The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region

capsular (CPS) polysaccharide, LPS biosynthesis genes, V. cholerae, surface polysaccharide, biogenesisBackground: In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. Results: The structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified. Conclusion: The co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations.Page 1 of 15 (page number not for citation purposes) BMC Microbiology Research article The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region Yuansha Chen1, Peter Bystricky2,6, Jacob Adeyeye2,7, Pinaki Panigrahi3, Afsar Ali1, Judith A Johnson4,5, CA Bush2, JG Morris Jr1 and OC Stine*1 Address: 1Department of Epidemiology and Preventive Medicine, University of Maryland, Baltimore, Baltimore, MD 21201, USA, 2Department of Chemistry and Biochemistry, University of Maryland, Baltimore County Baltimore, MD 21250, USA, 3Department of Pediatrics, University of Maryland, Baltimore, Baltimore, MD 21201, USA, 4Department of Pathology, University of Maryland, Baltimore, Baltimore, MD 21201, USA, 5Clinical Microbiology, Veterans Affairs Maryland Health Care System, Baltimore, MD 21201, USA, 6Slovak Medical University; Bratislava, SK- 83303, Slovakia and 7Department of Natural Sciences, Coppin State College, Baltimore, MD 21216, USA Email: Yuansha Chen - ychen@epi.umaryland.edu; Peter Bystricky - peter.bystricky@szu.sk; Jacob Adeyeye - jadeyeye@coppin.edu; Pinaki Panigrahi - ppanigrahi@peds.umaryland.edu; Afsar Ali - aali@epi.umaryland.edu; Judith A Johnson - Judy.Johnson2@va.gov; CA Bush - bush@chem.umbc.edu; JG Morris - Jmorris@epi.umaryland.edu; OC Stine* - ostin001@umaryland.edu * Corresponding author Abstract Background: In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. Results: The structure of the capsular (CPS) polysaccharide was determined by high resolution NMR spectroscopy and shown to be a complex structure with four residues in the repeating subunit. The gene cluster of capsule biogenesis was identified by transposon mutagenesis combined with whole genome sequencing data (GenBank accession DQ915177). The capsule gene cluster shared the same genetic locus as that of the O-antigen of lipopolysaccharide (LPS) biogenesis gene cluster. Other than V. cholerae O139, this is the first V. cholerae CPS for which a structure has been fully elucidated and the genetic locus responsible for biosynthesis identified. Conclusion: The co-location of CPS and LPS biosynthesis genes was unexpected, and would provide a mechanism for simultaneous emergence of new O and K antigens in a single strain. This, in turn, may be a key element for V. cholerae to evolve new strains that can escape immunologic detection by host populations. Background Vibrio cholerae has three forms of surface polysaccharide, although some strains do not express all three forms: a lipopolysaccharide (LPS) inserted in the outer membrane, a capsule composed of high molecular weight polysaccharide that forms a dense thick coat outside of the bacterial cells, and a loose slime-like exopolysaccharide. Unlike V. cholerae of serogroup O1, which causes cholera, most non- Published: 15 March 2007 BMC Microbiology 2007, 7:20 doi:10.1186/1471-2180-7-20 Received: 30 August 2006 Accepted: 15 March 2007 This article is available from: http://www.biomedcentral.com/1471-2180/7/20 © 2007 Chen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Microbiology 2007, 7:20 http://www.biomedcentral.com/1471-2180/7/20 Page 2 of 15 (page number not for citation purposes) O1 isolates have capsular polysaccharide (CPS) in addition to LPS. The LPS of V. cholerae is a protective antigen for cholera [1,2], with over 200 serogroups identified based on the O-antigen of the LPS. The O-antigen biogenesis loci of 4 serogroups (O1, O139. O22, O37) have been sequenced and characterized, and have been found to reside between two genes, gmhD and rjg, in the genome [3-7]. More than 85% of non-O1 V. cholerae isolates have a capsule that is critical for virulence in extraintestinal infections [8]. However, in contrast to E. coli, in which extensive work has been done on capsule structure and genetics (with associated classification into groups by Whitfield and Roberts [9]), structures and the genetics of CPS in V. cholerae are poorly understood. The one strain for which data on capsule structure and genetics are available is the newly emerged epidemic strain V. cholerae O139. This strain has a capsule that appears to have arisen from the replacement of the O1 antigen biosynthetic region with a new gene cluster in the genetic background of an O1 strain [5,6], resulting in emergence of a strain to which the human population did not yet have immunity. The capsule in O139 is unusual in that it shares the same repeating subunit as the O-antigen [10-12]. Therefore, the polysaccharide in O139 appears as both capsule and LPS and resembles the KLPS in the group 4 E. coli capsule [9]. There are limited studies on the genetics of polysaccharide biogenesis for the genus Vibrio. In V. vulnificus the CPS is a primary virulence factor and hence has been the target of more intensive study [13]. An operon including genes wza, wzb and wzc was identified as part of the CPS genes for V. vulnificus strain M06-24 [14,15], consistent with the presence of a group 1 capsule. The genetic loci for CPS were also identified in another strain of V. vulnificus 1003 [16]. A wzx/wzy system was present for polymerization and exporting the CPS. However, the genetic region responsible for LPS biosynthesis has not been identified in V. vulnificus. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. It also has clear implications for understanding the behavior of this species within human populations, as the ability to change these surface antigens to avoid host immunologic detection is a key feature underlying the ability of V. cholerare to survive. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S. Results and discussion Structure of the CPS Gas chromatography Carbohydrate analysis was done by gas chromatography of the trimethyl silyl methyl glycosides and the absolute configurations were determined by gas chromatography of the + and - 2-butyl glycosides as the trimethyl silyl derivatives. Both experiments were performed at the Complex Carbohydrate Research Center (CCRC) at the University of Georgia. The results indicated L-rhamnose (Rha), D-glucosamine (GlcNAc), D-glucuronic acid (GlcA) and D-galactose (Gal). The results of methylation analysis, also performed at CCRC, are given in Table 1. These results indicate that the major components of the hydrolyzed sample of methylated CPS sample represent 3- linked L-rhamnose, 4-linked D-glucosamine, 4- and 6- linked D-glucosamine and galactose in various linkages including 3-linked, 4-linked and 3,4 -linked. The D-glucuronic acid residue cannot be detected by this analysis because the uronic acids are converted to a sodium salt in the standard protocol [17]. Nuclear magnetic resonance (NMR) results NMR spectra of the native polysaccharide are complex, showing a number of peaks in the anomeric region that are not in simple stoichiometric ratios. Likewise, the acetyl methyl region (2.0 – 2.3 ppm) shows approximately 10 peaks not in simple ratios suggesting that the polysaccharide may be heterogeneously substituted with O-acetyl functions. Therefore the sample was treated with aqueous ammonium hydroxide, which is expected to cleave O-acetyl groups by mild base catalyzed hydrolysis. The NMR spectra of the resulting sample (de-O-acetyl polysaccharide) showed only two peaks in the acetyl methyl region (2.06 and 2.09 ppm) and a greatly simplified pattern in the anomeric region with four distinct signals in the C-H HSQC (heteronuclear single quantum Table 1: Results of methylation analysis. Glycosyl residue Percentage present 3-linked Rha 25 terminal Gal 4 3,4-linked Rha 2 3-linked Gal 13 4-linked Gal 8 4-linked Glc 3 2- and 4-linked Gal 3 3- and 4-linked Gal 4 3- and 6-linked Gal 4 4- and 6-linked Gal 1 Terminal GlcNAc 2 4-linked GlcNAc 17 4- and 6-linked GlcNAc 14 BMC Microbiology 2007, 7:20 http://www.biomedcentral.com/1471-2180/7/20 Page 3 of 15 (page number not for citation purposes) correlation) spectrum. We show below that the peaks at 2.06 and 2.09 arise from N-acetyl groups. The HSQC spectrum (Figure 1A&1B) indicates four sugar residues in the repeating subunit of the polysaccharide and the four signals were arbitrarily assigned identifying letters, A, B, C and D for the purpose of individual sugar identification using homonuclear 1H spin correlation. Experiments used to identify the sugar ring spin systems included COSY (correlation spectroscopy), TOCSY (total correlation spectroscopy), HMBC (heteronuclear multiple bond coherence) and NOESY (nuclear Overhauser spectroscopy). Residue C is identified as rhamnose by the characteristic methyl resonance of the 6-deoxy sugar at 1.33 ppm in combination with the equatorial configuration of H2 indicated by its small homonuclear coupling constants. The anomeric configuration is identified as β – by large NOESY peaks between H1, H3 and H5 as well as by 1JCH = 162 Hz. Residues B and D are identified as amino sugars by the characteristic chemical shift of C2 and as glucosamine by homonuclear coupling values. Residue B has the α-anomeric configuration as indicated by small JH1-H2 and by 1JCH = 172 Hz while residue D has the β – configuration as indicated by large JH1-H2 and by 1JCH = 158 Hz. The fourth residue, A, is identified as α-glucuronic acid. 1JCH for the anomeric signal is 168 Hz. and JH1-H2 is small, but the coupling constants of H3, H4 and H5 are all large as expected for the gluco configuration. No crosspeaks can be detected for H6 but an HMBC spectrum selective for the carbonyl region shows crosspeaks between both H4 and H5 and a carbonyl resonance at 176.7 ppm consistent with α-glucuronic acid. The central part and the anomeric region of the HSQC spectrum of the de-O-acetyl polysaccharide are shown in Figure 1A&1B and the complete 1H and 13C resonance assignments are given in Table 2. The glycosidic linkages between the four residues were determined from HMBC and NOESY data as indicated in Table 3. While no HMBC peaks could be observed for the D-B linkage, the nuclear Overhauser data clearly indicate a β-1–6 linkage and the downfield chemical shift of B-C6 at 68.9 ppm confirms this linkage assignment. The proposed structure of the tetrasaccharide repeating unit is given in Figure 2. Having determined the structure of the sugar backbone, we turned to the acetylated forms of the polysaccharide. While interpretation of their complex spectra was made difficult by heterogeneity, it was possible given the basic sugar structure. Base hydrolysis milder than that required to produce the de-O-acetyl polysaccharide yielded a sample with NMR spectra having peaks in stoichiometric ratios (mono-O-acetyl polysaccharide). The single Oacetyl group is assigned by carbonyl-selective HMBC to the 2-position of rhamnose (C), a position resistant to base hydrolysis due to the absence of a neighboring hydroxyl function. A complete set of homonuclear and heteronuclear NMR spectra of a sample of this form of the polysaccharide in H2O solution allowed assignment of signals of the amide protons of residues B and D confirming that they are N-acetyl amino sugars. The complete assignment of the NMR spectrum of the mono-O-acetyl polysaccharide is given in Table 4. The acetate methyl region of the NMR spectrum of the native, untreated, polysaccharide shows a number of peaks including those assignable to the amides of residue B and D and of the 2-O-acetyl group of residue C along with smaller peaks indicating partial O-acetylation at other positions. Using HMBC spectra, it was possible to correlate methyl proton signals, through carbonyl carbon resonances, to sugar ring protons indicating positions of acetyl substitution. In addition to the 2-O-acetyl of residue C, it was possible to identify an O-acetyl group on the 3-position of residue B to the extent of about 50% as indicated in Table 4. Anomeric resonances of residue A were split into three peaks in the native polysaccharide suggesting partial O-acetylation of that residue but the exact positions could not be definitively assigned. Although the chromatographic data indicate that the sample contains galactose, it is not part of the capsular polysaccharide. The NMR data of Figure 1 are consistent with four residues per repeating subunit and no sign of any galactose. Although the NMR spectra show minor peaks at approximately 10% level, none are characteristic of carbohydrates and they show no connection to the peaks assigned for the polysaccharide structure by NOESY or HMBC spectra. The structure proposed here for the NRT36S capsule repeating subunit is very similar to that reported for the V. cholerae O6 lipopolysaccharide [18]. The polysaccharide backbones are identical differing only in the degrees of Oacetylation. Bergstrom et al reported stoichiometric Oacetylation at both C2 of rhamnose and at C3 of α-Glc- NAc while our structure for native capsular polysaccharide of V. cholerae NRT36S is only partially substituted at C3 of α-GlcNAc along with lower degrees of acetylation at other positions. In spite of these differences, the NMR data reported by Bergstrom et al [18] are quite close to those reported for our native structure in Table 4. Transposon mutagenesis and mutant selection The conjugations between wild type Vibrio cholerae NRT36S and donor strain E. coli S17λpir/putKm-2 generated 20,615 mutants of NRT36S, each carrying a single copy of the transposon Km-2 in its genome. Among these mutants, 411 colonies displayed a translucent phenotype on LB agar. This phenotype suggests that genes involved in BMC Microbiology 2007, 7:20 http://www.biomedcentral.com/1471-2180/7/20 Page 4 of 15 (page number not for citation purposes) FPaignuelr Ae 1shows the HSQC spectrum of the de-O-acetylated capsular polysaccharide from NRT36S Panel A shows the HSQC spectrum of the de-O-acetylated capsular polysaccharide from NRT36S. The strong signal at 3.78, 60 ppm is a low molecular weight impurity. Panel B shows the signal from the anomeric region. The methyl group region is not shown. A. B. BMC Microbiology 2007, 7:20 http://www.biomedcentral.com/1471-2180/7/20 Page 5 of 15 (page number not for citation purposes) capsule biogenesis have been disrupted by the transposon [19]. DNA analysis of mutants Genomic DNA was isolated from the translucent mutants and analyzed by inverted polymerase chain reaction (PCR) and sequencing, identifying 13 unique insertion sites in 11 genes. Since NRT36S can also undergo spontaneous phase variation between transluscent and opaque colony morphologies, isolates with the 13 insertions were tested for complement resistance [19]. Nine insertions in eight genes were sensitive to serum killing and showed no reversion to the opaque morphology (Table 5). Isolates with insertions in the other 3 genes reverted to opaque colonies and were resistant to serum killing and therefore were excluded from further analysis. Only four of the stable mutant genes related to sugar modification and processing and were considered as putative structural genes for the biogenesis of the NRT36S capsule. The function of the other genes was unclear. One of the putative structural genes had a homolog in the fully sequenced genome of V. cholerae N16961, a serogroup O1 pandemic strain. Orf23, a homolog of VC0262, an UDP-glucose 4- epimerase (galE) was disrupted in translucent colony TR3. TR3 was restored to opaque phenotype and resistant to serum killing when complemented with gal E gene. The other three structural genes identified by transposon mutagenesis did not have homologs in the genome of V. cholerae N16961. In translucent colony TR17, a glycosyltransferase gene was disrupted and a rhamnosyltransferase gene was disrupted in both TR43 and TR287. An ABC transporter system integral membrane protein gene wzm was disrupted in TR296. The VC0262 homolog and the 3 other genes are typical of genes commonly found in polysaccharide biogenesis. Immuno blotting and size exclusion chromatography (SEC) SEC data indicated that the molecular weight of the NRT36S capsule is greater than 670k Dalton (Figure 3A&3B). The antiserum raised against the whole cell of V. cholerae NRT36S did not detect anything close to that molecular weight of CPS in the immuno blot (Figure 4), indicating that the antibodies did not react with the capsule. This result was consistent with the previous finding [20]. Nevertheless, the antibodies detected some polysaccharides that formed a ladder pattern in the molecular weight range of 20k to 40k Dalton (Figure 4). We believed these were the LPS. Interestingly, the amount of reactive LPS to the antibody was reduced in mutants TR3, TR17, and TR296. Analysis of the capsule preps by SEC from the mutants showed that three of the mutants, TR3, Tr287 and TR43 had completely lost the high molecular weight peaks corresponding to the capsule while in TR17 and TR296, the amount of capsule was significantly reduced (Figure 3B). Electron microscopy (EM) We evaluated thin sections of wild type V. cholerae NRT36S and several translucent mutants stained with polycationic ferritin by EM. Representative profiles are shown in Figure 5. As seen before ([19], NRT36S displayed a heavy, complete capsule surrounding the cell (Figure 5A). TR3 did not have a complete capsule, but had some patches of capsule materials (Figure 5B). Both of TR17 and TR296 had a much thinner capsule compared to Table 2: Complete assignment of the NMR spectra of the de-O-acetylated polysaccharide NRT36 (deOAc) ~50°C (321 K) H-1 H-2 H-3 H-4 H-5 H-6, H-6' C-1 C-2 C-3 C-4 C-5 C-6 A: α-D-GlcA 5.19 3.66 3.67 3.50 3.97 - 101.9 72.7 73.8 72.7 74.1 176.7 B: α-D-GlcNAc 5.01 3.93 4.05 3.64 4.16 4.09, 3.71 95.5 53.9 72.0 80.2 69.9 68.6 C: β-L-Rha 4.85 4.16 3.61 3.42 3.40 1.33 101.3 68.6 79.1 71.2 72.9 17.6 D: β-D-GlcNAc 4.42 3.78 3.67 3.67 3.49 3.83, 3.92 102.3 56.3 74.7 77.6 75.3 61.7 Table 3: Residue linkages for the capsular polysaccharide of V. cholerae NRT36S Linkage HMBC NOESY A-B AC1-BH4 AH1-BC4 B-C BH1-CC3 BH1-CH2 BH1-CH3 C-D CH1-DC4 CH1-DH4 D-B DH1-BH6 DH1-BH6' BMC Microbiology 2007, 7:20 http://www.biomedcentral.com/1471-2180/7/20 Page 6 of 15 (page number not for citation purposes) opaque NRT36S (Figure 5C&5D). EM pictures for all three mutants were consistent with the amounts of capsule observed by SEC (Figure 3B). Sequencing of the V. cholerae NRT36S genome V. cholerae NRT36S genome was sequenced by the company 454 Life Sciences. The sequencing runs have generated 1,082,967 reads and output 104,531,256 bases of sequence. The estimated coverage depth was 26X. The draft genome consisted of 184 contigs with total length of 3.9 million bases. The average GC (guanine-cytosine) content for the draft genome was 47.5%. The draft genome was annotated [21,22]. For the purpose of the discussion in this paper, only those features related to the polysaccharide biogenesis will be discussed. Genetics of the polysaccharide biogenesis O-antigen region In previous studies, the O-antigen biogenesis genes for V. cholerae had been identified to cluster at one locus in the genome, between genes gmhD and rjg [3,4,6,23]. After aligning the contigs of the draft genome of V. cholerae NRT36S to the fully sequenced genome of V. cholerae N16961 [24], we found that 3 contigs (contigs 34, 19, 78) of NRT 36S can partially align to the O-antigen region of N16961. Contig 34 contains gmhD gene and contig 78 co