCA→TTA | S→L |
1236 | 412 | CAG→CAC | Q→H | |||||||||||||
CECT 4602 | 128 (R) | 8 (R) | 64 (R) | 1 (S) | 248 | 83 | AGT→ATT | S→I | NC | NC | NC | NC | 254 | 85 | TCA→TTA | S→L |
CECT 4603, CECT 4606, CECT 4608, PD-5, PD-12, JE | 32 (R) | 2 (S) | <1 (S) | <1 (S) | 248 | 83 | AGT→ATT | S→I | NC | NC | NC | NC | NC | NC | NC | NC |
CECT 4862 | 64 (R) | 2 (S) | 2 (S) | <1 (S) | 249 | 83 | AGT→AGA | S→R | NC | NC | NC | NC | NC | NC | NC | NC |
A2, A4, A5, A6, A7, PD-1, PD-3 | 64-128 (R) | 2 (S) | 4 (S) | <1 (S) | 248 | 83 | AGT→ATT | S→I | NC | NC | NC | NC | 338 | 113 | GCA→GTA | A→V |
V1 | 128 (R) | 4 (S) | 4 (S) | <1 (S) | 248 | 83 | AGT→ATT | S→I | 1274 | 425 | GAG→GGG | E→G | NC | NC | NC | NC |
1314 | 438 | AAC→AAA | N→K |
TABLE 3.
Oligonucleotides used in this studyPrimer | Sequence | Annealing temp (°C) | Size (bp) |
---|---|---|---|
GyrAF | GGCAACGACTGGAATAAACC | 55.8 | 416 |
GyrAR | CAGCCATCAATCACTTCCGTC | ||
ParCF | CGCAAGTTCACCGAAGATGC | 56.6 | 411 |
ParCR | GGCATCCGCAACTTCACG | ||
GyrBF | CGACTTCTGGTGACGATGCG | 57.4 | 642 |
GyrBR | GACCGATACCACAACCTAGTG | ||
ParEF | GCCAGGTAAGTTGACCGATTG | 56.8 | 512 |
ParER | CACCCAGACCTTTGAATCGTTG |
6.
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9.
One- and Two-Locus Population Models With Differential Viability Between Sexes: Parallels Between Haploid Parental Selection and Genomic Imprinting 下载免费PDF全文
Alexey Yanchukov 《Genetics》2009,182(4):1117-1127
A model of genomic imprinting with complete inactivation of the imprinted allele is shown to be formally equivalent to the haploid model of parental selection. When single-locus dynamics are considered, an internal equilibrium is possible only if selection acts in the opposite directions in males and females. I study a two-locus version of the latter model, in which maternal and paternal effects are attributed to the single alleles at two different loci. A necessary condition for the allele frequency equilibria to remain on the linkage equilibrium surface is the multiplicative interaction between maternal and paternal fitness parameters. In this case the equilibrium dynamics are independent at both loci and results from the single-locus model apply. When fitness parameters are additive, analytic treatment was not possible but numerical simulations revealed that stable polymorphism characterized by association between loci is possible only in several special cases in which maternal and paternal fitness contributions are precisely balanced. As in the single-locus case, antagonistic selection in males and females is a necessary condition for the maintenance of polymorphism. I also show that the above two-locus results of the parental selection model are very sensitive to the inclusion of weak directional selection on the individual''s own genotypes.PARENTAL genetic effects refer to the influence of the mother''s and father''s genotypes on the phenotypes of their offspring, not attributable just to the transfer of genes. Examples have been documented across a wide range of areas of organism biology; see, for example, Wade (1998) and and22 in Rasanen and Kruuk (2007). Parental selection is a more formal concept used in theoretical modeling and concerns situations where the fitness of the offspring depends, besides other factors, on the genotypes of its parent(s) (generalizing from Kirkpatrick and Lande 1989).
Open in a separate windowParentheses in the first column indicate maternal genotype (parental selection model) or inactivation of the maternally derived allele (imprinting model). Whether selection occurs at the diploid (first column) or subsequent haploid (second column) stage does not change the resulting allele frequencies.
Open in a separate windowAnother well-known parent-of-origin phenomenon is genomic imprinting. Here, the level of expression of one of the alleles depends on which parent it is inherited from. Often it is difficult to tell apart the phenotypic patterns due to parental effects and genomic imprinting, and thus a problem arises in the process of identifying the candidate genes for such effects (Hager et al. 2008). Analytic methods (Weinberg et al. 1998; Santure and Spencer 2006; Hager et al. 2008) have been developed to quantify subtle differences between the two. In this article, I point out that a simple mathematical model, first suggested for genomic imprinting at a diploid locus, can be interpreted, without any formal changes, to describe parental selection on haploids.While there has been much progress in understanding the evolution of genomic imprinting (Hunter 2007), including advances in modeling (Spencer 2000, 2008), the population genetics theory of parental effects received less attention. Existing major-locus effect models of parental selection are single-locus, two-allele, and mostly concern uniparental (maternal) selection (Wright 1969; Spencer 2003; Gavrilets and Rice 2006; Santure and Spencer 2006), with only one specific case where the fitness effects of both parents interact studied by Gavrilets and Rice (2006). No attempt to extend this theory into multilocus systems has yet been made. Considering a two-locus model with both parents playing a role in selection on the offspring is called for by the observation that many maternal and paternal effects aim at the different traits or different life stages of their progeny. Among birds, for example, body condition soon after hatching is largely determined by the mother, while paternally transmitted sexual display traits develop much later in life (Price 1998). Such effects are therefore unlikely to be regulated within a single locus. Sometimes the effects are on the same trait, but still attributed to different loci: expression of gene Avy that causes the “agouti” phenotype (yellow fur coat and obesity) in mice is enhanced by maternal epigenetic modification (Rakyan et al. 2003), while paternal mutations at the other locus, MommeD4, contribute to a reverse phenotypic pattern in the offspring (Rakyan et al. 2003). The epigenetic state of the murine AxinFu allele is both maternally and paternally inherited (Rakyan et al. 2003).Focusing selection on haploids reduces the number of genotypes that need to be taken into account, while preserving the main properties of the multilocus system. Genes with haploid expression and a potential of parental effects can be found in two major taxonomic kingdoms. A notable candidate is Spam1 in mice, which is expressed during spermogenesis and encodes a factor that enables sperm to penetrate the egg cumulus (Zheng et al. 2001). This gene remains a target for effectively haploid selection, because its product is not shared via cytoplasm bridges between developing spermatides. Mutations at Spam1 alter performance of the male gametes that carry it and might indirectly, perhaps by altering the timing of fertilization, affect the fitness of the zygote. The highest estimated number of mouse genes expressed in the male gametes is currently 2375 (Joseph and Kirkpatrick 2004), and one might expect some of them to have similar paternal effects. Plants go through a profound haploid stage in their life cycles, and genes involved at this stage have an inevitable effect on the fitness of the future generations. In angiosperms, seed development is known to be controlled by both maternal (Chaudhury and Berger 2001; Yadegari and Drews 2004) and paternal (Nowack et al. 2006) effect genes, expressed, respectively, in female and male gametophytes.Under haploid selection, there can be no overdominance, and thus polymorphism is much more difficult to maintain than in diploid selection models (summarized in Feldman 1971). Nevertheless, differential or antagonistic selection between sexes can lead to a new class of stable internal equilibria in the diploid systems (Owen 1953; Bodmer 1965; Mandel 1971; Kidwell et al. 1977; Reed 2007), and I make use of this property in the haploid models developed below. In the experiment by Chippindale and colleagues (Chippindale et al. 2001), ∼75% of the total fitness variation in the adult stage of Drosophila melanogaster was negatively correlated between males and females, which suggests that a substantial portion of the fruit fly expressed genome is under sexually antagonistic selection. I assume that the effect of either parent on the fitness of the individual depends on the sex of the latter, which in respect to modeling is equivalent to the assumption of differential viability between the sexes in the progeny of the same parent(s). Biological systems that satisfy the latter assumptions can be found among colonial green algae: many members of the order Volvocales are haploid except for the short zygotic stage, and during sexual reproduction, they are also dioecious and anisogametic. I return to this example in the discussion. The possibility that genes expressed in animal gametes may be under antagonistic selection between sexes has been discussed (Bernasconi et al. 2004). For example, a (hypothetical) mutation increasing the ATP production in mitochondria would be beneficial in sperm, because of the increased mobility of the latter, but neutral or detrimental in the egg, due to a higher level of oxidative damage to DNA (Zeh and Zeh 2007).My main purpose was to derive conditions for existence and stability of the internal equilibria of the model(s). I begin with a simple one-locus case, which can be analyzed explicitly, and show how these one-locus results can be extended to the case of two recombining loci with multiplicative fitness. Then, I assume an additive relation between the maternal and paternal effect parameters and study the special cases where parental effects are symmetric. 相似文献
TABLE 1
Frequencies of genotypes and fitness parameterizations in model 1Gametes/haploids | Frequency before selection | Fitness
| ||
---|---|---|---|---|
Zygote | Male | Female | ||
(A)A | A | pfpm | 1 − α | 1 − δ |
(A)a | 1/2 A 1/2 a | pf(1 − pm) | 1 | 1 |
(a)A | 1/2 a 1/2 A | (1 − pf)pm | 1 − α | 1 − δ |
(a)a | A | (1 − pf)(1 − pm) | 1 | 1 |
TABLE 2
Offspring genotypic proportions from different mating types, sorted among four phenotypic groups/combinations of maternal and paternal effects: model 2Offspring genotypes/phenotypes
| |||||||||
---|---|---|---|---|---|---|---|---|---|
Parental genotypes
| Paternal (φ = 1)
| Joint (φ = 4)
| |||||||
Male | Female | AB | Ab | aB | Ab | AB | Ab | aB | ab |
AB | AB | 1 | |||||||
Ab | |||||||||
aB | |||||||||
ab | (1−r)/2 | r/2 | r/2 | (1−r)/2 | |||||
Ab | AB | ||||||||
Ab | 1 | ||||||||
aB | r/2 | (1−r)/2 | (1−r)/2 | r/2 | |||||
ab | |||||||||
Offspring genotypes/phenotypes
| |||||||||
Parental genotypes
| Maternal (φ = 2)
| None (φ = 3)
| |||||||
Male | Female | AB | Ab | aB | Ab | AB | Ab | aB | ab |
aB | AB | ||||||||
Ab | r/2 | (1 − r)/2 | (1 − r)/2 | r/2 | |||||
aB | 1 | ||||||||
ab | |||||||||
ab | AB | (1 − r)/2 | (1 − r)/2 | ||||||
Ab | |||||||||
aB | |||||||||
ab | 1 |
10.
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).
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).
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
TABLE 1.
Peptide-bearing phage eluted from ANDVPhage | % Inhibition (SD)a | P valueb |
---|---|---|
Phage bearing the following peptides eluted with anti-Gn antibody 6B9/F5 | ||
Group 1 (<30% inhibition) | ||
CDQRTTRLC | 8.45 (15.34) | 0.0002 |
CPHDPNHPC | 9.94 (7.72) | 0.333 |
CQSQTRNHC | 11.76 (13.25) | 0.0001 |
CLQDMRQFC | 13.26 (9.92) | 0.0014 |
CLPTDPIQC | 15.70 (14.05) | 0.0005 |
CPDHPFLRC | 16.65 (15.22) | 0.8523 |
CSTRAENQC | 17.56 (16.50) | 0.0004 |
CPSHLDAFC | 18.98 (20.06) | 0.0017 |
CKTGHMRIC | 20.84 (7.47) | 0.0563 |
CVRTPTHHC | 20.89 (27.07) | 0.1483 |
CSGVINTTC | 21.57 (19.61) | 0.0643 |
CPLASTRTC | 21.65 (5.98) | 0.004 |
CSQFPPRLC | 22.19 (8.26) | 0.0004 |
CLLNKQNAC | 22.34 (7.78) | 0.001 |
CKFPLNAAC | 22.89 (6.15) | 0.0001 |
CSLTPHRSC | 23.63 (16.74) | 0.0563 |
CKPWPMYSC | 23.71 (6.68) | 0.0643 |
CLQHDALNC | 24.01 (7.60) | 1 |
CNANKPKMC | 24.67 (11.67) | 0.0004 |
CPKHVLKVC | 25.30 (28.36) | 0.0003 |
CTPDKKSFC | 26.91 (11.15) | 0.399 |
CHGKAALAC | 27.22 (32.53) | 0.005 |
CNLMGNPHC | 28.08 (21.35) | 0.0011 |
CLKNWFQPC | 28.64 (18.49) | 0.0016 |
CKEYGRQMC | 28.76 (29.33) | 0.0362 |
CQPSDPHLC | 29.44 (31.22) | 0.0183 |
CSHLPPNRC | 29.70 (17.37) | 0.0061 |
Group 2 (30-59% inhibition) | ||
CSPLLRTVC | 33.05 (20.26) | 0.0023 |
CHKGHTWNC | 34.17 (12.50) | 0.0795 |
CINASHAHC | 35.62 (13.03) | 0.3193 |
CWPPSSRTC | 36.75 (26.95) | 0.0006 |
CPSSPFNHC | 37.78 (7.11) | 0.0001 |
CEHLSHAAC | 38.47 (7.60) | 0.0115 |
CQDRKTSQC | 38.74 (9.12) | 0.1802 |
CTDVYRPTC | 38.90 (25.03) | 0.006 |
CGEKSAQLC | 39.11 (27.52) | 0.0013 |
CSAAERLNC | 40.13 (6.33) | 0.0033 |
CFRTLEHLC | 42.07 (5.01) | 0.0608 |
CEKLHTASC | 43.60 (27.92) | 0.1684 |
CSLHSHKGC | 45.11 (49.81) | 0.0864 |
CNSHSPVHC | 45.40 (28.80) | 0.0115 |
CMQSAAAHC | 48.88 (44.40) | 0.5794 |
CPAASHPRC | 51.84 (17.09) | 0.1935 |
CKSLGSSQC | 53.90 (13.34) | 0.0145 |
Group 3 (60-79% inhibition) | ||
CPSNVNNIC | 61.11 (25.41) | 0.1245 |
Negative control | 0 (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) | ||
CHPGSSSRC | 1.01 (7.03) | 0.0557 |
CSLSPLGRC | 10.56 (13.62) | 0.7895 |
CTARYTQHC | 12.86 (3.83) | 0.3193 |
CHGVYALHC | 12.91 (7.32) | 0.0003 |
CLQHNEREC | 16.79 (13.72) | 0.0958 |
CHPSTHRYC | 17.23 (14.53) | 0.0011 |
CPGNWWSTC | 19.34(9.91) | 0.1483 |
CGMLNWNRC | 19.48 (19.42) | 0.0777 |
CPHTQFWQC | 20.44 (13.65) | 0.0008 |
CTPTMHNHC | 20.92 (11.68) | 0.0001 |
CDQVAGYSC | 21.79 (23.60) | 0.0063 |
CIPMMTEFC | 24.33 (9.28) | 0.2999 |
CERPYSRLC | 24.38 (9.09) | 0.0041 |
CPSLHTREC | 25.06 (22.78) | 0.1202 |
CSPLQIPYC | 26.30 (34.29) | 0.4673 |
CTTMTRMTC (×2) | 29.27 (8.65) | 0.0001 |
Group 2 (30-59% inhibition) | ||
CNKPFSLPC | 30.09 (5.59) | 0.4384 |
CHNLESGTC | 31.63 (26.67) | 0.751 |
CNSVPPYQC | 31.96 (6.51) | 0.0903 |
CSDSWLPRC | 32.95 (28.54) | 0.259 |
CSAPFTKSC | 33.40 (10.64) | 0.0052 |
CEGLPNIDC | 35.63 (19.90) | 0.0853 |
CTSTHTKTC | 36.28 (13.42) | 0.132 |
CLSIHSSVC | 36.40 (16.44) | 0.8981 |
CPWSTQYAC | 36.81 (32.81) | 0.5725 |
CTGSNLPIC | 36.83 (31.64) | 0.0307 |
CSLAPANTC | 39.73 (4.03) | 0.1664 |
CGLKTNPAC | 39.75 (16.98) | 0.2084 |
CRDTTPWWC | 40.08 (18.52) | 0.0004 |
CHTNASPHC | 40.26 (4.77) | 0.5904 |
CTSMAYHHC | 41.89 (8.61) | 0.259 |
CSLSSPRIC | 42.13 (29.75) | 0.2463 |
CVSLEHQNC | 45.54 (6.55) | 0.5065 |
CRVTQTHTC | 46.55 (8.45) | 0.3676 |
CPTTKSNVC | 49.28 (14.00) | 0.3898 |
CSPGPHRVC | 49.50 (42.60) | 0.0115 |
CKSTSNVYC | 51.20 (4.60) | 0.0611 |
CTVGPTRSC | 57.30 (11.31) | 0.0176 |
Group 3 (60-79% inhibition) | ||
CPMSQNPTC | 65.60 (13.49) | 0.014 |
CPKLHPGGC | 71.88 (27.11) | 0.0059 |
Negative control | 0.26 (4.53) | |
6C5/D12 (5 μg/ml) | 22.62 (8.40) | |
ReoPro (80 μg/ml) | 80.02 (76.64) |
TABLE 2.
Synthetic cyclic peptides inhibit ANDV infectionTarget | Sample | % Inhibition bya:
| |
---|---|---|---|
Peptide-bearing phage | Synthetic peptide | ||
Gn | CMQSAAAHC | 48.88 (44.40) | 59.66 (11.17) |
Gc | CTVGPTRSC | 57.30 (11.31) | 46.47 (7.61) |
Gn | CPSNVNNIC | 61.11 (25.41) | 44.14 (10.74) |
Gn | CEKLHTASC | 43.60 (27.92) | 34.87 (9.26) |
Gc | CPKLHPGGC | 71.88 (27.11) | 30.95 (7.73)b |
Gn | CSLHSHKGC | 45.11 (49.81) | 29.79 (9.34) |
Gc | CPMSQNPTC | 65.60 (13.49) | 18.19 (8.55)b |
Gn | CKSLGSSQC | 53.90 (13.34) | 18.10 (7.55)b |
Gn | CNSHSPVHC | 45.40 (28.80) | 15.52 (10.48) |
Gn | CPAASHPRC | 51.84 (17.09) | 0 (10.72)b |
Integrin β3 | ReoPro | 80.10 (7.72) | |
Gn | 6B9/F5 antibody | 42.72 (6.75) | |
Gc | 6C5/D12 antibody | 31.04 (7.81) |
11.
Lisa Jacobsen Lisa Durso Tyrell Conway Kenneth W. Nickerson 《Applied and environmental microbiology》2009,75(13):4633-4635
Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table (Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table (Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table (Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table Table22.
Open in a separate window
Open in a separate windowaEight LB controls were run, two for each set of LB experiments: SDS, CTAB, benzalkonium chloride (BAC), and pH stress.bGrowth was measured as either +++, +, or 0 (good, poor, and none, respectively), with +++ being the growth achieved on the LB control plates. “Variable” means that two or three replicates did not agree. All experiments were done at 37°C.c“Anaerobic” refers to use of an Oxoid anaerobic chamber. Aerobic and anaerobic growth data are presented together when the results were identical and separately when the results were not the same or the anaerobic set had not been done. LB plates were measured after 1 (aerobic) or 2 (anaerobic) days, and the M63 plates were measured after 2 or 3 days.dCTAB used at 0.05, 0.2%, and 0.4%.eM63 defined medium (3) was supplemented with glucose, gluconate, or glucuronate, all at 0.2%.fIdentical results were obtained with and without 0.0001% thiamine.gND, not determined. 相似文献
TABLE 1.
E. coli strains used in this studyE. coli strain (n) | Source |
---|---|
ECOR strains (72) | Thomas Whittman |
Laboratory adapted (6) | |
K-12 Davis | Paul Blum |
CG5C 4401 | Paul Blum |
K-12 Stanford | Paul Blum |
W3110 | Paul Blum |
B | Tyler Kokjohn |
AB 1157 | Tyler Kokjohn |
O157:H7 (10) | |
FRIK 528 | Andrew Benson |
ATCC 43895 | Andrew Benson |
MC 1061 | Andrew Benson |
C536 | Tim Cebula |
C503 | Tim Cebula |
C535 | Tim Cebula |
ATCC 43889 | William Cray, Jr. |
ATCC 43890 | William Cray, Jr. |
ATCC 43888 | Willaim Cray, Jr. |
ATCC 43894 | William Cray, Jr. |
TABLE 2.
Physiological comparison of 88 strains of Escherichia coliGrowth medium or condition | Oxygenc | No. of strains with type of growthb
| |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ECOR strains (n = 72)
| Laboratory strains (n = 6)
| O157:H7 strains (n = 10)
| |||||||||||
Good | Poor | None | Variable | Good | Poor | None | Variable | Good | Poor | None | Variable | ||
LB controla | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
1% SDS | Aerobic | 69 | 3 | 0 | 0 | 6 | 0 | 0 | 0 | 8 | 0 | 0 | 2 |
5% SDS | Aerobic | 68 | 4 | 0 | 0 | 6 | 0 | 0 | 0 | 8 | 2 | 0 | 0 |
1% SDS | Anaerobic | 53 | 15 | 4 | 0 | 2 | 3 | 1 | 0 | 1 | 7 | 0 | 2 |
5% SDS | Anaerobic | 0 | 68 | 4 | 0 | 0 | 4 | 2 | 0 | 0 | 7 | 0 | 4 |
CTABd (all) | Both | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 |
0.05% BAC | Aerobic | 3 | 11 | 58 | 2 | 0 | 2 | 2 | 2 | 0 | 0 | 9 | 1 |
0.2% BAC | Aerobic | 0 | 1 | 71 | 0 | 1 | 0 | 5 | 0 | 0 | 0 | 10 | 0 |
0.05% BAC | Anaerobic | 2 | 3 | 67 | 0 | 0 | 1 | 5 | 0 | 0 | 0 | 9 | 1 |
0.2% BAC | Anaerobic | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 |
pH 6.5 | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 6 | Both | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 5 | Both | 70 | 2 | 0 | 0 | 6 | 0 | 0 | 0 | 9 | 0 | 0 | 1 |
pH 4.6 | Both | 70 | 2 | 0 | 0 | 6 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
pH 4.3 | Aerobic | 14 | 0 | 1 | 57 | 3 | 1 | 2 | 0 | 3 | 2 | 0 | 5 |
pH 4.3 | Anaerobic | 69 | 3 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 1 | 0 | 0 |
pH 4.1 or 4.2 | Aerobic | 0 | 0 | 72 | 0 | NDg | ND | ||||||
pH 4.0 | Both | 0 | 0 | 72 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 9 | 1 |
M63 with supplemente | |||||||||||||
Glucose | Aerobicf | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucose | Anaerobicf | 70 | 0 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Gluconate | Both | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucuronate | Aerobic | 68 | 2 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
Glucuronate | Anaerobic | 69 | 1 | 2 | 0 | 5 | 0 | 1 | 0 | 9 | 0 | 1 | 0 |
12.
13.
14.
Andrea Jesenská Marta Monincová Táňa Koudeláková Khomaini Hasan Radka Chaloupková Zbyněk Prokop Arie Geerlof Ji?í Damborsky 《Applied and environmental microbiology》2009,75(15):5157-5160
This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.Haloalkane dehalogenases (EC 3.8.1.5.) acting on halogenated aliphatic hydrocarbons catalyze carbon-halogen bond cleavage, leading to an alcohol, a halide ion, and a proton as the reaction products (7). Haloalkane dehalogenases originating from various bacterial strains have potential for application in bioremediation technologies (4, 6, 22), construction of biosensors (2), decontamination of warfare agents (17), and synthesis of optically pure compounds (19). Recent evolutionary study of haloalkane dehalogenase sequences revealed the existence of three subfamilies, denoted HLD-I, HLD-II, and HLD-III (3). In contrast to subfamilies HLD-I and HLD-II, the subfamily HLD-III is currently lacking experimentally characterized proteins. We have therefore focused on the isolation and study of two selected representatives of the HLD-III subfamily, DrbA and DmbC.The drbA gene was amplified by PCR using the cosmid pircos.a3g10 originating from marine bacterium Rhodopirellula baltica SH1, and the dmbC gene was amplified from DNA originating from obligatory pathogen Mycobacterium bovis 5033/66. Six-histidine tails were added to the C termini of DrbA and DmbC in a cloning step, enabling single-step purification using Ni-nitrilotriacetic acid resin. Haloalkane dehalogenase DrbA was expressed under the T7 promoter and purified, with a resulting yield of 0.1 mg of protein per gram of cell mass. Haloalkane dehalogenase DmbC was obtained by expression in Mycobacterium smegmatis, with a yield of 0.07 mg of purified protein per gram of cell mass.The correct folding and secondary structures of the newly prepared enzymes were verified by circular dichroism (CD) spectroscopy. Far-UV CD spectra were recorded for DrbA and DmbC enzymes and other, related haloalkane dehalogenases. All enzymes tested exhibited CD spectra with two negative features at 208 and 222 nm and one positive peak at 195 nm, which are characteristic of α-helical content (Fig. (Fig.1).1). This suggested that both new enzymes, DrbA and DmbC, were folded correctly. However, DmbC exhibited more intense negative maxima which differed from other haloalkane dehalogenases in the θ222/θ208 ratio. This finding indicated a slight variation in the arrangement of secondary structure elements of the DmbC enzyme. Thermally induced denaturations of DrbA and DmbC were tested in parallel. Both enzymes showed changes in ellipticity during increasing temperature. The melting temperatures calculated from these curves were 45.8 ± 0.4°C for DmbC and 39.4 ± 0.1°C for DrbA. The thermostability results obtained for DrbA and DmbC were in good agreement with the range of melting temperatures determined for other, related haloalkane dehalogenases.Open in a separate windowFIG. 1.Far-UV CD spectra of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Protein concentration used for far-UV CD spectrum measurement was 0.2 mg/ml.The sizes of the purified proteins were estimated by electrophoresis under native conditions conducted using a 10% polyacrylamide gel (Fig. (Fig.2).2). More precise determination of the sizes of DrbA and DmbC was achieved by gel filtration chromatography performed on Sephacryl S-500 HR (GE Healthcare, Uppsala, Sweden), calibrated with blue dextran 2000, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (240 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa) (Fig. (Fig.3A).3A). Both DrbA and DmbC were eluted from the column in the fraction prior to blue dextran, indicating that both enzymes form oligomeric complexes of a size larger than 2,000 kDa (Fig. 3B and C). The haloalkane dehalogenases which have been biochemically characterized so far form monomers, except for DbjA isolated from Bradyrhizobium japonicum USDA110 (21), which shows monomeric, dimeric, and tetrameric forms according to the pH of the buffer (R. Chaloupkova, submitted for publication).Open in a separate windowFIG. 2.Native protein electrophoresis of DrbA and DmbC. Lane 1, carbonic anhydrase (29 kDa); lane 2, ovalbumin (43 kDa); lane 3, bovine albumin (67 kDa); lane 4, conalbumin (75 kDa); lane 5, catalase (240 kDa); lane 6, ferritin (440 kDa); lane 7, DrbA; lane 8, DmbC.Open in a separate windowFIG. 3.Gel filtration chromatogram of DrbA and DmbC. (A) The following calibration kit samples (0.5 ml of a concentration of 2 mg/ml protein loaded) were analyzed using 50 mM Tris-HCl with 150 mM NaCl, pH 7.5, as elution buffer: blue dextran (line 1, 9.6-ml fraction), thyroglobulin (line 2, 15.95-ml fraction), ferritin (line 3, 16.78-ml fraction), ovalbumin (line 4, 18.55-ml fraction), and RNase A (line 5, 20.08-ml fraction). (B and C) Haloalkane dehalogenase DrbA eluted in the 9.03-ml fraction (B), and haloalkane dehalogenase DmbC in the 9.31-ml fraction (C).The substrate specificities of DrbA and DmbC were investigated with a set of 30 selected chlorinated, brominated, and iodinated hydrocarbons. Standardized specific activities related to 1-chlorobutane (summarized in Table Table1)1) were compared with the activity profiles of other haloalkane dehalogenases (Fig. (Fig.4).4). DrbA and DmbC displayed similar activity patterns, with catalytic activities approximately two orders of magnitude lower than those of other known haloalkane dehalogenases (1, 5, 8-11, 13-16, 18, 20, 23). HLD-III subfamily enzymes showed a restricted specificity range and a preference for iodinated short-chain hydrocarbons. Both phenomena may be related to the composition of the catalytic pentad Asp-His-Asp+Asn-Trp, which is unique to the members of the HLD-III subfamily (3). The preference for substrates carrying an iodine substituent can be related to a pair of halide-binding residues and their spatial arrangement with the catalytic triad. These residues make up the catalytic pentad, playing a critical role in substrate binding, formation of the transition states, and the reaction intermediates of the dehalogenation reaction (12).Open in a separate windowFIG. 4.Substrate specificity profiles of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Activities were determined using a consistent set of 30 halogenated substrates (see Table Table1).1). Data were standardized by dividing each value by the sum of all activities determined for individual enzymes in order to mask the differences in absolute activities. Specific activities (in μmol·s−1·mg−1) with 1-chlorobutane are 0.0003 (DrbA), 0.0001 (DmbC), 0.0003 (DatA), 0.0133 (DbjA), 0.0010 (DbeA), 0.0128 (DhaA), 0.0231 (LinB), 0.0171 (DmbA), and 0.0117 (DhlA).
Open in a separate windowaNA, no activity detected.Substrates 1-iodobutane and 1,3-diiodopropane, identified as the best substrates for haloalkane dehalogenases DrbA and DmbC, were used for measuring the dependency of enzyme activity on temperature and for determination of the pH optima. DrbA exhibited the highest activity with 1-iodobutane at 50°C, although above this temperature, the enzyme rapidly became inactivated. DmbC showed the highest activity toward 1,3-diiodopropane at 40°C, which is similar to the temperature determined with the haloalkane dehalogenases DmbA and DmbB (45°C), isolated from the same species (10). Irrespective of the reaction temperature, DrbA showed the maximum activity at pH 9.15. DrbA kept 80% of its activity throughout a relatively wide range of pH values (pH 7.00 and 9.91) compared to DmbC, which showed a sharp maximum at pH 8.30. The Michaelis-Menten kinetics of DrbA and DmbC determined by isothermal titration microcalorimetry were investigated with 1-iodobutane, which is an iodinated analogue of 1-chlorobutane routinely used for characterization of haloalkane dehalogenases. The low magnitudes of the Michaelis constants (Km = 0.063 ± 0.003 mM for DrbA and 0.018 ± 0.001 mM for DmbC) suggest a high affinity of both enzymes for 1-iodobutane. The catalytic constants determined with 1-iodobutane (kcat = 0.128 ± 0.002 s−1 for DrbA and 0.0715 ± 0.0004 s−1 for DmbC) suggest that the low specific activities observed during substrate screening are not due to poor affinity but are instead due to a low conversion rate.The biochemical characteristics of purified DrbA and DmbC suggest that these proteins represent novel enzymes differing from previously characterized haloalkane dehalogenases by (i) their unique ability to form oligomers and (ii) low levels of dehalogenating activity with typical substrates of haloalkane dehalogenases. This study further illustrates how genome sequencing projects and phylogenetic analyses contribute to the identification of novel enzymes. Characterization of DrbA and DmbC, belonging to the subfamily HLD-III, partially filled a gap in the knowledge of the haloalkane dehalogenase family and provided an additional insight into evolutionary relationships among its members. 相似文献
TABLE 1.
Specific activities of haloalkane dehalogenases DrbA and DmbC toward a set of 30 halogenated hydrocarbonsaSubstrate | DrbA
| DmbC
| ||
---|---|---|---|---|
Sp act (nmol product·s−1· mg−1 protein) | Relative activity (%) | Sp act (nmol product·s−1· mg−1 protein) | Relative activity (%) | |
1-Chlorobutane | 0.291 | 100 | 0.122 | 100 |
1-Chlorohexane | 0.129 | 44 | 0.122 | 100 |
1-Bromobutane | 0.081 | 28 | 1.221 | 1,000 |
1-Bromohexane | 0.181 | 62 | 0.977 | 800 |
1-Iodopropane | 0.143 | 49 | 2.198 | 1,800 |
1-Iodobutane | 0.506 | 174 | 2.564 | 2,100 |
1-Iodohexane | 0.095 | 33 | 0.244 | 200 |
1,2-Dichloroethane | NA | NA | NA | NA |
1,3-Dichloropropane | NA | NA | 0.012 | 10 |
1,5-Dichloropentane | NA | NA | 0.061 | 50 |
1,2-Dibromoethane | 0.098 | 34 | 0.855 | 700 |
1,3-Dibromopropane | NA | NA | 5.007 | 4,100 |
1-Bromo-3-chloropropane | 0.001 | 0 | 1.465 | 1,200 |
1,3-Diiodopropane | 0.358 | 123 | 6.716 | 5,500 |
2-Iodobutane | 0.028 | 9 | NA | NA |
1,2-Dichloropropane | NA | NA | NA | NA |
1,2-Dibromopropane | 0.148 | 51 | 0.244 | 200 |
2-Bromo-1-chloropropane | 0.091 | 31 | 0.488 | 400 |
1,2,3-Trichloropropane | NA | NA | NA | NA |
Bis-(2-chloroethyl) ether | NA | NA | NA | NA |
Chlorocyclohexane | NA | NA | NA | NA |
Bromocyclohexane | 0.026 | 9 | NA | NA |
(1-Bromomethyl)-cyclohexane | NA | NA | 0.089 | 73 |
1-Bromo-2-chloroethane | 0.167 | 57 | 0.111 | 91 |
Chlorocyclopentane | NA | NA | NA | NA |
4-Bromobutyronitrile | 0.200 | 69 | 0.444 | 364 |
1,2,3-Tribromopropane | NA | NA | 0.222 | 182 |
3-Chloro-2-methyl propene | NA | NA | NA | NA |
2,3-Dichloropropene | 0.276 | 95 | NA | NA |
1,2-Dibromo-3-chloropropane | 0.010 | 3 | 0.044 | 36 |
15.
16.
Banumathi Sankaran Shilah A. Bonnett Kinjal Shah Scott Gabriel Robert Reddy Paul Schimmel Dmitry A. Rodionov Valérie de Crécy-Lagard John D. Helmann Dirk Iwata-Reuyl Manal A. Swairjo 《Journal of bacteriology》2009,191(22):6936-6949
GTP cyclohydrolase I (GCYH-I) is an essential Zn2+-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn2+-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn2+ starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn2+-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.The Zn2+-dependent enzyme GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo tetrahydrofolate (THF) biosynthesis pathway (Fig. (Fig.1)1) (38). THF is an essential cofactor in one-carbon transfer reactions in the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (38), and formylmethionyl-tRNA in bacteria (7). Recently, it has also been shown that GCYH-I is required for the biosynthesis of the 7-deazaguanosine-modified tRNA nucleosides queuosine and archaeosine produced in Bacteria and Archaea (44), respectively, as well as the 7-deazaadenosine metabolites produced in some Streptomyces species (33). GCYH-I is encoded in Escherichia coli by the folE gene (28) and catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (55), a complex reaction that begins with hydrolytic opening of the purine ring at C-8 of GTP to generate an N-formyl intermediate, followed by deformylation and subsequent rearrangement and cyclization of the ribosyl moiety to generate the pterin ring in THF (Fig. (Fig.1).1). Notably, the enzyme is dependent on an essential active-site Zn2+ that serves to activate a water molecule for nucleophilic attack at C-8 in the first step of the reaction (2).Open in a separate windowFIG. 1.Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH4) pathway (Fig. (Fig.1),1), an essential cofactor in the biosynthesis of tyrosine and neurotransmitters, such as serotonin and l-3,4-dihydroxyphenylalanine (3, 52). Recently, a distinct class of GCYH-I enzymes, GCYH-IB (encoded by the folE2 gene), was discovered in microbes (26% of sequenced Bacteria and most Archaea) (12), including several clinically important human pathogens, e.g., Neisseria and Staphylococcus species. Notably, GCYH-IB is absent in eukaryotes.The distribution of folE (gene product renamed GCYH-IA) and folE2 (GCYH-IB) in bacteria is diverse (12). The majority of organisms possess either a folE (65%; e.g., Escherichia coli) or a folE2 (14%; e.g., Neisseria gonorrhoeae) gene. A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table Table1),1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13, 44).
TABLE 1.
Distribution and candidate Zur-dependent regulation of alternative GCYH-I genes in bacteriaaOpen in a separate windowaGenes that are preceded by candidate Zur binding sites.bZur-regulated cluster is on the virulence plasmid pLVPK.cExamples of organisms with no folE genes are in boldface type.dZn-dependent regulation of B. subtilis folE2 by Zur was experimentally verified (17).Expression of the Bacillus subtilis folE2 gene, yciA, is controlled by the Zn2+-dependent Zur repressor and is upregulated under Zn2+-limiting conditions (17). This led us to propose that the GCYH-IB family utilizes a metal other than Zn2+ to allow growth in Zn2+-limiting environments, a hypothesis strengthened by the observation that an archaeal ortholog from Methanocaldococcus jannaschii has recently been shown to be Fe2+ dependent (22). To test this hypothesis, we investigated the physiological role of GCYH-IB in B. subtilis, an organism that contains both isozymes, as well as the metal dependence of B. subtilis GCYH-IB in vitro. To gain a structural understanding of the metal dependence of GCYH-IB, we determined high-resolution crystal structures of Zn2+- and Mn2+-bound forms of the N. gonorrhoeae ortholog. Notably, although the GCYH-IA and -IB enzymes belong to the tunneling-fold (T-fold) superfamily, there are significant differences in their global and active-site architecture. These studies shed light on the physiological significance of the alternative folate biosynthesis isozymes in bacteria exposed to various metal environments, and offer a structural understanding of the differential metal dependence of GCYH-IA and -IB. 相似文献17.
18.
19.
Katharine J. Bar Hui Li Annie Chamberland Cecile Tremblay Jean Pierre Routy Truman Grayson Chuanxi Sun Shuyi Wang Gerald H. Learn Charity J. Morgan Joseph E. Schumacher Barton F. Haynes Brandon F. Keele Beatrice H. Hahn George M. Shaw 《Journal of virology》2010,84(12):6241-6247
Recent studies indicate that sexual transmission of human immunodeficiency virus type 1 (HIV-1) generally results from productive infection by only one virus, a finding attributable to the mucosal barrier. Surprisingly, a recent study of injection drug users (IDUs) from St. Petersburg, Russia, also found most subjects to be acutely infected by a single virus. Here, we show by single-genome amplification and sequencing in a different IDU cohort that 60% of IDU subjects were infected by more than one virus, including one subject who was acutely infected by at least 16 viruses. Multivariant transmission was more common in IDUs than in heterosexuals (60% versus 19%; odds ratio, 6.14; 95% confidence interval [CI], 1.37 to 31.27; P = 0.008). These findings highlight the diversity in HIV-1 infection risks among different IDU cohorts and the challenges faced by vaccines in protecting against this mode of infection.Elucidation of virus-host interactions during and immediately following the transmission event is one of the great challenges and opportunities in human immunodeficiency virus (HIV)/AIDS prevention research (14-16, 31, 34, 45). Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference based on a model of random virus evolution (18-20, 43) have allowed for the identification of transmitted/founder viruses that actually cross from donor to recipient, leading to productive HIV type 1 (HIV-1) infection. Our laboratory and others have made the surprising finding that HIV-1 transmission results from productive infection by a single transmitted/founder virus (or virally infected cell) in ∼80% of HIV-infected heterosexuals and in ∼60% of HIV-infected men who have sex with men (MSM) (1, 13, 18, 24). These studies thus provided a precise quantitative estimate for the long-recognized genetic bottleneck in HIV-1 transmission (6, 11-13, 17, 25, 28, 30, 35, 38, 42, 47-49) and a plausible explanation for the low acquisition rate per coital act and for graded infection risks associated with different exposure routes and behaviors (15, 36).In contrast to sexual transmission of HIV-1, virus transmission resulting from injection drug use has received relatively little attention (2, 3, 29, 42) despite the fact that injection drug use-associated transmission accounts for as many as 10% of new infections globally (26, 46). We hypothesized that SGA strategies developed for identifying transmitted/founder viruses following mucosal acquisition are applicable to deciphering transmission events following intravenous inoculation and that, due to the absence of a mucosal barrier, injection drug users (IDUs) exhibit a higher frequency of multiple-variant transmission and a wider range in numbers of transmitted viruses than do acutely infected heterosexual subjects. We obtained evidence in support of these hypotheses from the simian immunodeficiency virus (SIV)-Indian rhesus macaque infection model, where we showed that discrete low-diversity viral lineages emanating from single or multiple transmitted/founder viruses could be identified following intravenous inoculation and that the rectal mucosal barrier to infection was 2,000- to 20,000-fold greater than with intravenous inoculation (19). However, we also recognized potentially important differences between virus transmission in Indian rhesus macaques and virus transmission in humans that could complicate an IDU acquisition study. For example, in the SIV macaque model, the virus inocula can be well characterized genetically and the route and timing of virus exposure in relation to plasma sampling precisely defined, whereas in IDUs, the virus inoculum is generally undefined and the timing of virus infection only approximated based on clinical history and seroconversion testing (8). In addition, IDUs may have additional routes of potential virus acquisition due to concomitant sexual activity. Finally, there is a paucity of IDU cohorts for whom incident infection is monitored sufficiently frequently and clinical samples are collected often enough to allow for the identification and enumeration of transmitted/founder viruses. To address these special challenges, we proposed a pilot study of 10 IDU subjects designed to determine with 95% confidence if the proportion of multivariant transmissions in IDUs was more than 2-fold greater than the 20% frequency established for heterosexual transmission (1, 13, 18, 24). A secondary objective of the study was to determine whether the range in numbers of transmitted/founder viruses in IDUs exceeded the 1-to-6 range observed in heterosexuals (1, 13, 18, 24). To ensure comparability among the studies, we employed SGA-direct amplicon sequencing approaches, statistical methods, and power calculations identical to those that we had used previously to enumerate transmitted/founder viruses in heterosexual and MSM cohorts (1, 13, 18, 20, 24).We first surveyed investigators representing acute-infection cohorts in the United States, Canada, Russia, and China; only one cohort—the Montreal Primary HIV Infection Cohort (41)—had IDU clinical samples and clinical data available for study. The Montreal cohort of subjects with acute and early-stage HIV-1 infection was established in 1996 and recruits subjects from both academic and private medical centers throughout the city. Injection drug use is an important contributing factor to Montreal''s HIV burden, with IDUs comprising approximately 20% of the city''s AIDS cases and 35% of the cohort (21, 40, 41). A large proportion of Montreal''s IDUs use injection cocaine, with 50 to 69% of subjects reporting cocaine as their injection drug of choice (4, 5, 9, 22, 23).Subjects with documented serological evidence of recent HIV-1 infection and a concurrent history of injection drug use were selected for study. These individuals had few or no reported risk factors for sexual HIV-1 acquisition. Clinical history and laboratory tests of HIV-1 viremia and antibody seroconversion were used to determine the Fiebig clinical stage (8) and to estimate the date of infection (Table (Table1).1). One subject was determined to be in Fiebig stage III, one subject was in Fiebig stage IV, five subjects were in Fiebig stage V, and three subjects were in Fiebig stage VI. We performed SGA-direct amplicon sequencing on stored plasma samples and obtained a total of 391 3′ half-genomes (median, 25 per subject; range, 19 to 167). Nine of these sequences contained large deletions or were G-to-A hypermutated and were excluded from subsequent analysis. Sequences were aligned, visually inspected using the Highlighter tool (www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html), and analyzed by neighbor-joining (NJ) phylogenetic-tree construction. A composite NJ tree of full-length gp160 env sequences from all 10 subjects (Fig. (Fig.1A)1A) revealed distinct patient-specific monophyletic lineages, each with high bootstrap support and separated from the others by a mean genetic distance of 10.79% (median, 11.29%; range, 3.00 to 13.42%). Maximum within-patient env gene diversity ranged from 0.23% to 3.34% (Table (Table1).1). Four subjects displayed distinctly lower within-patient maximum env diversities (0.23 to 0.49%) than the other six subjects (1.48% to 3.34%). The lower maximum env diversities in the former group are consistent with infection either by a single virus or by multiple closely related viruses, while the higher diversities can be explained only by transmission of more than one virus based on empirical observations (1, 13, 18, 24) and mathematical modeling (18, 20).Open in a separate windowFIG. 1.NJ trees and Highlighter plots of HIV-1 gp160 env sequences. (A) Composite tree of 382 gp160 env sequences from all study subjects. The numerals at the nodes indicate bootstrap values for which statistical support exceeded 70%. (B) Subject ACT54869022 sequences suggest productive infection by a single virus (V1). (C) Subject HDNDRPI032 sequences suggest productive infection by as many as three viruses. (D) Subject HDNDRPI001 sequences suggest productive infection by at least five viruses with extensive interlineage recombination. Sequences are color coded to indicate viral progeny from distinct transmitted/founder viruses. Recombinant virus sequences are depicted in black. Methods for SGA, sequencing, model analysis, Highlighter plotting, and identification of transmitted/founder virus lineages are described elsewhere (18, 20, 24, 44). The horizontal scale bars represent genetic distance. nt, nucleotide.
Open in a separate windowaM, male; F, female.bNumbers of days postinfection were estimated on the basis of serological markers, clinical symptoms, or a history of a high-risk behavior leading to virus exposure.cDiversity measurements determined by PAUP* analysis.dThe model prediction of the maximum achievable env diversity 100 days after transmission is 0.60% (95% CI, 0.54 to 0.68%). Diversity values exceeding this range imply transmission and productive infection by more than one virus. Diversity values less than 0.54% can be explained by transmission of one virus or of multiple closely related viruses (18).eModel described in Keele et al. (18).fMinimum estimate of transmitted/founder viruses.gND, not determined.An example of productive clinical infection by a single virus is shown in phylogenetic tree and Highlighter plots from subject ACT54869022 (Fig. (Fig.1B).1B). A similar phylogenetic pattern of single-variant transmission was found in 4 of 10 IDU subjects (Table (Table1).1). Examples of multivariant transmission are shown for subject HDNDRPI032, for whom there was evidence of infection by 3 transmitted/founder viruses (Fig. (Fig.1C)1C) and for subject HDNDRPI001, for whom there was evidence of infection by at least 5 transmitted/founder viruses (Fig. (Fig.1D).1D). One IDU subject, HDNDRPI034, had evidence of multivariant transmission to an extent not previously seen in any of 225 subjects who acquired their infection by mucosal routes (1, 13, 18, 24) or in any of 13 IDUs, as recently reported by Masharsky and colleagues (29). We greatly extended the depth of our analysis in this subject to include 163 3′ half-genome sequences in order to increase the sensitivity of detection of low-frequency viral variants. Power calculations indicated that a sample size of 163 sequences gave us a >95% probability of sampling minor variants comprising as little as 2% of the virus population. By this approach, we found evidence of productive infection by at least 16 genetically distinct viruses (Fig. (Fig.2).2). Fourteen of these could be identified unambiguously based on the presence of discrete low-diversity viral lineages, each consisting of between 2 and 48 sequences. Two additional unique viral sequences with long branch lengths (3F8 and G10) exhibited diversity that was sufficiently great to indicate a distinct transmission event as opposed to divergence from other transmitted/founder lineages (see the legend to Fig. Fig.2).2). It is possible that still other unique sequences from this subject also represented transmitted/founder viruses, but we could not demonstrate this formally. We also could not determine if all 16 (or more) transmission events resulted from a single intravenous inoculation or from a series of inoculations separated by hours or days; however, it is likely that all transmitted viruses in this subject resulted from exposure to plasma from a single infected individual, since the maximum env diversity was only 3.34% (Fig. (Fig.1A).1A). It is also likely that transmission occurred within a brief window of time, since the period from transmission to the end of Fiebig stage III is typically only about 25 days (95% CI, 22 to 37 days) (18, 20) and the diversity observed in all transmitted/founder viral lineages in subject HDNDRPI034 was exceedingly low, consistent with model predictions for subjects with very recent infections (18, 20).Open in a separate windowFIG. 2.NJ tree and Highlighter plot of HIV-1 3′ half-genome sequences from subject HDNDRPI034. Sequences emanating from 16 transmitted/founder viruses are color coded. Fourteen transmitted/founder viral lineages comprised of 2 or more identical or nearly identical sequences could be readily distinguished from recombinant sequences (depicted in black), which invariably appeared as unique sequences containing interspersed segments shared with other transmitted/founder virus lineages. The two sequences with the longest branch lengths (3F8 and G10) were interpreted to represent rare progeny of discrete transmitted/founder viruses because their unique polymorphisms far exceeded the maximum diversity estimated to occur in the first 30 days of infection (0.22%; CI, 0.15 to 0.31%) (18) and far exceeded the diversity observed within the other transmitted/founder virus lineages. The horizontal scale bar represents genetic distance.Lastly, we compared the multiplicity of HIV-1 transmission in the Montreal IDU subjects with that of non-IDU subjects for whom identical SGA methods had been employed. In this combined-cohort analysis, we found the frequency of multiple-variant transmission in heterosexuals to be 19% (34 of 175) and in MSM 38% (19 of 50) (Table (Table2)2) (24). The current study was powered to detect a >2-fold difference in multivariant transmission between IDUs and heterosexual subjects; in fact, we observed a 3-fold-higher frequency of multiple-variant transmission in Montreal IDUs (6 of 10 subjects [60%]) than in heterosexuals (odds ratio, 6.14; 95% CI, 1.37 to 31.27; Fisher exact test, P = 0.008) and a 1.5-fold-higher frequency in Montreal IDUs than in MSM (odds ratio, 2.41; 95% CI, 0.50 to 13.20; P = 0.294, not significant). In addition, we found that the range of numbers of transmitted/founder viruses was greater in IDUs (range, 1 to 16 viruses; median, 3) than in either heterosexuals (range, 1 to 6 viruses; median, 1) or MSM (range, 1 to 10 viruses; median, 1). The finding of larger numbers of transmitted/founder viruses in IDUs was not simply the result of more intensive sampling, since the numbers of sequences analyzed in all studies were comparable. Moreover, it is notable that in studies reported elsewhere, we sampled as many as 239 sequences by SGA or as many as 500,000 sequences by 454 pyrosequencing from four acutely infected MSM subjects and in each case found evidence of productive clinical infection by only a single virus (24; W. Fischer, B. Keele, G. Shaw, and B. Korber, unpublished). These results thus suggest that IDUs may be infected by more viruses and by a greater range of viruses than is the case following mucosal transmission. On this count, our findings differ from those reported by Masharsky and coworkers for an IDU cohort from St. Petersburg, Russia (29). Their study found a low frequency of multiple virus transmissions (31%), not significantly different from that of acutely infected heterosexuals, and a low number of transmitted/founder viruses (range, 1 to 3 viruses; median, 1). Because the SGA methods employed in both studies were identical, the numbers of sequences analyzed per subject were comparable (median of 25 sequences in Montreal versus 33 in St. Petersburg), and because the discriminating power of the SGA-direct sequencing method was sufficient to distinguish transmitted/founder viruses differing by as few as 3 nucleotides, or <0.1% of nucleotides (Fig. (Fig.2,2, compare lineages V4 and V5), it is unlikely that differences in the genetic diversity of HIV-1 in the two IDU populations explain the differences in findings between the two studies. Instead, we suspect that the explanation lies in the small cohort sizes (10 versus 13 subjects) and the particular risk behaviors of the IDUs in each cohort. The Russian cohort is heavily weighted toward heroine use, whereas the Montreal cohort is weighted toward injection cocaine use, the latter being associated with more frequent drug administration and the attendant infection risks of needle sharing (4).
Open in a separate windowaFisher''s exact test of multiple-variant transmission in heterosexuals versus in IDUs.bFisher''s exact test of multiple-variant transmission in MSM versus in IDUs.The results from the present study indicate that transmission of HIV-1 to IDUs can be associated with a high frequency of multiple-variant transmission and a broad range in the numbers of transmitted viruses. This wide variation in the multiplicity of HIV-1 infection in IDUs is likely due to the absence of a mucosal barrier to virus transmission (12, 19) and differences in the virus inocula (27, 29, 32, 39). The findings substantiate concerns raised in recent HIV-1 vaccine efficacy trials that different vaccine candidates may be more efficacious in preventing infection by some exposure routes than by others (7, 10, 33, 37). They further suggest that biological comparisons of molecularly cloned transmitted/founder viruses responsible for vaginal, rectal, penile, and intravenous infection could facilitate a mechanistic understanding of HIV-1 transmission and vaccine prevention (24, 44). 相似文献
TABLE 1.
Subject demographics and HIV-1 envelope analysis resultsSubject identifier | Age (yr) | Sexa | Fiebig stage | Estimated no. of days postinfectionb | CD4 count | Plasma viral load (log) | No. of SGA amplicons | Diversity of env genes (%)c | No. of transmitted/ founder viruses | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Mean | Interquartile range | Maximumd | Model predictione | Phylogenetic estimatef | ||||||||
HDNDRPI034 | 47 | M | III | 29 | 240 | 7.88 | 163 | 1.07 | 0.55 | 3.34 | >1 | 16 |
HDNDRPI029 | 18 | F | IV | 48 | 440 | 4.34 | 29 | 0.16 | 0.15 | 0.49 | 1 | 1 |
HTM385 | 24 | M | V | 62 | 406 | 5.37 | 22 | 0.12 | 0.08 | 0.27 | 1 | 1 |
CQLDR03 | 42 | M | V | 66 | NDg | 5.01 | 21 | 0.08 | 0.08 | 0.23 | 1 | 1 |
HDNDRPI001 | 36 | M | V | 28 | 690 | 5.94 | 25 | 0.90 | 0.63 | 1.91 | >1 | 5 |
HTM319 | 39 | M | V | 68 | 520 | 4.43 | 25 | 0.77 | 0.46 | 1.54 | >1 | 3 |
HDNDRPI032 | 37 | M | V | 73 | 1,040 | 3.53 | 19 | 1.48 | 2.99 | 3.34 | >1 | 3 |
ACTDM580208 | 39 | M | VI | 93 | 387 | 4.53 | 30 | 1.17 | 0.97 | 2.64 | >1 | 3 |
ACT54869022 | 28 | M | VI | 68 | 723 | 3.43 | 27 | 0.07 | 0.04 | 0.24 | 1 | 1 |
PSL024 | 46 | M | VI | 82 | 340 | 4.46 | 21 | 0.82 | 0.63 | 1.57 | >1 | 3 |
TABLE 2.
Multiplicity of HIV-1 infection in IDU, heterosexual, and MSM subjectsCohort | Reference | Virus subtype | Total no. of subjects | Single-variant transmission | Multiple-variant transmission | P value | Odds ratio | 95% CI | Median | Range | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|
No. of subjects | % of total | No. of subjects | % of total | |||||||||
Heterosexuals | Keele et al. (18) | B | 79 | 65 | 82.30 | 14 | 17.70 | 1 | 1-4 | |||
Abrahams et al. (1) | C | 69 | 54 | 78.30 | 15 | 21.70 | 1 | 1-5 | ||||
Haaland et al. (13) | A or C | 27 | 22 | 81.50 | 5 | 18.50 | 1 | 1-6 | ||||
Total | 175 | 141 | 80.60 | 34 | 19.40 | 0.008a | 6.14 | 1.37-31.27 | 1 | 1-6 | ||
MSM | Keele et al. (18) | B | 22 | 13 | 59.10 | 9 | 40.90 | 1 | 1-6 | |||
Li et al. (24) | B | 28 | 18 | 64.30 | 10 | 35.70 | 1 | 1-10 | ||||
Total | 50 | 31 | 62.00 | 19 | 38.00 | 0.294b | 2.41 | 0.50-13.20 | 1 | 1-10 | ||
IDUs | Bar | B | 10 | 4 | 40.00 | 6 | 60.00 | 3 | 1-16 |