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1.
Lichenysins are surface-active lipopeptides with antibiotic properties produced nonribosomally by several strains of Bacillus licheniformis. Here, we report the cloning and sequencing of an entire 26.6-kb lichenysin biosynthesis operon from B. licheniformis ATCC 10716. Three large open reading frames coding for peptide synthetases, designated licA, licB (three modules each), and licC (one module), could be detected, followed by a gene, licTE, coding for a thioesterase-like protein. The domain structure of the seven identified modules, which resembles that of the surfactin synthetases SrfA-A to -C, showed two epimerization domains attached to the third and sixth modules. The substrate specificity of the first, fifth, and seventh recombinant adenylation domains of LicA to -C (cloned and expressed in Escherichia coli) was determined to be Gln, Asp, and Ile (with minor Val and Leu substitutions), respectively. Therefore, we suppose that the identified biosynthesis operon is responsible for the production of a lichenysin variant with the primary amino acid sequence l-Gln–l-Leu–d-Leu–l-Val–l-Asp–d-Leu–l-Ile, with minor Leu and Val substitutions at the seventh position.Many strains of Bacillus are known to produce lipopeptides with remarkable surface-active properties (11). The most prominent of these powerful lipopeptides is surfactin from Bacillus subtilis (1). Surfactin is an acylated cyclic heptapeptide that reduces the surface tension of water from 72 to 27 mN m−1 even in a concentration below 0.05% and shows some antibacterial and antifungal activities (1). Some B. subtilis strains are also known to produce other, structurally related lipoheptapeptides (Table (Table1),1), like iturin (32, 34) and bacillomycin (3, 27, 30), or the lipodecapeptides fengycin (50) and plipastatin (29).
Open in a separate windowaFA, β-hydroxy fatty acid. The β-hydroxy group forms an ester bond with the carboxy group of the C-terminal amino acid. bFA, β-hydroxy fatty acid. The β-hydroxy group forms an ester bond with the carboxy group of Asp5. cFA, β-amino fatty acid. The β-amino group forms a peptide bond with the carboxy group of the C-terminal amino acid. dOnly the following combinations of amino acid 1 and 5 are allowed: Gln-Asp or Glu-Asn. eWhere an alternative amino acid may be present in a structure, the alternative is also presented. In addition to B. subtilis, several strains of Bacillus licheniformis have been described as producing the lipopeptide lichenysin (14, 17, 23, 26, 51). Lichenysins can be grouped under the general sequence l-Glx–l-Leu–d-Leu–l-Val–l-Asx–d-Leu–l-Ile/Leu/Val (Table (Table1).1). The first amino acid is connected to a β-hydroxyl fatty acid, and the carboxy-terminal amino acid forms a lactone ring to the β-OH group of the lipophilic part of the molecule. In contrast to the lipopeptide surfactin, lichenysins seem to be synthesized during growth under aerobic and anaerobic conditions (16, 51). The isolation of lichenysins from cells growing on liquid mineral salt medium on glucose or sucrose basic has been studied intensively. Antimicrobial properties and the ability to reduce the surface tension of water have also been described (14, 17, 26, 51). The structural elucidation of the compounds revealed slight differences, depending on the producer strain. Various distributions of branched and linear fatty acid moieties of diverse lengths and amino acid variations in three defined positions have been identified (Table (Table11).In contrast to the well-defined methods for isolation and structural characterization of lichenysins, little is known about the biosynthetic mechanisms of lichenysin production. The structural similarity of lichenysins and surfactin suggests that the peptide moiety is produced nonribosomally by multifunctional peptide synthetases (7, 13, 25, 49, 53). Peptide synthetases from bacterial and fungal sources describe an alternative route in peptide bond formation in addition to the ubiquitous ribosomal pathway. Here, large multienzyme complexes affect the ordered recognition, activation, and linking of amino acids by utilizing the thiotemplate mechanism (19, 24, 25). According to this model, peptide synthetases activate their substrate amino acids as aminoacyl adenylates by ATP hydrolysis. These unstable intermediates are subsequently transferred to a covalently enzyme-bound 4′-phosphopantetheinyl cofactor as thioesters. The thioesterified amino acids are then integrated into the peptide product through a stepwise elongation by a series of transpeptidations directed from the amino terminals to the carboxy terminals. Peptide synthetases have not only awakened interest because of their mechanistic features; many of the nonribosomally processed peptide products also possess important biological and medical properties.In this report we describe the identification and characterization of a putative lichenysin biosynthesis operon from B. licheniformis ATCC 10716. Cloning and sequencing of the entire lic operon (26.6 kb) revealed three genes, licA, licB, and licC, with structural patterns common to peptide synthetases and a gene designated licTE, which codes for a putative thioesterase. The modular organization of the sequenced genes resembles the requirements for the biosynthesis of the heptapeptide lichenysin. Based on the arrangement of the seven identified modules and the tested substrate specificities, we propose that the identified genes are involved in the nonribosomal synthesis of the portion of the lichenysin peptide with the primary sequence l-Gln–l-Leu–d-Leu–l-Val–l-Asp–d-Leu–l-Ile (with minor Val and Leu substitutions). 相似文献
TABLE 1
Lipoheptapeptide antibiotics of Bacillus spp.Lipopeptide | Organism | Structure | Reference |
---|---|---|---|
Lichenysin A | B. licheniformis | FAa-L-Glu-L-Leu-D-Leu-L-Val-L-Asn-D-Leu-L-Ile | 51, 52 |
Lichenysin B | FAa-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu | 23, 26 | |
Lichenysin C | FAa-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Ile | 17 | |
Lichenysin D | FAa-L-Gln-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Ile | This work | |
Surfactant 86 | B. licheniformis | FAa-L-Glxd-L-Leu-D-Leu-L-Val-L-Asxd-D-Leu-L-Ilee | 14, 15 |
L-Val | |||
Surfactin | B. subtilis | FAa-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu | 1, 7, 49 |
Esperin | B. subtilis | FAb-L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leue | 45 |
L-Val | |||
Iturin A | B. subtilis | FAc-L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser | 32 |
Iturin C | FAc-L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asne-L-Asne | 34 | |
D-Ser-L-Thr | |||
Bacillomycin L | B. subtilis | FAc-L-Asp-D-Tyr-D-Asn-L-Ser-L-Gln-D-Proe-L-Thr | 3 |
D-Ser- | |||
Bacillomycin D | FAc-L-Asp-D-Tyr-D-Asn-L-Pro-L-Glu-D-Ser-L-Thr | 30, 31 | |
Bacillomycin F | FAc-L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Thr | 27 |
2.
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 |
---|---|---|---|---|
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 |
3.
4.
5.
Lyle R. McKinnon Rupert Capina Harold Peters Mark Mendoza Joshua Kimani Charles Wachihi Anthony Kariri Makobu Kimani Meika Richmond Sandy Koesters Kiazyk Keith R. Fowke Walter Jaoko Ma Luo T. Blake Ball Francis A. Plummer 《Journal of virology》2009,83(23):12636-12642
HLA-B*57-mediated selection pressure leads to a typical escape pathway in human immunodeficiency virus type 1 (HIV-1) CD8 epitopes such as TW10. Whether this T242N pathway is shared by all clades remains unknown. We therefore assessed the nature of HLA-B*57 selection in a large, observational Kenyan cohort where clades A1 and D predominate. While T242N was ubiquitous in clade D HLA-B*57+ subjects, this mutation was rare (15%) in clade A1. Instead, P243T and I247L were selected by clade A1-infected HLA-B*57 subjects but not by HLA-B*5801+ subjects. Our data suggest that clade A1 consensus proline at Gag residue 243 might represent an inherent block to T242N escape in clade A1. We confirmed immunologically that P243T and I247L likely represent escape mutations. HLA-B*57 evolution also differed between clades in the KF11 and IW9 epitopes. A better understanding of clade-specific evolution is important for the development of HIV vaccines in regions with multiple clades.Human immunodeficiency virus type 1 (HIV-1) displays extreme genetic diversity, with nine clades (subtypes) described in group M, and frequent genomic recombination among and within the clades (7, 44). HIV is also capable of rapid evolution, which can lead to mutational escape from immune control (43). Escape from CD8+ T-cell responses occurs frequently in HIV-1 infection through mutations that affect epitope processing, HLA class I binding, and/or T-cell receptor recognition (23). In early HIV-1 infection, the majority of amino acid substitutions are associated HLA class I alleles (1). The timing and consequences of mutational escape from CD8+ T-cell responses vary considerably (8, 22).HLA-B*57, and to a lesser extent HLA-B*5801, has been associated with slower progression to AIDS in several studies (18, 27, 39), and HLA-B*5701 was associated with a lower viremia set point in a genome-wide association study (16). Several attributes of HLA-B*57-restricted CD8+ T-cell responses may contribute to their protectiveness, including dominant responses in acute infection (2), recognition of protective epitopes in HIV-1 p24 (33), better recognition of epitope variation (45), and retention of proliferative capability in chronic infection (24).HLA-B*57/5801 also exert powerful selection pressure on HIV to avoid CD8+ T-cell recognition. This was first demonstrated in the HLA-B*57-restricted TW10 epitope (TSTLQEQIGW [Gag240-249]), which accounts for >30% of overall HIV-specific CD8+ T-cell responses in acutely infected HLA-B*57+ subjects (3). Escape in this epitope usually occurs early in infection, which coincidently is when HLA-B*57 is most protective (18). In clade B and C infections, >75 to 100% of HLA-B*57/5801+ subjects develop the T242N escape mutation, while HLA-B*57/5701-negative subjects rarely display polymorphism at this residue (5, 9, 10, 15, 32, 35, 38, 41). When T242N is transmitted to HLA-B*57/5801-negative subjects, it rapidly reverts to the consensus, suggesting that T242N is associated with a fitness defect (32, 35).While CD8+ T-cell cross-clade recognition has been tested extensively (6, 11, 19, 36, 48), few studies have addressed the possibility of clade-specific escape from CD8+ T-cell responses. This may be especially relevant where clade consensus sequences differ in immunologically relevant epitopes. Here we demonstrate in a large Kenyan cohort substantial differences in HLA-B*57/B*5801-mediated selection among HIV clades.Participants were enrolled from a Nairobi, Kenya-based cohort, and the relevant ethical review boards approved the study. HLA typing was performed as described previously (34). CD4 counts were measured longitudinally at biannual visits. Multiple and other clade infections were excluded. The HIV-1 p24 gene was amplified from proviral HIV DNA or RNA using a nested PCR approach and sequenced, and viral subtyping was carried out as described previously (42). Previously described HLA-B*57 epitopes IW9 (ISPRTLNAW), KF11 (KAFSPEVIPMF), and TW10 (TSTLQEQIGW) and selected variants were tested in immunological assays and described where relevant. Gamma interferon enzyme-linked immunospot (ELISPOT) assays were performed as described previously (37) using blood samples from HLA-B*57+ and -B*5801+ subjects. All peptides were tested at concentrations of 10 μg, 1 μg, 0.1 μg, and 0.01 μg/ml. Responses were considered positive if they were more than two times higher than that of the negative control and were measured at ≥100 spot-forming units ml−1. Fisher''s exact test and chi-square analyses were used to determine differences among groups in categorical analyses. Mann-Whitney U tests were used to compare response magnitudes and disease progression between groups.We confirmed the protective effects of HLA-B*57 in clade A1 infection (mean of 9.9 years versus 7.8 years until CD4 counts were <200, P = 0.041) (Fig. (Fig.1).1). Slow progressors were overrepresented in HLA-B*57+ clade A1+ subjects (52.2%) compared to both HLA-B*5801+ clade A1+ (13.3%, P = 0.02) and HLA-B*57/5801-negative clade A1+ (27.8%, P = 0.028) subjects (Fig. (Fig.1b).1b). In contrast to what has been shown for other clades (2, 27), protection was not observed for clade A1-infected HLA-B*5801+ subjects (mean of 6.5 years versus 7.8 years until CD4 counts were <200, P > 0.3) (Fig. (Fig.11).Open in a separate windowFIG. 1.HLA-B*57, but not HLA-B*5801, is associated with a lower rate of disease progression in clade A1-infected subjects than that of the overall cohort. (a) Number of years from cohort entry until sequential CD4 counts fell below 200/μl. (b) Slow progressors (>10 years with CD4 counts of >200) were also more common in HLA-B*57+ clade A1-infected subjects than in those expressing HLA-B*5801 or neither.Stratification of TW10 (Gag240-249) proviral sequences on the basis of HLA allele and clade revealed several differences in selection between clades A1 and D (Fig. (Fig.2a).2a). We observed the expected T242N substitution in 100% of HLA-B*57+ clade D-infected subjects (7/7), compared to only 14.7% variability at Gag residue 242 in HLA-B*57/5801-negative subjects (13/88, P = 3.26 × 10−9) (Fig. (Fig.2b).2b). In contrast, T242N was found infrequently in clade A1-infected HLA-B*57+ subjects (15%, 5/33, P = 0.0004). Instead, variants containing the mutations P243T and I247L were more frequently observed (both observed in 11/33 subjects). Overall, variation at residues 243 and 247 was more common in HLA-B*57+ subjects (51% and 15%, respectively; P = 2.92 × 10−6) than in HLA-B*57/5801-negative clade A1+ subjects (33% and 9%, respectively; P = 0.0008). Selection at both residues 243 and 247 was observed only in 2/33 HLA-B*57+ subjects, suggesting that these mutations are independent. Selection at residue 248, observed in clade B infection (32), was not evident in either clade A1 or D. While I247X selection has been described in other clades at low frequencies and in elite controllers (21, 40), HLA-B*57-mediated selection at Gag residue 243 has not yet been described. In summary, the T242N mutation, which is typical of other clades, does not appear to be the primary escape mutant in clade A1.Open in a separate windowFIG. 2.HLA-B*57-mediated selection in TW10 differs between clade A1 and clade D. (a) TW10 sequences were stratified by HLA-B*57, HLA-B*5801, or other alleles (HLA-B*57/5801−) and compared between clades A1 and D, based on the clade B consensus TW10 sequence. Each subject is represented by one sequence, and the numbers of subjects with a given sequence are shown in parentheses. A summary of variation from the TW10 consensus at Gag residues 242, 243, 247, and 248 is shown for HLA-B*57+ (b) and -B*5801+ (c) subjects. (b and c) Clade D is shown at the top, and clade A1 is shown at the bottom. Variation is shown in dark gray, and consensus is shown in light gray. (d) The proportions of clade A1-infected subjects with selection at Gag residue 242 only, and those with selection at residues 242 and 243 in combination, are shown. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.Previous studies have suggested that HLA-B*5801 places selection pressure on TW10, similar to that of HLA-B*57 (35). Similar to clades B and C, selection of T242N was evident in HLA-B*5801+ clade D-infected subjects (TW10 variation in 8/11 HLA-B*5801+ subjects versus 13/88 HLA-B*57/5801-negative subjects; P = 0.0069) (Fig. (Fig.2c).2c). Limited T242N selection was observed in clade A1-infected HLA-B*5801+ subjects, and in contrast to HLA-B*57, there were no HLA-B*5801-associated substitutions at residues 243 and 247 in clade A1 (P values of 0.75 and 0.29, respectively) (Fig. (Fig.2c).2c). In summary, these data suggest that in addition to HLA-B*5801 not being associated with protection in clade A1 (Fig. (Fig.1),1), HLA-B*5801 does not select the HLA-B*57-associated clade A1 TW10 escape mutations.Inclusion of all clade A1 sequences with T242X substitutions (regardless of the HLA allele) reveals that in every case (10/10), there is an accompanying residue 243 mutation. Polymorphisms at these sites correlate very strongly (P = 3.71 × 10−8) (Fig. (Fig.2d).2d). Together, these data suggest that residue 242 polymorphism in clade A1 is incompatible with proline at residue 243, which is the clade A1 consensus.We next assessed the immunological implications of novel clade A1 variants in HLA-B*57+ (n = 12) and -B*5801+ (n = 6) subjects infected primarily by clade A1. Clade A1-infected subjects commonly made anamnestic, low-avidity responses to TW10. The majority of HLA-B*57+ subjects who recognized clade A1 TW10 did not respond to P243T or I247L in ELISPOT assays (Fig. (Fig.3),3), supporting the hypothesis that these represent escape mutations. Those who did recognize P243T and I247L had lower magnitude responses than those who recognized clade A1 TW10 at the 10-μg/ml peptide concentration (P of 0.0005 for both) (Fig. (Fig.3a).3a). Similarly, these variants were not well recognized by CD8+ T cells from HLA-B*5801+ subjects (Fig. (Fig.3b).3b). For two HLA-B*57+ subjects, P243T and I247L responses had lower avidity than clade A1 TW10 responses (Fig. (Fig.3c).3c). These data support the hypothesis that P243T and I247L likely represent escape mutations.Open in a separate windowFIG. 3.Peptides with novel TW10 clade A1-selected mutations are poorly recognized in ex vivo gamma interferon ELISPOT avidity assays, suggestive of escape mutations. ELISPOT responses to TW10 and variants at 10 μg/ml peptide by HLA-B*57+ (a) and HLA-B*5801+ (b) subjects are shown. (c) The functional avidity of TW10 and variants for two HLA-B*57+ subjects is shown, suggesting that P243T and I247L are less recognized than TW10, particularly at lower peptide concentrations. Sequence names are described in the text. (d) Elispot responses to A1 TW10 and T242N correlated at 10 μg/ml. SFU/m, spot-forming units/million PBMCs.Recognition of the clade B/D consensus (TSTLQEQIGW) was diminished compared to that of clade A1 TW10. However, despite the presumed absence of this variant in these subjects'' autologous sequences, the clade B/D escape variant (TSNLQEQIGW [T242N]) was recognized at a magnitude similar to that of the consensus clade A1 TW10 (r = 0.71, P = 0.0099) (Fig. (Fig.3d).3d). No responses to T242N/G248A were observed (not shown), as described previously (32). These data suggest that clade A1 and B TW10, and their escape variants, are immunologically distinct from one another.We next assessed whether clade-specific selection was evident in other immunodominant HLA-B*57 p24 epitopes that are commonly targeted in chronic clade B infection (2). In clade D IW9 (ISPRTLNAW [Gag147-155]), variants containing the escape variant I147L (14) were more common in HLA-B*57+ subjects than in HLA-B*57/5801-negative subjects (86% and 30%, respectively; P = 0.0055) (Table (Table1).1). However, this variant was not selected in clade A1 (variation in 30% versus 21% subjects; P value was not significant), where leucine is the consensus. Interestingly, ELISPOT data indicated substantial cross-reactivity between 147L and 147I in clade A1-infected subjects (10 μg/ml, r = 0.987, P < 0.0001) (data not shown), suggesting that infection with an escape variant from one clade (clade D) does not necessarily preclude recognition of this epitope in another one (clade A1). Other amino acids (F, M, and P) were common in HLA-B*57+ subjects at residue 147 (>30% versus 3% in HLA-B*57/5801-negative subjects, P = 3.85 × 10−6). Although the immunological consequences are unknown, these HLA-associated substitutions could represent novel escape variants.
Open in a separate windowaThe first row of epitope sequences shows the consensus sequences.bND, not done.In addition, a substitution at Gag residue 146 (A146P) represents an IW9 processing escape mutation in clades B and C (14), and this mutation was also selected by HLA-B*57 in both clades A1 and D (Table (Table1).1). In clade A1, substitutions at Gag residue 146 (primarily P and T) were more frequent in HLA-B*57+ subjects than in HLA-B*57/5801-negative subjects (13/33 and 10/221, respectively; P = 1.42 × 10−7) (Table (Table1).1). Therefore, although the consensus at residue 146 differs among clades, here escape at residue 146 occurs in HLA-B*57+ subjects infected by clades A1, B, C, and D.For KF11 (KAFSPEVIPMF [Gag162-172]), HLA-B*57-associated variation from the consensus was more common in clade A1 (67% versus 21%, P = 2.44 × 10−7) than in clade D (43% versus 17%, P = 0.012). Previously described A163G and A163G/S165N variants (13, 20) were most common in clade A1 (Table (Table1).1). In addition, the novel K162X substitution was present in clade A1. HLA-B*5703 and -B*5701 have previously been shown to display differences in KF11 selection (20, 47), and our data indicate that HLA-B*5702 also differs from HLA-B*5703 in terms of KF11 selection. While the KF11 consensus is present in the majority of HLA-B*5702+ subjects, it is rare in HLA-B*5703+ clade A1 infection (8/9 versus 2/22, P = 4.74 × 10−5).Mounting evidence suggests that HLA alleles are a major force in viral evolution (26). We show that in clade A1 p24, HLA-B*57 selection in three epitopes differs from earlier clade B and C data in several important aspects, while clade D selection resembles what has previously been shown. This included a low frequency of T242N in clade A1 TW10, with selection being more common at Gag residues 243 and 247, more extensive KF11 escape, and selection of different amino acids in IW9. Overall, selection was evident in the majority of HLA-B*57+ subjects (>90% of clade A1-infected subjects had selection in more than one epitope, and >75% of them had selection in more than two) (Table (Table1).1). Parallel escape in multiple epitopes demonstrates the need to avoid the pressure of CD8+ T-cell responses.One possible mechanism underlying the differences in TW10 selection is that TSNPQEQIGW (never observed) (Fig. (Fig.2d)2d) is not feasible virologically, such that T242N is possible only in conjunction with a preexisting residue 243 mutation (TSNXQEQIGW, observed in 15% of HLA-B*57+ subjects) (underlining shows mutation). One would expect to observe T242N at a higher frequency, given its dominance in HLA-B*57+ subjects infected by other clades. Therefore, while T242N has been implicated in HLA-B*57-mediated protection, this mutation is rare in clade A1. Because HLA-B*57 remains protective in clade A1, that protection may be mediated by novel mechanisms.Because TW10 is commonly recognized by 86% of clade A1 subjects, it is evident that clade A1 TW10 can bind HLA-B*57. We therefore hypothesize that TSTPQEQIGW may affect the interaction between epitope and cognate T-cell receptors, which in turn influences which escape mutations are optimal. This hypothesis is supported by our immunological data showing cross-reactivity between clade A1 TW10 and TSNLQEQIGW (underlining shows mutation), which imply that mutation at residue 242 may not lead to effective escape in clade A1 (Fig. (Fig.3d3d).In contrast to other clades (including clade D), HLA-B*5801 does not appear to place selection pressure on clade A1 TW10. A previous study in Rwanda similarly showed that in clade A1, HLA-B*5703 but not HLA-B*5801 was associated with lower HIV viral loads (30). Therefore, HLA-B*5801 was associated with neither protection nor selection in clade A1 TW10. Our data also show that HLA-B*5702- and -B*5703-mediated KF11 selection differs, despite these alleles differing at only one codon. Similar findings have been published for HLA-B7 supertype alleles (31). These data highlight the differences in immunological pressure within HLA supertype alleles, even though these alleles often present the same epitopes to the immune system.Previous reports have suggested that HIV evolution can differ among clades for a variety of reasons. HLA-B*1503 differed in its protectiveness in clade B- and clade C-infected cohorts, and the apparent mechanism is broader recognition of subdominant epitopes, which remain intact due to limited selection where HLA-B*1503 is less common (17). Similarly, Yu et al. showed that differences in KF11 evolution between clades B and C were largely the result of differences in immunological features of HLA-B*5701- and -B*5703-restricted responses, with the latter allele being more frequent in clade C-infected populations (47). The temporality of selection can also differ between clades; while TW10 and IW9 selection is similar between clade B and C, the order in which they are selected is opposite (12). Our data show that virological factors (i.e., sequence differences) can also lead to clade-specific escape. Other reports have found few differences in evolution among clades, including no differences in HLA-A2 Gag SL9 escape among clades A, B, and D (25), so the presence of clade-specific evolution will depend on the epitope and allele under study.Recent reports have suggested that Gag-specific CD8+ T-cell responses are protective in HIV infection (28), possibly because escape in Gag comes at a fitness cost. In support of this, infection by strains containing multiple Gag escape mutations was associated with lower set point viremia independent of HLA alleles in the recipients (21). One of the first demonstrations of Gag escape with fitness cost was T242N selection and reversion (32), and this substitution dominates in clade B- and clade C-infected HLA-B*57+ subjects in numerous cohorts (5, 9, 10, 15, 32, 35, 38, 41). Our data show that while clade D follows clades B and C, HLA-B*57-mediated evolution in clade A1 differs not only in TW10 but also in other p24 epitopes. Knowledge of clade-specific escape pathways will be important for vaccines that aim to cover multiple clades, particularly where clades differ in immunologically critical epitopes. 相似文献
TABLE 1.
p24 sequences in HLA-B*57+ subjects infected by clades A1 and DClade | HLA-B allele | Subject no. | No. of years infected prior to sampleb | Polymorphism at residue S146 | Epitope sequencea | ||
---|---|---|---|---|---|---|---|
IW9 | KF11 | TW10 | |||||
LSPRTLNAW | KAFSPEVIPMF | TSTPQEQIGW | |||||
A1 | 5701 | 1665 | >130 | N | F-------- | ----------- | ---------- |
5702 | 139 | 21 | N | --------- | ----------- | --NI------ | |
59 | ND | P | --------- | ----------- | --NT---LA- | ||
41 | >122 | T | --------- | ----------- | --NV------ | ||
1419 | >41 | N | --------- | ----------- | ---------- | ||
616 | >102 | T | --------- | ----------- | -------LA- | ||
1647 | 18 | - | P-------- | ----------- | ---S---LQ- | ||
613 | >33 | P | --------- | ----------- | ---T------ | ||
718 | >71 | P | M-------- | ----------- | ---T---L-- | ||
1315 | >58 | P | --------- | -G--------- | ---TS---L-- | ||
5703 | 2125 | >0 | A | --------- | ---N------- | --NL------ | |
561 | >78 | P | --------- | ---N------- | ---------- | ||
1926 | >39 | P | --------- | ---c------- | ---T------ | ||
1778 | >13 | - | --------- | ----------- | ---------- | ||
1609 | 0 | T | --------- | ----------- | -------LQ- | ||
525 | >135 | - | --------- | -G-n------- | -------L-- | ||
1368 | >57 | - | M-------- | -G-n------- | ---T------ | ||
509 | >150 | T | F-------- | -G-N------- | ---------- | ||
260 | >163 | T | F-------- | -G-N------- | -------L-- | ||
111 | >133 | - | --------- | -G-N------- | -------LQ- | ||
1111 | >34 | - | --------- | -G-N------- | -------LQ- | ||
1638 | >27 | - | --------- | -G-N------- | ---T------ | ||
2101 | >0 | P | --------- | -G--------- | --NT------ | ||
532 | >28 | - | --------- | -G--------- | --------A- | ||
1669 | >23 | P | P-------- | -G--------- | --------A- | ||
703 | >71 | T | F-------- | -G--------- | ---------- | ||
1741 | >11 | T | F-------- | -G--------- | ---------- | ||
1452 | >37 | P | --------- | -G--------- | ---------- | ||
30 | >121 | P | --------- | R---------- | --------A- | ||
1122 | >39 | P | --------- | RG-Q------- | ---------- | ||
995 | >80 | P | F-------- | RG-Q------- | ---T------ | ||
1564 | >27 | P | --------- | RG--------- | ---T------ | ||
5707 | 330 | >164 | T | --------- | -G-N------- | ---T------ | |
D | 5701 | 1859 | P | --------- | ----------- | --NL------ | |
1852 | P | --------- | ----------- | --NL---VA- | |||
5703 | 1756 | P | --------- | ----------- | --NL------ | ||
1423 | P | -T------- | ----------- | --NL------ | |||
1188 | P | I-------- | -G-N------- | --NL----A- | |||
1894 | P | --------- | -N--------- | --NL----R- | |||
199 | P | -T------- | -S--------- | --NL---V-- |
6.
7.
Feras F. Lafi John A. Fuerst Lars Fieseler Cecilia Engels Winnie Wei Ling Goh Ute Hentschel 《Applied and environmental microbiology》2009,75(17):5695-5699
Poribacteria were found in nine sponge species belonging to six orders of Porifera from three oceans. Phylogenetic analysis revealed four distinct poribacterial clades, which contained organisms obtained from several different geographic regions, indicating that the distribution of poribacteria is cosmopolitan. Members of divergent poribacterial clades were also found in the same sponge species in three different sponge genera.Recently, a novel bacterial phylum, termed “Poribacteria,” was discovered, and members of this phylum have been found exclusively in sponges (2). Phylogenetic analyses of 16S rRNA genes indicated that poribacteria are evolutionarily deeply branching organisms and related to a superphylum composed of Planctomycetes, Verrucomicrobia, and Chlamydia (11). Poribacterial 16S rRNA genes contain 13 of 15 planctomycete signature nucleotides, but a level of sequence divergence of more than 25% compared to any other bacterial phylum, including the Planctomycetes, justifies the status of this taxon as an independent phylum. A consistent treeing pattern is difficult to resolve in comparative phylogenetic sequence analyses, making the poribacteria an unusual line of phylogenetic descent. In addition to their divergent status as a separate phylum on the basis of the 16S rRNA sequence, poribacteria are also divergent because they may have a compartmentalized cell structure, a cell plan they share only with members of the phyla Planctomycetes and Verrucomicrobia (2). They are also of interest for understanding the potential contribution of obligate sponge-associated bacteria to the sponges harboring them and as an example of a yet-to-be-cultured group of bacteria associated with invertebrate tissue apparently exclusively but for unknown reasons. This study aimed to further explore the presence and diversity of poribacteria in different marine demosponge genera using samples from around the world.The Mediterranean sponges were collected by scuba divers offshore at Banyuls sur Mer, France (42°29′N, 03°08′E). The Caribbean sponges were collected offshore at Little San Salvador Island, Bahamas (24°32′N, 75°55′W). The eastern Pacific sponge Aplysina fistularis was collected offshore at San Diego, CA (32°51′N, 117°15′W). The western Pacific sponge Theonella swinhoei was collected offshore at Palau (07°23′N, 134°38′E). All non-Great Barrier Reef (non-GBR) sponges were collected between May and July 2000, and once individual sponge specimens were brought to the surface, they were frozen in liquid nitrogen on board ship and stored at −80°C until microbiological processing (9). The GBR marine sponges were collected off Heron Island Research Station (23°27′S, 151°5′E) in April 2002 (5). Pseudoceratina clavata was collected by scuba divers at a depth of 14 m, and Rhabdastrella globostellata was collected at a depth of ca. 0.5 m after a reef walk consisting of a few hundred meters. The samples were immediately placed in plastic bags and brought to Heron Island Research Station, where they were stored at −80°C until processing. Sponge DNA was extracted as described previously (2, 5).Total sponge-derived genomic DNA was screened by PCR for the presence of poribacteria using a 16S rRNA gene primer set. Poribacterial 16S rRNA genes were amplified by employing a pair of Poribacteria-specific primers, POR389f (5′-ACG ATG CGA CGC CGC GTG-3′) and POR1130r (5′-GGC TCG TCA CCA GCG GTC-3′) (2). The poribacterial PCR products that were ca. 740 bp long derived from one sponge individual were cloned into the pGEM-T Easy vector (Promega, Madison, WI). Clone inserts were digested with restriction endonucleases MspI and HaeIII (New England Biolabs, Inc., United States), characterized to obtain restriction profiles and unique profiles, and sequenced. The compiled partial 16S rRNA gene sequences were then analyzed using BLASTN to select the most closely related poribacterial reference sequences.The sequences exhibiting levels of similarity of less than 97% were used for further analysis. Poribacterial 16S rRNA gene sequences were aligned using the ARB software package (7). The resulting alignment was imported into PAUP (10) and analyzed by using distance, maximum parsimony, and maximum likelihood algorithms together with bootstrap resamplings (3,000, 3,000, and 200 resamplings, respectively), and the resulting bootstrap values were applied to nodes on the ARB neighbor-joining tree. Signature sequences were detected using the ARB software package. A signature sequence is defined here as a short sequence that is present in a group of poribacterial sequences in a phylogenetic clade but is not found in any other clade in the poribacterial tree.Analysis of the 16S rRNA gene clone library sequences generated from sponge tissues revealed the presence of poribacteria in sponge individuals belonging to the orders Verongida, Astrophorida, Dictyoceratida, Haplosclerida, Lithistida, and Homosclerophorida, while poribacteria could not be detected in sponges belonging to the orders Hadromerida and Agelasida. In the order Halichondrida, poribacteria were detected in Xestospongia muta but not in Haliclona sp. Altogether, nine sponge species were added to the list of Poribacteria-containing sponges (Table (Table1).1). Three distinct clades were observed that were clearly supported by bootstrap values greater than 75 with every tree-building algorithm applied (Fig. (Fig.1),1), and one clade (clade I) was supported by bootstrap values of 64, 98, and 71 in distance, maximum parsimony, and maximum likelihood trees, respectively. Similarity calculations using approximately 740-bp amplified poribacterial 16S rRNA gene fragments and other poribacterial sequences from the NCBI database showed that the dissimilarity between clades was consistent with their separation in phylogenetic trees. For example, the levels of dissimilarity between members of clade I and clade II were 3 to 8%, while the levels of dissimilarity between members of clades I and III and between members of clades I and IV were 10 to 14% and 11 to 15%, respectively.Open in a separate windowFIG. 1.Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing relationships of poribacterial clones from different global regions. The poribacterial clones on the right are additional clones belonging to the same clades as strains in the tree at the same level. Bootstrap confidence values of >75% for distance, maximum parsimony, and maximum likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba; C, Aplysina cavernicola; F, Aplysina fistularis; L, Aplysina lacunosa; S, Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella globostellata; T, Theonella swinhoei; X, Xestospongia muta. Scale bar = 0.1 nucleotide substitution per site.
Open in a separate windowaMED, Mediterranean Sea; BAH, Bahamas; WPAC, western Pacific Ocean; EPAC, eastern Pacific Ocean.bThe presence of poribacteria was evaluated by sequencing and phylogenetic analysis of amplified PCR products. +, present; −, absent.cNA, not applicable.Within each clade in the phylum Poribacteria, there were higher similarity values, including 94 to 100% among members of clade I, 94 to 99% among members of clade II, 96 to 99% among members of clade III, and 96 to 99% among members of clade IV. When members of the the phylum Poribacteria were compared to members of the Planctomycetes (Fig. (Fig.1),1), the 16S rRNA genes exhibited levels of sequence dissimilarity of up to 38%, consistent with the conclusion of Fieseler et al. concerning the separate phylum level status of poribacteria based on a similarity value of <75%. A phylogenetic correlation between sponge phylogeny and poribacterial phylogeny is not evident, since, for example, clones from A. fistularis and Aplysina aerophoba occurred in both clade I and clade II and one clone from A. aerophoba also occurred in clade III, while clones from P. clavata and R. globostellata occurred in clades I, II, and III but not in clade IV. Clades I and II included poribacterial clones derived from all sponge species occurring in all of the widely separated geographic regions examined in this study (Fig. (Fig.2).2). Clade III represented poribacterial clones derived from sponge species obtained in the eastern Pacific region, GBR, and the Bahamas but not in the Mediterranean region. The majority of poribacterial clones in clade IV were derived from sponge species obtained in the Bahamas, and one clade IV clone was obtained from a sponge species collected in the Mediterranean region.Open in a separate windowFIG. 2.Neighbor-joining phylogenetic tree for poribacterial clones based on Poribacteria-specific PCR products (740 bp) of the 16S rRNA gene, showing the internal relationships of and occurrence of clade I members in distinct sponge species representing cosmopolitan geographic regions. For an explanation of the colors, see Fig. Fig.1.1. Bootstrap confidence values of >75% for distance, maximum parsimony, and maximum likelihood algorithm analyses are indicated by filled circles at nodes, and open circles indicate unsupported nodes. Prefixes for clones: A, Aplysina aerophoba; C, Aplysina cavernicola; F, Aplysina fistularis; L, Aplysina lacunosa; S, Ircinia sp.; P, Plakortis sp.; PC, Pseudoceratina clavata; RG, Rhabdastrella globostellata; T, Theonella swinhoei; X, Xestospongia muta. Scale bar = 0.1 nucleotide substitution per site. Clones PC15, L8, T6, C2, P3, S2, and X1 were removed from this analysis to allow better branch resolution.Poribacterial clones from different sponges from widely separated marine habitats belonged to at least four major clades with similarities ranging from 94 and 96%. For clade III (Fig. (Fig.1),1), we detected a signature sequence characteristic of poribacterial clones from the GBR sponges R. globostellata and P. clavata. This signature sequence (CCA GTT AGC TTG ACG GTA) (Table (Table2)2) at E. coli positions 469 to 487 targeted 10 sequences, 5 of which were from GBR marine sponges generated in this study (clones RG68, RG112, RG105, PC96, and PC8). Another five poribacterial sequences were detected in an unpublished study investigating the microbial diversity in GBR sponges. This signature sequence indicates a specific geographic presence of poribacteria belonging to clade IV in the GBR region. In addition, a sequence (GAG TGT GAA ATG GCT TGG at E. coli positions 599 to 617) characteristic of clade IV was found in 11 sequences derived from sponges from the Bahamas and one sequence (A7) from a Mediterranean sponge.
Open in a separate windowaAsterisks indicate poribacterial clones derived from the GBR sponge R. globostellata in a separate study.bThe internal sequence (indicated by equals signs) of each pori_SSIII clone is CCAGUUAGCUUGACGGUA, and that of each pori_SSIV clone is GAGUGUGAAAUGGCUUGG.Based on the data presented here, Poribacteria appears to be a bacterial phylum that is specifically found in several demosponge genera of the phylum Porifera (Table (Table1).1). To our knowledge, this is the only case of a bacterial phylum specifically associated with a marine invertebrate phylum. Certain phylum members appear to be widely distributed among sponges belonging to different species and in different geographic regions, forming sponge-specific lineages (3), but these are individual species level or at most genus level clades in a subdivision of a phylum rather than in a whole phylum.PCR analyses of seawater samples collected in this study (Table (Table1)1) and searches using nucleotide sequence databases of seawater metagenomes were negative for poribacteria. This is consistent with the concept that Poribacteria is a sponge-specific phylum. Within the sponges poribacteria are distributed among members of distinct demosponge orders that occur in various geographic locations, indicating that there is wide distribution of poribacteria among marine demosponges. Very similar 16S rRNA clone sequences that cluster in clade I were found in sponges from all geographic regions sampled in this study, including locations in the Northern and Southern hemispheres (Fig. (Fig.2).2). Similarly, clade II contains poribacterial clones from the Mediterranean Aplysina species and from GBR Pseudoceratina and Rhabdastrella species. This appears to contrast, albeit at a lower level of resolution, with results suggesting that bacterial populations are endemic in different geographic regions, e.g., with the findings that marine bacterioplankton communities include few cosmopolitan operational taxonomic units (8), that fluorescent Pseudomonas genotypes from soil are endemic at different geographic sites (1), and that hyperthermophilic Sulfolobus archaea from different geothermal areas are genetically divergent (12). Judging the endemicity of populations in different geographic regions may depend on the taxonomic scale used to distinguish populations (1). In this study we provide evidence that at least some clades may be relatively characteristic of particular regions, e.g., GBR clade III (Table (Table2).2). It is remarkable that in the case of the sponge species R. globostellata and P. clavata from a single geographic region (GBR), the microbial communities include representatives of distantly related poribacterial clades II and III, whose sequences exhibit levels of dissimilarity ranging from 10 to 13%. In another case poribacteria belonging to clades I, II, and IV were found in a single host, A. aerophoba, from the Mediterranean. Thus, members of widely divergent poribacterial clades occur in the same specimen in sponges in widely separated geographic regions in the world''s oceans. Three different sponge species belonging to three different genera exhibit this phenomenon.The morphology and life strategy of sponges have remained unchanged for the past 580 million years, as judged by the dramatic similarity of the morphologies of Precambrian fossils to the morphologies of recent sponges (6). Adaptation of the poribacteria to this niche might have taken place early in evolution before the various sponge orders separated from each other. It seems likely that poribacteria diverged from other bacterial phyla long before evolution of the metazoans as part of the fan-like radiation by which all bacterial phyla appear to have arisen (4). This bacterial radiation may have resulted in the divergence of the clades that we have observed for the poribacteria, but there is no indication of cospeciation between host sponges and the poribacteria.In summary, poribacteria exhibit considerable diversity and are classified into four phylogenetic clades. Poribacteria seem to be widely distributed among many different marine demosponge genera, and further studies are needed to explain the nature of the poribacterium-sponge interaction. 相似文献
TABLE 1.
Distribution of poribacteria in different demosponge ordersSponge species or seawater | Order | Geographic locationa | Presence of poribacteriab | Reference |
---|---|---|---|---|
Aplysina aerophoba | Verongida | MED | + | 2 |
Aplysina lacunosa | Verongida | BAH | + | 2 |
Aplysina fistularis | Verongida | EPAC or BAH | + | 2 |
Aplysina insularis | Verongida | BAH | + | 2 |
Verongula gigantea | Verongida | BAH | + | 2 |
Smenospongia aurea | Dictyoceratida | BAH | + | 2 |
Aplysina cauliformis | Verongida | BAH | + | This study |
Aplysina archeri | Verongida | BAH | + | This study |
Aplysina cavernicola | Verongida | MED | + | This study |
Pseudoceratina clavata | Verongida | WPAC | + | This study |
Rhabdastrella globostellata | Astrophorida | WPAC | + | This study |
Ircinia sp. | Dictyoceratida | BAH | + | This study |
Xestospongia muta | Haplosclerida | BAH | + | This study |
Theonella swinhoei | Lithistida | EPAC | + | This study |
Plakortis sp. | Homosclerophorida | BAH | + | This study |
Chondrilla nucula | Hadromerida | BAH | − | 2 |
Agelas wiedenmayeri | Agelasida | BAH | − | 2 |
Agelas cerebrum | Agelasida | BAH | − | This study |
Axinella polypoides | Halichondrida | MED | − | This study |
Ptilocaulis sp. | Halichondrida | BAH | − | 2 |
Dysidea avara | Dictyoceratida | MED | − | This study |
Haliclona sp. | Haplosclerida | MED | − | This study |
Ectyoplasia ferox | Poecilosclerida | BAH | − | 2 |
Seawater | NAc | MED | − | This study |
TABLE 2.
Poribacterial signature sequences for clades III and IV, including a GBR-specific signature sequence (pori_SSIII) and a signature sequence specific to 11 of 12 sequences from the Bahamas (pori_SSIV)Signature sequence | Name | Full namea | E. coli position | Sequenceb |
---|---|---|---|---|
pori_SSIII | Pla101P | Pla101P* | 469 | GGUGAUAAG-==================-CCAUAGUA |
Pla131P | Pla131P* | 469 | GGUGAUAAG-==================-CCAUAGUA | |
Pla134P | Pla134P* | 469 | GGUGAUAAG-==================-CCAUAGUA | |
Pla50P | Pla50P* | 469 | GGUGAUAAG-==================-CCAUAGUA | |
Pla82P | Pla82P* | 469 | GGUGAUAAG-==================-CCAUAGUA | |
PO68 | Pori clone RGPo68 | 469 | GGUGAUAAG-==================-GAGAAAAG | |
PO112 | Pori clone RGPo112 | 469 | GGUGAUAAU-==================-CCAUAGUA | |
PO105 | Pori clone RGPo105 | 469 | GGUGAUAAG-==================-CCAUAGUA | |
PO96 | Uncultured Pori clone | 469 | GGUGAUAAG-==================-CCAUAGUA | |
PCPO8 | Pori clone PCPo8 | 469 | GGUGAUAAG-==================-CCAUAGUA | |
pori_SSIV | AY485286 | Uncultured Pori clone | 599 | ACAUUAGUC-==================-CUCAACCA |
AY485285 | Uncultured Pori clone | 599 | ACAUNAGUC-==================-CUCAACNA | |
AY485284 | Uncultured Pori clone | 599 | ACAUUAGUC-==================-CUCAACCA | |
AY485281 | Uncultured Pori bacterium | 599 | ACAUUAGUC-==================-CUCAACCA | |
A7 | A7 | 599 | AUAUUAGUC-==================-CUCAACCA | |
F2 | F2 | 599 | ACAUAAGUC-==================-CUCAACCA | |
L16 | L16 | 599 | ACAUUAGUC-==================-CUCAACCA | |
P20 | P20 | 599 | ACAUAAGUC-==================-CUCAACCA | |
P38 | P38 | 599 | ACAUUAGUC-==================-CUCAACCA | |
S6 | S6 | 599 | AUAUUAGUC-==================-CUCAACCA | |
S10 | S10 | 599 | AUAUUAGUC-==================-CUCAACCA | |
X18 | X18 | 599 | ACAUUAGUC-==================-CUCAACCA |
8.
The Norway spruce genome provides key insights into the evolution of plant genomes, leading to testable new hypotheses about conifer, gymnosperm, and vascular plant evolution.In the past year a burst of plant genome sequences have been published, providing enhanced phylogenetic coverage of green plants (Figure (Figure1)1) and inclusion of new agricultural, ecological, and evolutionary models. Collectively, these sequences are revealing some extraordinary structural and evolutionary attributes in plant genomes. Perhaps most surprising is the exceptionally high frequency of whole-genome duplication (WGD): nearly every genome that has been analyzed has borne the signature of one or more WGDs, with particularly notable events having occurred in the common ancestors of seed plants, of angiosperms, and of core eudicots (the latter ''WGD'' represents two WGDs in close succession) [1,2]. Given this tendency for plant genomes to duplicate and then return to an essentially diploid genetic system (an example is the cotton genomes, which have accumulated the effects of perhaps 15 WGDs [3]), the conservation of genomes in terms of gene number, chromosomal organization, and gene content is astonishing. From the publication of the first plant genome, Arabidopsis thaliana [4], the number of inferred genes has been between 25,000 and 30,000, with many gene families shared across all land plants, although the number of members and patterns of expansion and contraction vary. Furthermore, conserved synteny has been detected across the genomes of diverse angiosperms, despite WGDs, diploidization, and millions of years of evolution.Open in a separate windowFigure 1Simplified phylogeny of land plants, showing major clades and their component lineages. Asterisks indicate species (or lineage) for which whole-genome sequence (or sequences) is (are) available. Increases and decreases in genome size are shown by arrows.Despite the proliferation of genome sequences available for angiosperms, genome-level data for both ferns (and their relatives, collectively termed monilophytes; Figure Figure1)1) and gymnosperms have been conspicuously lacking - until recently, with the publication of the genome sequence of the gymnosperm Norway spruce (Picea abies) [5]. The large genome sizes for both monilophytes and gymnosperms have discouraged attempts at genome sequencing and assembly, whereas the smaller genome size of angiosperms has resulted in more genome sequences being available (Table (Table1)1) [6]. Because of this limited phylogenetic sample, our understanding of the timing and phylogenetic positions of WGDs, the core number of plant genes, possible conserved syntenic regions, and patterns of expansion and contraction of gene families across both tracheophytes (vascular plants) and across all land plants is imperfect. This sampling problem is particularly acute in analyses of the genes and genomes of seed plants; many hundreds of genes are present in angiosperms that are not present in mosses or lycophytes, but whether these genes arose in the common ancestor of seed plants or of angiosperms cannot be determined without a gymnosperm genome sequence. The Norway spruce genome therefore offers tremendous power, not only for understanding the structure and evolution of conifer genomes, but also as a reference for interpreting gene and genome evolution in angiosperms.
Open in a separate windown/a, not applicable. Data based on [6]. 相似文献
Table 1
Genome sizes in land plantsLineage | Range (1C; pg) | Mean |
---|---|---|
Gymnosperms | ||
Conifers | ||
Pinaceae | 9.5-36.0 | 23.7 |
Cupressaceae | 8.3-32.1 | 12.8 |
Sciadopitys | 20.8 | n/a |
Gnetales | ||
Ephedraceae | 8.9-15.7 | 8.9 |
Gnetaceae | 2.3-4.0 | 2.3 |
Cycadaceae | 12.6-14.8 | 13.4 |
Ginkgo biloba | 11.75 | n/a |
Monilophytes | ||
Ophioglossaceae | 10.2-65.6 | 31.05 |
Equisetaceae | 12.9-304 | 22.0 |
Psilotum | 72.7 | n/a |
Leptosporangiate ferns | ||
Polypodiaceae | 7.5-19.7 | 7.5 |
Aspleniaceae | 4.1-9.1 | 6.2 |
Athyriaceae | 6.3-9.3 | 7.6 |
Dryopteridaceae | 6.8-23.6 | 11.7 |
Water ferns | ||
Azolla | 0.77 | n/a |
Angiosperms | ||
Oryza sativa | 0.50 | n/a |
Amborella trichopoda | 0.89 | n/a |
Arabidopsis thaliana | 0.16 | n/a |
Zea mays | 2.73 | n/a |
9.
This study investigated the added value, i.e. discriminative and concurrent validity and reproducibility, of an eye-hand coordination test relevant to table tennis as part of talent identification. Forty-three table tennis players (7–12 years) from national (n = 13), regional (n = 11) and local training centres (n = 19) participated. During the eye-hand coordination test, children needed to throw a ball against a vertical positioned table tennis table with one hand and to catch the ball correctly with the other hand as frequently as possible in 30 seconds. Four different test versions were assessed varying the distance to the Total National Regional Local Total 43 13 11 19 Boys 26 8 8 10 Girls 17 5 3 9 Age (years) 10.4±1.4 10.9±1.5 10.4±1.5 10.1±1.4 7 year olds 1 - - 1 8 year olds 5 1 1 3 9 year olds 3 - 3 - 10 year olds 12 3 2 7 11 year olds 11 5 1 5 12 year olds 11 4 4 3 Length (cm) 149±11 150±12 150±12 148±10 Weight (kg ) 38±8 37±7 37±7 38±9 Right-handed 35 9 9 17 Left-handed 8 4 2 2 Training (hours*week-1) 6 (0–20) 11 (7–20) 7 (4–11) 2 (0–3) Competition (points) 173 (−52–430) 297 (144–430) 188 (72–317) 36 (−52–130)