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1
Joseph, Lavin A., and Anita C. Wright. "Expression of Vibrio vulnificus Capsular Polysaccharide Inhibits Biofilm Formation." Journal of Bacteriology 186, no. 3 (February 1, 2004): 889–93. http://dx.doi.org/10.1128/jb.186.3.889-893.2004.
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ABSTRACT Vibrio vulnificus is a human pathogen that produces lethal septicemia in susceptible persons, and the primary virulence factor for this organism is capsular polysaccharide (CPS). The role of the capsule in V. vulnificus biofilms was examined under a variety of conditions, by using either defined CPS mutants or spontaneous CPS expression phase variants derived from multiple strains. CPS expression was shown to inhibit attachment and biofilm formation, which contrasted with other studies describing polysaccharides as integral to biofilms in related species.
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Thurlow, Lance R., Vinai Chittezham Thomas, and Lynn E. Hancock. "Capsular Polysaccharide Production in Enterococcus faecalis and Contribution of CpsF to Capsule Serospecificity." Journal of Bacteriology 191, no. 20 (August 14, 2009): 6203–10. http://dx.doi.org/10.1128/jb.00592-09.
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ABSTRACT Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis.
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Trzcinski, Krzysztof, Claudette M. Thompson, and Marc Lipsitch. "Construction of Otherwise Isogenic Serotype 6B, 7F, 14, and 19F Capsular Variants of Streptococcus pneumoniae Strain TIGR4." Applied and Environmental Microbiology 69, no. 12 (December 2003): 7364–70. http://dx.doi.org/10.1128/aem.69.12.7364-7370.2003.
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ABSTRACT The polysaccharide capsule is the primary virulence factor in Streptococcus pneumoniae. There are at least 90 serotypes of S. pneumoniae, identified based on the immunogenicity of different capsular sugars. The aim of this study was to construct pneumococcal strains that are isogenic except for capsular type. Serotype 4 strain TIGR4 was rendered unencapsulated by recombinational replacement of the capsular polysaccharide synthesis (cps) locus with the bicistronic Janus cassette (C. K. Sung, J. P. Claverys, and D. A. Morrison, Appl. Environ. Microbiol. 67:5190-5196, 2001). In subsequent transformation with chromosomal DNA, the cassette was replaced by the cps locus derived from a strain of a different serotype, either 6B, 7F, 14, or 19F. To minimize the risk of uncontrolled recombinational replacements in loci other than cps, the TIGRcps::Janus strain was“ backcross” transformed three times with chromosomal DNA of subsequently constructed capsular type transformants. Capsular serotypes were confirmed in all new capsule variants by the Quellung reaction. Restriction fragment length polymorphism (RFLP) analysis of the cps locus confirmed the integrity of the cps region transformed into the TIGR strain, and RFLP of the flanking regions confirmed their identities with the corresponding regions of the recipient. Transformants had in vitro growth rates greater than or equal to that of TIGR4. All four strains were able to colonize C57BL/6 mice (female, 6 weeks old) for at least 7 days when mice were intranasally inoculated with 6 × 106 to 8 × 106 CFU. The constructed capsular variants of TIGR4 are suitable for use in studies on the role of S. pneumoniae capsular polysaccharide in immunity, colonization, and pathogenesis.
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Mavroidi, Angeliki, David M. Aanensen, Daniel Godoy, Ian C. Skovsted, Margit S. Kaltoft, Peter R. Reeves, Stephen D. Bentley, and Brian G. Spratt. "Genetic Relatedness of the Streptococcus pneumoniae Capsular Biosynthetic Loci." Journal of Bacteriology 189, no. 21 (August 31, 2007): 7841–55. http://dx.doi.org/10.1128/jb.00836-07.
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ABSTRACT Streptococcus pneumoniae (the pneumococcus) produces 1 of 91 capsular polysaccharides (CPS) that define the serotype. The cps loci of 88 pneumococcal serotypes whose CPS is synthesized by the Wzy-dependent pathway were compared with each other and with additional streptococcal polysaccharide biosynthetic loci and were clustered according to the proportion of shared homology groups (HGs), weighted for the sequence similarities between the genes encoding the shared HGs. The cps loci of the 88 pneumococcal serotypes were distributed into eight major clusters and 21 subclusters. All serotypes within the same serogroup fell into the same major cluster, but in six cases, serotypes within the same serogroup were in different subclusters and, conversely, nine subclusters included completely different serotypes. The closely related cps loci within a subcluster were compared to the known CPS structures to relate gene content to structure. The Streptococcus oralis and Streptococcus mitis polysaccharide biosynthetic loci clustered within the pneumococcal cps loci and were in a subcluster that also included the cps locus of pneumococcal serotype 21, whereas the Streptococcus agalactiae cps loci formed a single cluster that was not closely related to any of the pneumococcal cps clusters.
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Hsieh, Yu-Chi, Shu-Mei Liang, Wan-Ling Tsai, Yee-Hsiung Chen, Teh-Yung Liu, and Chi-Ming Liang. "Study of Capsular Polysaccharide from Vibrioparahaemolyticus." Infection and Immunity 71, no. 6 (June 2003): 3329–36. http://dx.doi.org/10.1128/iai.71.6.3329-3336.2003.
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ABSTRACT The leading cause of food poisoning in both Taiwan and Japan is Vibrio parahaemolyticus infection, whose mechanism of enteropathogenesis is still unclear. To evaluate whether surface components are responsible for the intestinal adhesion of V. parahaemolyticus, we have developed a novel method for isolating the capsular polysaccharide (CPS) from V. parahaemolyticus (serotype O4:K8). We found that culturing of V. parahaemolyticus in broth for 1 week or more changed the colony form of the bacteria on an agar plate from opaque to translucent. The translucent colonies of V. parahaemolyticus contained little CPS and exhibited a much lower level of adherence to epithelial cells (Int-407) than the opaque colonies of the bacteria. Incubation of V. parahaemolyticus in medium supplemented with bile increased the levels of CPS and adherence. Treatment of V. parahaemolyticus with anti-CPS but not anti-LPS serum decreased the level of bacterial adherence. In addition, purified CPS bound to epithelial cells in a dose-dependent manner. Intranasal administration of CPS to mice in the presence of adjuvants such as immunostimulatory sequence oligodeoxynucleotides or cholera toxin elicited CPS-specific mucosal and systemic immune responses. These results indicate that CPS plays an important role in the adherence of V. parahaemolyticus to its target cells and may be considered a potential target for the development of a vaccine against this pathogen.
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Gilbert, Christophe, Karen Robinson, Richard W. F. Le Page, and Jeremy M. Wells. "Heterologous Expression of an Immunogenic Pneumococcal Type 3 Capsular Polysaccharide in Lactococcus lactis." Infection and Immunity 68, no. 6 (June 1, 2000): 3251–60. http://dx.doi.org/10.1128/iai.68.6.3251-3260.2000.
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ABSTRACT In order to develop a new system for the analysis of capsular biosynthetic pathways we have explored the possibility of expressing type 3 capsular polysaccharide (CPS) from the pathogenStreptococcus pneumoniae in Lactococcus lactis, an unencapsulated lactic acid bacterium being developed as a vaccine delivery vehicle for mucosal immunization. Only three of the four type 3 CPS biosynthesis genes were found to be necessary for the abundant formation (120 mg liter−1) of an extracellular type 3 CPS in L. lactis, implying a role for the type 3-specific synthase in the extracellular transport of the CPS or implying the existence of an alternative export system in L. lactis. The authenticity of the expressed heterologous polysaccharide was established by chemical and immunological analyses. Proton and carbon nuclear magnetic resonance spectroscopy of CPSs purified from L. lactis and S. pneumoniae showed that the two CPS structures were identical. When mice were immunized intraperitoneally with 3.5 × 106 CFU of live recombinant lactococci expressing a total of approximately 0.5 μg of type 3 CPS, the immune responses elicited appeared identical to those observed in mice inoculated with 0.5 μg of type 3 CPS purified from S. pneumoniae. These findings show that L. lactis is a useful host in which to study the role and function of genes involved in the production of bacterial capsules. Additionally, L. lactis shows potential as a host for the safe production of capsule antigens and as a vaccine delivery vehicle for polysaccharide antigens.
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Wright, Anita C., Jan L. Powell, Mike K. Tanner, Lynne A. Ensor, Arthur B. Karpas, J. Glenn Morris, and Marcelo B. Sztein. "Differential Expression of Vibrio vulnificus Capsular Polysaccharide." Infection and Immunity 67, no. 5 (May 1, 1999): 2250–57. http://dx.doi.org/10.1128/iai.67.5.2250-2257.1999.
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ABSTRACT Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificusM06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression (P = 0.026) was observed for cells grown at 30°C than for those at 37°C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.
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Van Calsteren, Marie-Rose, Fleur Gagnon, Cynthia Calzas, Guillaume Goyette-Desjardins, Masatoshi Okura, Daisuke Takamatsu, Marcelo Gottschalk, and Mariela Segura. "Structure determination ofStreptococcus suisserotype 14 capsular polysaccharide." Biochemistry and Cell Biology 91, no. 2 (April 2013): 49–58. http://dx.doi.org/10.1139/bcb-2012-0036.
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The capsular polysaccharide (CPS) of Streptococcus suis serotype 14 was purified, chemically modified, and characterized. Sugar and absolute configuration analyses gave the following CPS composition: d-Gal, 3; d-Glc, 1; d-GlcNAc, 1; d-Neu5Ac, 1. The Sambucus nigra lectin, which recognizes the Neu5Ac(α2–6)Gal/GalNAc sequence, showed binding to the native CPS. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. It was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analyses,1H and13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [6)[Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)]Gal(β1–3)Gal(β1–4)Glc(β1–]n. S. suis serotype 14 CPS has an identical sialic acid-containing side chain as serotype 2 CPS, but differs by the absence of rhamnose in its composition. The same side chain is also present in group B Streptococcus type Ia CPS, except that in the latter sialic acid is 2,3- rather than 2,6-linked to the following galactose. A correlation between the S. suis CPS sequence and genes of the serotype 14 cps locus encoding putative glycosyltransferases and polymerase responsible for the biosynthesis of the repeating unit is proposed.
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Van Calsteren, Marie-Rose, Fleur Gagnon, Sonia Lacouture, Nahuel Fittipaldi, and Marcello Gottschalk. "Structure determination ofStreptococcus suisserotype 2 capsular polysaccharide." Biochemistry and Cell Biology 88, no. 3 (June 2010): 513–25. http://dx.doi.org/10.1139/o09-170.
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The capsular polysaccharide (CPS) of Streptococcus suis serotype 2 was isolated, purified, chemically modified, and characterized. Sugar and absolute configuration analyses of the CPS gave the following composition: d-Gal, 3; d-Glc, 1; d-GlcNAc, 1; d-Neu5Ac, 1; l-Rha, 1. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. The CPS was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analysis,1H and13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [4)[Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)]Gal(β1–4)[Gal(α1–3)]Rha(β1–4)Glc(β1-]n. The backbone sequence was found to be identical to that of Streptococcus agalactiae or group B Streptococcus (GBS) type VIII and Streptococcus pneumoniae type 23F. The S. suis CPS shares the sequence Neu5Ac-Gal-GlcNAc-Gal in common with GBS types Ia, Ib, II, III, and IV CPSs but differs from them by the presence of rhamnose and the fact that sialic acid is 2,6- rather than 2,3-linked to the following Gal. A correlation between the S. suis CPS sequence and genes of the serotype 2 cps locus encoding putative enzymes responsible for the biosynthesis of the repeating unit was tentatively established.
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Reckseidler-Zenteno, Shauna L., Duber-Frey Viteri, Richard Moore, Erica Wong, Apichai Tuanyok, and Donald E. Woods. "Characterization of the type III capsular polysaccharide produced by Burkholderia pseudomallei." Journal of Medical Microbiology 59, no. 12 (December 1, 2010): 1403–14. http://dx.doi.org/10.1099/jmm.0.022202-0.
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Burkholderia pseudomallei has been shown to produce more than one capsular polysaccharide (CPS). Analysis of the B. pseudomallei genome has revealed that the organism contains four CPS operons (I–IV). One of these operons (CPS III) was selected for further study. Comparative sequencing analysis revealed that the genes encoding CPS III are present in B. pseudomallei and Burkholderia thailandensis but not in Burkholderia mallei. In this study, CPS III was not found to contribute to the virulence of B. pseudomallei. Strains containing mutations in CPS III had the same LD50 value as the wild-type when tested in an animal infection model. Production of CPS III was shown to be induced in water but inhibited in 30 % normal human serum using a lux reporter fusion assay. Microarray analysis of capsule gene expression in infected hamsters revealed that the genes encoding CPS III were not significantly expressed in vivo compared with the genes encoding the previously characterized mannoheptose capsule (CPS I), which is an important virulence factor in B. pseudomallei. Glycosyl-composition analysis by combined GC/MS indicated that the CPS III genes are involved in the synthesis of a capsule composed of galactose, glucose, mannose and xylose.
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Schiano di Visconte, Gino, Michael J. Allen, and Andrew Spicer. "Novel Capsular Polysaccharide from Lobochlamys segnis." Polysaccharides 2, no. 1 (March 3, 2021): 121–37. http://dx.doi.org/10.3390/polysaccharides2010009.
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In recent years there has been a significant effort from food, nutraceutical, cosmeceutical, pharmaceutical, and biomedical industries to discover and develop new natural ingredients. Microalgae have been recognised as potential sources of high value chemicals, with most attention focused upon antioxidants, pigments, and specialty oils. An under-exploited group of biochemicals produced by microalgae are extracellular polymeric substances (EPS). Lobochlamys segnis (formerly called Chlamydomonas segnis) was previously reported to produce a large amount of capsular polysaccharide (CPS) closely related to hyaluronan (HA) under stress conditions. In this work, the purified CPS was characterised and shown to have an average molecular mass (Mn) of about 3.7 MDa, and displayed a highly branched random coil structure unlike the simple repeating linear HA polysaccharide. Chemical analysis showed the presence of galactose, glucuronic acid, and glucose sugars confirming that the product has a different composition to that of HA. Mixotrophic growth and stress conditions were identified and improved upon with a pH control system using acetic acid solution to induce efficient production of CPS. Extraction and purification conditions were also identified exploiting the high Mn of the product. The CPS showed thickening properties and both significant antioxidant capacity and reducing power, which could have commercial applications. This is the first report on the characterization of this novel polysaccharide with non-Newtonian properties similar to HA.
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Bertin, Clothilde, Corinne Pau-Roblot, Josiane Courtois, Lucía Manso-Silván, Florence Tardy, François Poumarat, Christine Citti, Pascal Sirand-Pugnet, Patrice Gaurivaud, and François Thiaucourt. "Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster." Applied and Environmental Microbiology 81, no. 2 (November 14, 2014): 676–87. http://dx.doi.org/10.1128/aem.02892-14.
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ABSTRACTMycoplasmas of theMycoplasma mycoidescluster are all ruminant pathogens.Mycoplasma mycoidessubsp.mycoidesis responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role forMycoplasma mycoidessubsp.mycoidespolysaccharides in pathogenicity.Mycoplasma mycoidessubsp.mycoides-secreted EPS was recently characterized as a β(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of theM. mycoidescluster:M. capricolumsubsp.capripneumoniae,M. capricolumsubsp.capricolum,M. leachii, andM. mycoidessubsp.capri(including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β(1→2)-glucopyranose (glucan) inM. capricolumsubsp.capripneumoniaeandM. leachii. Monoclonal antibodies specific for this glucan and for theMycoplasma mycoidessubsp.mycoides-secreted galactan were used to detect the two polysaccharides. WhileM. mycoidessubsp.capristrains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains ofM. capricolumsubsp.capripneumoniaeandM. leachiiproduced glucan CPS and EPS, whereas glucan production and localization varied amongM. capricolumsubsp.capricolumstrains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notablygalEandglf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides.
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Corcionivoschi, N., M. Clyne, A. Lyons, A. Elmi, O. Gundogdu, B. W. Wren, N. Dorrell, A. V. Karlyshev, and B. Bourke. "Campylobacter jejuni Cocultured with Epithelial Cells Reduces Surface Capsular Polysaccharide Expression." Infection and Immunity 77, no. 5 (March 9, 2009): 1959–67. http://dx.doi.org/10.1128/iai.01239-08.
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ABSTRACT The host cell environment can alter bacterial pathogenicity. We employed a combination of cellular and molecular techniques to study the expression of Campylobacter jejuni polysaccharides cocultured with HCT-8 epithelial cells. After two passages, the amount of membrane-bound high-molecular-weight polysaccharide was considerably reduced. Microarray profiling confirmed significant downregulation of capsular polysaccharide (CPS) locus genes. Experiments using conditioned media showed that sugar depletion occurred only when the bacterial and epithelial cells were cocultured. CPS depletion occurred when C. jejuni organisms were exposed to conditioned media from a different C. jejuni strain but not when exposed to conditioned media from other bacterial species. Proteinase K or heat treatment of conditioned media under coculture conditions abrogated the effect on the sugars, as did formaldehyde fixation and cycloheximide treatment of host cells or chloramphenicol treatment of the bacteria. However, sugar depletion was not affected in flagellar export (fliQ) and quorum-sensing (luxS) gene mutants. Passaged C. jejuni showed reduced invasiveness and increased serum sensitivity in vitro. C. jejuni alters its surface polysaccharides when cocultured with epithelial cells, suggesting the existence of a cross talk mechanism that modulates CPS expression during infection.
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Hilton, Tamara, Tom Rosche, Brett Froelich, Benjamin Smith, and James Oliver. "Capsular Polysaccharide Phase Variation in Vibrio vulnificus." Applied and Environmental Microbiology 72, no. 11 (August 25, 2006): 6986–93. http://dx.doi.org/10.1128/aem.00544-06.
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ABSTRACT Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.
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Nakhamchik, Alina, Caroline Wilde, and Dean A. Rowe-Magnus. "Identification of a Wzy Polymerase Required for Group IV Capsular Polysaccharide and Lipopolysaccharide Biosynthesis in Vibrio vulnificus." Infection and Immunity 75, no. 12 (October 8, 2007): 5550–58. http://dx.doi.org/10.1128/iai.00932-07.
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ABSTRACT The estuarine bacterium Vibrio vulnificus is a human and animal pathogen. The expression of capsular polysaccharide (CPS) is essential for virulence. We used a new mini-Tn10 delivery vector, pNKTXI-SceI, to generate a mutant library and identify genes essential for CPS biosynthesis. Twenty-one acapsular mutants were isolated, and the disrupted gene in one mutant, coding for a polysaccharide polymerase (wzy), is described here. A wecA gene initiating glycosyltransferase was among the genes identified in the region flanking the wzy gene. This, together with the known structure of the CPS, supports a group IV capsule designation for the locus; however, its overall organization mirrored that of group I capsules. This new arrangement may be linked to our finding that the CPS region appears to have been recently acquired by horizontal transfer. Alcian Blue staining and immunoblotting with antisera against the wild-type strain indicated that the wzy::Tn10 mutant failed to produce CPS and was attenuated relative to the wild type in a septicemic mouse model. Interestingly, immunoblotting revealed that the mutant was also defective in lipopolysaccharide (LPS) production. However, the core-plus-one O-antigen pattern typical of wzy mutations was apparent. CPS production, LPS production, and virulence were restored following complementation with the wild-type wzy gene. Hence, Wzy participates in both CPS and LPS biosynthesis and is required for virulence in strain 27562. To our knowledge, this is the first functional demonstration of a Wzy polysaccharide polymerase in V. vulnificus and is the first to show a link between LPS and CPS biosynthesis.
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Güvener, Zehra Tüzün, and Linda L. McCarter. "Multiple Regulators Control Capsular Polysaccharide Production in Vibrio parahaemolyticus." Journal of Bacteriology 185, no. 18 (September 15, 2003): 5431–41. http://dx.doi.org/10.1128/jb.185.18.5431-5441.2003.
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ABSTRACT Vibrio parahaemolyticus, a biofouling marine bacterium and human pathogen, undergoes phase variation displaying translucent (TR) and opaque (OP) colony morphologies. Prior studies demonstrated that OP colonies produce more capsular polysaccharide (CPS) than TR colonies and that opacity is controlled by the Vibrio harveyi LuxR-type transcriptional activator OpaR. CPS has also been shown to be regulated by the scrABC signaling pathway, which involves a GGDEF-EAL motif-containing sensory protein. The present study identifies cps genes and examines their regulation. Transposon insertions in the cps locus, which contains 11 genes, abolished opacity. Such mutants failed to produce CPS and were defective in pellicle formation in microtiter wells and in a biofilm attachment assay. Reporter fusions to cpsA, the first gene in the locus, showed ∼10-fold-enhanced transcription in the OP (opaR +) strain compared to a TR (ΔopaR) strain. Two additional transcriptional regulators were discovered. One potential activator, CpsR, participates in the scrABC GGDEF-EAL-signaling pathway; CpsR was required for the increased cps expression observed in scrA ΔopaR strains. CpsR, which contains a conserved module found in members of the AAA+ superfamily of ATP-interacting proteins, is homologous to Vibrio cholerae VpsR; however, unlike VpsR, CpsR was not essential for cps expression. CpsS, the second newly identified regulator, contains a CsgD-type DNA-binding domain and appears to act as a repressor. Mutants with cpsS defects have greatly elevated cps transcription; their high level of cpsA expression was CpsR dependent in ΤR strains and primarily OpaR dependent in OP strains. Thus, a network of positive and negative regulators modulates CPS production in V. parahaemolyticus.
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Wright, Anita C., Jan L. Powell, James B. Kaper, and J. Glenn Morris. "Identification of a Group 1-Like Capsular Polysaccharide Operon for Vibrio vulnificus." Infection and Immunity 69, no. 11 (November 1, 2001): 6893–901. http://dx.doi.org/10.1128/iai.69.11.6893-6901.2001.
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ABSTRACT Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V.vulnificus CPS locus, which included an upstreamops element, a wza gene (wza Vv), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wzagene product is required for transport of CPS to the cell surface inEscherichia coli. Polar transposon mutations inwza Vv eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza Vv was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions inwza Vv and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V.vulnificus.
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Kaszowska, Marta, Grazyna Majkowska-Skrobek, Pawel Markwitz, Cédric Lood, Wojciech Jachymek, Anna Maciejewska, Jolanta Lukasiewicz, and Zuzanna Drulis-Kawa. "The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae." International Journal of Molecular Sciences 22, no. 21 (October 26, 2021): 11562. http://dx.doi.org/10.3390/ijms222111562.
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Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.
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Shi, Fang, Tomoyuki Harada, Yohsuke Ogawa, Hiroshi Ono, Mayumi Ohnishi-Kameyama, Toru Miyamoto, Masahiro Eguchi, and Yoshihiro Shimoji. "Capsular Polysaccharide of Erysipelothrix rhusiopathiae, the Causative Agent of Swine Erysipelas, and Its Modification with Phosphorylcholine." Infection and Immunity 80, no. 11 (September 4, 2012): 3993–4003. http://dx.doi.org/10.1128/iai.00635-12.
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ABSTRACTThe capsule has been implicated in the virulence of the swine pathogenErysipelothrix rhusiopathiae, a rod-shaped, intracellular Gram-positive bacterium that has a unique phylogenetic position in the phylumFirmicutesand is a close relative ofMollicutes(mycoplasma species). In this study, we analyzed the genetic locus and composition of the capsular polysaccharide (CPS) of the Fujisawa strain ofE. rhusiopathiae. Genome analysis of the Fujisawa strain revealed that the genetic locus for capsular polysaccharide synthesis (cps) is located next to anlicoperon, which is involved in the incorporation and expression of phosphorylcholine (PCho). Reverse transcription-PCR analysis showed thatcpsandlicare transcribed as a single mRNA, indicating that the loci form an operon. Using the cell surface antigen-specific monoclonal antibody (MAb) ER21 as a probe, the capsular materials were isolated from the Fujisawa strain by hot water extraction and treatment with DNase, RNase, pronase, andN-acetylmuramidase SG, followed by anion-exchange and gel filtration chromatography. The materials were then analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The CPS ofE. rhusiopathiaeis heterogeneous and consists of the major monosaccharides galacturonic acid, galactose, mannose, glucose, arabinose, xylose, andN-acetylglucosamine and some minor monosaccharides containing ribose, rhamnose, andN-acetylgalactosamine. In addition, the capsule is modified by PCho, which comigrates with the capsular materials, as determined by Western immunoblotting, and colocalizes on the cell surface, as determined by immunogold electron microscopy. Virulence testing of PCho-defective mutants in mice demonstrated that PCho is critical for the virulence of this organism.
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V. Volozhantsev, Nikolay, Anna M. Shpirt, Alexander I. Borzilov, Ekaterina V. Komisarova, Valentina M. Krasilnikova, Alexander S. Shashkov, Vladimir V. Verevkin, and Yuriy A. Knirel. "Characterization and Therapeutic Potential of Bacteriophage-Encoded Polysaccharide Depolymerases with β Galactosidase Activity against Klebsiella pneumoniae K57 Capsular Type." Antibiotics 9, no. 11 (October 25, 2020): 732. http://dx.doi.org/10.3390/antibiotics9110732.
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Bacteriophages and phage enzymes are considered as possible alternatives to antibiotics in the treatment of infections caused by antibiotic-resistant bacteria. Due to the ability to cleave the capsular polysaccharides (CPS), one of the main virulence factors of Klebsiella pneumoniae, phage depolymerases, has potential in the treatment of K. pneumoniae infections. Here, we characterized in vivo two novel phage-encoded polysaccharide depolymerases as therapeutics against clinical isolates of K. pneumoniae. The depolymerases Dep_kpv79 and Dep_kpv767 encoded by Klebsiella phages KpV79 (Myoviridae; Jedunavirus) and KpV767 (Autographiviridae, Studiervirinae, Przondovirus), respectively, were identified as specific β-galactosidases that cleave the K. pneumoniae K57 type CPS by the hydrolytic mechanism. They were found to be highly effective at combating sepsis and hip infection caused by K. pneumoniae in lethal mouse models. Here, 80–100% of animals were protected against death by a single dose (e.g., 50 μg/mouse) of the enzyme injected 0.5 h after infection by K. pneumoniae strains of the K57 capsular type. The therapeutic effect of the depolymerases is because they strip the capsule and expose the underlying bacterium to the immune attack such as complement-mediated killing. These data provide one more confirmation that phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.
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Guirguis, N., R. Schneerson, A. Bax, W. Egan, J. B. Robbins, J. Shiloach, I. Orskov, F. Orskov, and A. el Kholy. "Escherichia coli K51 and K93 capsular polysaccharides are crossreactive with the group A capsular polysaccharide of Neisseria meningitidis. Immunochemical, biological, and epidemiological studies." Journal of Experimental Medicine 162, no. 6 (December 1, 1985): 1837–51. http://dx.doi.org/10.1084/jem.162.6.1837.
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Eleven Escherichia coli strains, crossreactive with the capsular polysaccharide (CPS) of Neisseria meningitidis group A (GrA), were detected among 645 stool isolates from healthy families in Cairo, Egypt. 10 of these strains were of the O107:K93:H27 or O107:K93:SP serotypes and may be considered descendents of a single bacterium or as a clone. The remaining crossreactive strain was of the O7:K51:H18 serotype. None of the 11 strains produced enterotoxins and none were enteroinvasive. The purified CPS of these E. coli strains, as well as a polysaccharide (PS) from B. pumilis, strain Sh17, precipitated with equine GrA (H49) antiserum. A partial identity between the E. coli K93, K51 and Sh17 PS on the one hand and the GrA CPS on the other was observed by double immunodiffusion when reacted against the H49 antiserum. Four K93 strains and one K51 strain were found among 320 E. coli strains from patients at the Clinical Center, National Institutes of Health, and three K93 strains were found in 105 stool samples from children in Copenhagen. The data from these three surveys suggest that these crossreactive E. coli are common organisms and could serve as a stimulus for "natural" GrA CPS antibodies. Quantitative precipitation analysis showed that K51, K93, and Sh17 PS precipitated 25, 46.8, and 50% of H49 antibodies, respectively. Absorption of H49 antiserum with the GrA CPS removed its precipitating activity with the E. coli K93, K51, and Sh17 PS. Absorption of H49 antiserum with either K51 CPS or Sh17 PS removed the homologous crossreactivity only, whereas K93 CPS absorbed both K93 and K51 reactivities. Antibodies, raised by intravenous injection of formalinized E. coli K93 or K51 cells into rabbits, precipitated with GrA CPS and were bactericidal against GrA meningococci. The crossreaction between the E. coli K93 and the GrA CPS was unexpected since these two CPS are compositionally so dissimilar.
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Mathews, Christine E., Eric L. Brown, Perla J. Martinez, Upasana Bagaria, Moon H. Nahm, Robert L. Burton, Susan P. Fisher-Hoch, Joseph B. McCormick, and Shaper Mirza. "Impaired Function of Antibodies to Pneumococcal Surface Protein A but Not to Capsular Polysaccharide in Mexican American Adults with Type 2 Diabetes Mellitus." Clinical and Vaccine Immunology 19, no. 9 (July 3, 2012): 1360–69. http://dx.doi.org/10.1128/cvi.00268-12.
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ABSTRACTThe goal of the study was to determine baseline protective titers of antibodies toStreptococcus pneumoniaesurface protein A (PspA) and capsular polysaccharide in individuals with and individuals without type 2 diabetes mellitus. A total of 561 individuals (131 individuals with diabetes and 491 without) were screened for antibodies to PspA using a standard enzyme-linked immunosorbent assay (ELISA). A subset of participants with antibodies to PspA were retested using a WHO ELISA to determine titers of antibodies to capsular polysaccharide (CPS) (serotypes 4, 6B, 9V, 14, 18C, 19A, 19F, and 23F). Functional activity of antibodies was measured by assessing their ability to enhance complement (C3) deposition on pneumococci and promote killing of opsonized pneumococci. Titers of antibodies to protein antigens (PspA) were significantly lower in individuals with diabetes than controls without diabetes (P= 0.01), and antibodies showed a significantly reduced complement deposition ability (P= 0.02). Both antibody titers and complement deposition were negatively associated with hyperglycemia. Conversely, titers of antibodies to capsular polysaccharides were either comparable between the two groups or were significantly higher in individuals with diabetes, as was observed for CPS 14 (P= 0.05). The plasma specimens from individuals with diabetes also demonstrated a higher opsonophagocytic index against CPS serotype 14. Although we demonstrate comparable protective titers of antibodies to CPS in individuals with and individuals without diabetes, those with diabetes had lower PspA titers and poor opsonic activity strongly associated with hyperglycemia. These results suggest a link between diabetes and impairment of antibody response.
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Shu, Hung-Yu, Chang-Phone Fung, Yen-Ming Liu, Keh-Ming Wu, Ying-Tsong Chen, Ling-Hui Li, Tze-Tze Liu, Ralph Kirby, and Shih-Feng Tsai. "Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates." Microbiology 155, no. 12 (December 1, 2009): 4170–83. http://dx.doi.org/10.1099/mic.0.029017-0.
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Klebsiella pneumoniae is an enteric pathogen causing community-acquired and hospital-acquired infections in humans. Epidemiological studies have revealed significant diversity in capsular polysaccharide (CPS) type and clinical manifestation of K. pneumoniae infection in different geographical areas of the world. We have sequenced the capsular polysaccharide synthesis (cps) region of seven clinical isolates and compared the sequences with the publicly available cps sequence data of five strains: NTUH-K2044 (K1 serotype), Chedid (K2 serotype), MGH78578 (K52 serotype), A1142 (K57 serotype) and A1517. Among all strains, six genes at the 5′ end of the cps clusters that encode proteins for CPS transportation and processing at the bacterial surface are highly similar to each other. The central region of the cps gene clusters, which encodes proteins for polymerization and assembly of the CPS subunits, is highly divergent. Based on the collected sequence, we found that either the wbaP gene or the wcaJ gene exists in a given K. pneumoniae strain, suggesting that there is a major difference in the CPS biosynthesis pathway and that the K. pneumoniae strains can be classified into at least two distinct groups. All isolates contain gnd, encoding gluconate-6-phosphate dehydrogenase, at the 3′ end of the cps gene clusters. The rmlBADC genes were found in CPS K9-positive, K14-positive and K52-positive strains, while manC and manB were found in K1, K2, K5, K14, K62 and two undefined strains. Our data indicate that, while overall genomic organization is similar between different pathogenic K. pneumoniae strains, the genetic variation of the sugar moiety and polysaccharide linkage generate the diversity in CPS molecules that could help evade host immune attack.
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To, Ho, Kaho Teshima, Michiha Kon, Saori Yasuda, Yuta Akaike, Kazumoto Shibuya, Shinya Nagai, and Chihiro Sasakawa. "Characterization of nontypeable Actinobacillus pleuropneumoniae isolates." Journal of Veterinary Diagnostic Investigation 32, no. 4 (June 9, 2020): 581–84. http://dx.doi.org/10.1177/1040638720931469.
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Two Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia in Japan were positive in the capsular serovar 15–specific PCR assay, but nontypeable (NT) in the agar gel precipitation (AGP) test. Nucleotide sequence analysis of gene clusters involved in the biosynthesis of capsular polysaccharide (CPS) and lipopolysaccharide O-polysaccharide (O-PS) revealed that both clusters contained transposable element IS Apl1 of A. pleuropneumoniae belonging to the IS30 family. Immunoblot analysis revealed that these 2 isolates could not produce O-PS. We conclude that the IS Apl1 of A. pleuropneumoniae can interfere in the biosynthesis of both CPS and O-PS.
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Lin, Tien-Huang, Su-Hua Huang, Chien-Chen Wu, Hsin-Ho Liu, Tzyy-Rong Jinn, Yeh Chen, and Ching-Ting Lin. "Inhibition ofKlebsiella pneumoniaeGrowth and Capsular Polysaccharide Biosynthesis byFructus mume." Evidence-Based Complementary and Alternative Medicine 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/621701.
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Klebsiella pneumoniaeis the predominant pathogen isolated from liver abscess of diabetic patients in Asian countries. With the spread of multiple-drug-resistantK. pneumoniae, there is an increasing need for the development of alternative bactericides and approaches to block the production of bacterial virulence factors. Capsular polysaccharide (CPS), especially from the K1 and K2 serotypes, is considered the major determinant forK. pneumoniaevirulence. We found that extracts of the traditional Chinese medicineFructus mumeinhibited the growth ofK. pneumoniaestrains of both serotypes. Furthermore,Fructus mumedecreased the mucoviscosity, and the CPS produced in a dose-dependent manner, thus reducing bacterial resistance to serum killing. Quantitative reverse transcription polymerase chain reaction analyses showed thatFructus mumedownregulated the mRNA levels ofcpsbiosynthesis genes in both serotypes, possibly by increasing the intracellular iron concentration inK. pneumoniae. Moreover, citric acid, a major organic acid inFructus mumeextracts, was found to have an inhibitory effect on growth and CPS biosynthesis inK. pneumoniae. Taken together, our results indicate thatFructus mumenot only possesses antibacterial activity against highly virulentK. pneumoniaestrains but also inhibits bacterial CPS biosynthesis, thereby facilitating pathogen clearance by the host immune system.
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Nahm, Moon H., Terry Brissac, Mogens Kilian, Jiri Vlach, Carlos J. Orihuela, Jamil S. Saad, and Feroze Ganaie. "Pneumococci Can Become Virulent by Acquiring a New Capsule From Oral Streptococci." Journal of Infectious Diseases 222, no. 3 (September 7, 2019): 372–80. http://dx.doi.org/10.1093/infdis/jiz456.
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Abstract Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model.
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Troxler, Lukas J., Joel P. Werren, Thierry O. Schaffner, Nadezda Mostacci, Peter Vermathen, Martina Vermathen, Daniel Wüthrich, et al. "Carbon source regulates polysaccharide capsule biosynthesis in Streptococcus pneumoniae." Journal of Biological Chemistry 294, no. 46 (October 8, 2019): 17224–38. http://dx.doi.org/10.1074/jbc.ra119.010764.
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The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is <10% that of glucose. Our findings on the effects of carbon sources on CPS production in different S. pneumoniae serotypes may contribute to a better understanding of pneumococcal diseases and could inform future therapeutic approaches.
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Chatzidaki-Livanis, Maria, Melissa K. Jones, and Anita C. Wright. "Genetic Variation in the Vibrio vulnificus Group 1 Capsular Polysaccharide Operon." Journal of Bacteriology 188, no. 5 (March 1, 2006): 1987–98. http://dx.doi.org/10.1128/jb.188.5.1987-1998.2006.
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ABSTRACT Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show “phase variation” to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.
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Pettis, Gregg S., and Aheli S. Mukerji. "Structure, Function, and Regulation of the Essential Virulence Factor Capsular Polysaccharide of Vibrio vulnificus." International Journal of Molecular Sciences 21, no. 9 (May 5, 2020): 3259. http://dx.doi.org/10.3390/ijms21093259.
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Vibrio vulnificus populates coastal waters around the world, where it exists freely or becomes concentrated in filter feeding mollusks. It also causes rapid and life-threatening sepsis and wound infections in humans. Of its many virulence factors, it is the V. vulnificus capsule, composed of capsular polysaccharide (CPS), that plays a critical role in evasion of the host innate immune system by conferring antiphagocytic ability and resistance to complement-mediated killing. CPS may also provoke a portion of the host inflammatory cytokine response to this bacterium. CPS production is biochemically and genetically diverse among strains of V. vulnificus, and the carbohydrate diversity of CPS is likely affected by horizontal gene transfer events that result in new combinations of biosynthetic genes. Phase variation between virulent encapsulated opaque colonial variants and attenuated translucent colonial variants, which have little or no CPS, is a common phenotype among strains of this species. One mechanism for generating acapsular variants likely involves homologous recombination between repeat sequences flanking the wzb phosphatase gene within the Group 1 CPS biosynthetic and transport operon. A considerable number of environmental, genetic, and regulatory factors have now been identified that affect CPS gene expression and CPS production in this pathogen.
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Hufnagel, Markus, Andrea Kropec, Christian Theilacker, and Johannes Huebner. "Naturally Acquired Antibodies against Four Enterococcus faecalis Capsular Polysaccharides in Healthy Human Sera." Clinical Diagnostic Laboratory Immunology 12, no. 8 (August 2005): 930–34. http://dx.doi.org/10.1128/cdli.12.8.930-934.2005.
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ABSTRACT Healthy human sera (HHS) contain naturally acquired enterococcal antibodies which promote neutrophil-mediated killing. The target antigens remain unknown. The present study used a capsular polysaccharide (CPS)-enzyme-linked immunosorbent assay (ELISA) to investigate whether the HHS antibodies of 12 healthy donors bound to the CPS of four E. faecalis serotypes (CPS-A to CPS-D) and then employed an opsonic-killing assay to determine if these antibodies mediated phagocyte-dependent killing. All HHS contained immunoglobulin G (IgG) and IgM antibodies directed against capsular polysaccharides of the four serotypes. Absorption of the sera with homologous and heterologous strains showed a majority of antibodies to be cross-reactive among the prototype strains. The susceptibility of the four prototype strains to opsonic killing varied. Opsonic killing of CPS-A and CPS-B strains was significantly higher than killing of CPS-C and CPS-D strains. Absorption studies revealed that the opsonic killing of HHS was only partially type specific, with cross-reactivity between CPS-A and CPS-B strains and between CPS-C and CPS-D strains. These data indicate that healthy individuals possess opsonic antibodies specific for CPS-A and CPS-B but only low titers of opsonic antibodies against CPS-C and CPS-D. Titers of opsonic antibodies did not correlate with antibody titers measured by ELISA. Whether this lack of correlation is due to the low frequency of opsonic antibodies or to increased resistance to the opsonophagocytic killing of some serotypes remains to be determined.
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Shchurova, Anastasia S., Mikhail M. Shneider, Nikolay P. Arbatsky, Alexander S. Shashkov, Alexander O. Chizhov, Yuriy P. Skryabin, Yulia V. Mikhaylova, et al. "Novel Acinetobacter baumannii Myovirus TaPaz Encoding Two Tailspike Depolymerases: Characterization and Host-Recognition Strategy." Viruses 13, no. 6 (May 25, 2021): 978. http://dx.doi.org/10.3390/v13060978.
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Acinetobacter baumannii, one of the most significant nosocomial pathogens, is capable of producing structurally diverse capsular polysaccharides (CPSs) which are the primary receptors for A. baumannii bacteriophages encoding polysaccharide-degrading enzymes. To date, bacterial viruses specifically infecting A. baumannii strains belonging to more than ten various capsular types (K types) were isolated and characterized. In the present study, we investigate the biological properties, genomic organization, and virus–bacterial host interaction strategy of novel myovirus TaPaz isolated on the bacterial lawn of A. baumannii strain with a K47 capsular polysaccharide structure. The phage linear double-stranded DNA genome of 93,703 bp contains 178 open reading frames. Genes encoding two different tailspike depolymerases (TSDs) were identified in the phage genome. Recombinant TSDs were purified and tested against the collection of A. baumannii strains belonging to 56 different K types. One of the TSDs was demonstrated to be a specific glycosidase that cleaves the K47 CPS by the hydrolytic mechanism.
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Cheng, H. Y., Y. S. Chen, C. Y. Wu, H. Y. Chang, Y. C. Lai, and H. L. Peng. "RmpA Regulation of Capsular Polysaccharide Biosynthesis in Klebsiella pneumoniae CG43." Journal of Bacteriology 192, no. 12 (April 9, 2010): 3144–58. http://dx.doi.org/10.1128/jb.00031-10.
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ABSTRACT Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5′ noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur.
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Wen, Zhensong, Odeniel Sertil, Yongxin Cheng, Shanshan Zhang, Xue Liu, Wen-Ching Wang, and Jing-Ren Zhang. "Sequence Elements Upstream of the Core Promoter Are Necessary for Full Transcription of the Capsule Gene Operon in Streptococcus pneumoniae Strain D39." Infection and Immunity 83, no. 5 (March 2, 2015): 1957–72. http://dx.doi.org/10.1128/iai.02944-14.
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Streptococcus pneumoniaeis a major bacterial pathogen in humans. Its polysaccharide capsule is a key virulence factor that promotes bacterial evasion of human phagocytic killing. WhileS. pneumoniaeproduces at least 94 antigenically different types of capsule, the genes for biosynthesis of almost all capsular types are arranged in the same locus. The transcription of the capsular polysaccharide (cps) locus is not well understood. This study determined the transcriptional features of thecpslocus in the type 2 virulent strain D39. The initial analysis revealed that thecpsgenes are cotranscribed from a major transcription start site at the −25 nucleotide (G) upstream ofcps2A, the first gene in the locus. Using unmarked chromosomal truncations and a luciferase-based transcriptional reporter, we showed that the full transcription of thecpsgenes not only depends on the core promoter immediately upstream ofcps2A, but also requires additional elements upstream of the core promoter, particularly a 59-bp sequence immediately upstream of the core promoter. Unmarked deletions of these promoter elements in the D39 genome also led to significant reduction in CPS production and virulence in mice. Lastly, commoncpsgene (cps2ABCD) mutants did not show significant abnormality incpstranscription, although they produced significantly less CPS, indicating that the CpsABCD proteins are involved in the encapsulation ofS. pneumoniaein a posttranscriptional manner. This study has yielded important information on the transcriptional characteristics of thecpslocus inS. pneumoniae.
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Morelli, Laura, Damiano Cancogni, Marta Tontini, Alberto Nilo, Sara Filippini, Paolo Costantino, Maria Rosaria Romano, Francesco Berti, Roberto Adamo, and Luigi Lay. "Synthesis and immunological evaluation of protein conjugates ofNeisseria meningitidisX capsular polysaccharide fragments." Beilstein Journal of Organic Chemistry 10 (October 13, 2014): 2367–76. http://dx.doi.org/10.3762/bjoc.10.247.
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A vaccine to prevent infections from the emergingNeisseria meningitidisX (MenX) is becoming an urgent issue. Recently MenX capsular polysaccharide (CPS) fragments conjugated to CRM197as carrier protein have been confirmed at preclinical stage as promising candidates for vaccine development. However, more insights about the minimal epitope required for the immunological activity of MenX CPS are needed. We report herein the chemical conjugation of fully synthetic MenX CPS oligomers (monomer, dimer, and trimer) to CRM197. Moreover, improvements in some crucial steps leading to the synthesis of MenX CPS fragments are described. Following immunization with the obtained neoglycoconjugates, the conjugated trimer was demonstrated as the minimal fragment possessing immunogenic activity, even though significantly lower than a pentadecamer obtained from the native polymer and conjugated to the same protein. This finding suggests that oligomers longer than three repeating units are possibly needed to mimic the activity of the native polysaccharide.
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Kossaczka, Zuzana, Joseph Shiloach, Virginia Johnson, David N. Taylor, Richard A. Finkelstein, John B. Robbins, and Shousun C. Szu. "Vibrio cholerae O139 Conjugate Vaccines: Synthesis and Immunogenicity of V. cholerae O139 Capsular Polysaccharide Conjugates with Recombinant Diphtheria Toxin Mutant in Mice." Infection and Immunity 68, no. 9 (September 1, 2000): 5037–43. http://dx.doi.org/10.1128/iai.68.9.5037-5043.2000.
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ABSTRACT Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulatedV. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to ≤50. Treatment with 2-ME reduced the titers of 17 of 20 patients to ≤50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.
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Yang, Jinghua, Moon H. Nahm, C. Allen Bush, and John O. Cisar. "Comparative Structural and Molecular Characterization of Streptococcus pneumoniae Capsular Polysaccharide Serogroup 10." Journal of Biological Chemistry 286, no. 41 (August 22, 2011): 35813–22. http://dx.doi.org/10.1074/jbc.m111.255422.
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Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1–6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1–2 or wcrF-dependent α1–4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1–3-linked Galf branches linked to GalNAc, only CPS10A had additional β1–6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1–3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1–6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.
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Chaffin, Donald O., Stephen B. Beres, Harry H. Yim, and Craig E. Rubens. "The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon." Journal of Bacteriology 182, no. 16 (August 15, 2000): 4466–77. http://dx.doi.org/10.1128/jb.182.16.4466-4477.2000.
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ABSTRACT Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cpsIIIFGHIJKL,neuIIIB, and neuIIIC. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cpsIIIH, the putative CPS polymerase gene. Expression of cpsIIIH in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cpsIaH andcpsIIIH encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.
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Tzeng, Yih-Ling, Anup K. Datta, Cristy A. Strole, Michael A. Lobritz, Russell W. Carlson, and David S. Stephens. "Translocation and Surface Expression of Lipidated Serogroup B Capsular Polysaccharide in Neisseria meningitidis." Infection and Immunity 73, no. 3 (March 2005): 1491–505. http://dx.doi.org/10.1128/iai.73.3.1491-1505.2005.
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ABSTRACT The capsule of N. meningitidis serogroup B, (α2→8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3′ end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.
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Grau, Brenda L., Margaret C. Henk, Katherine L. Garrison, Brett J. Olivier, Randall M. Schulz, Kathy L. O'Reilly, and Gregg S. Pettis. "Further Characterization of Vibrio vulnificus Rugose Variants and Identification of a Capsular and Rugose Exopolysaccharide Gene Cluster." Infection and Immunity 76, no. 4 (January 22, 2008): 1485–97. http://dx.doi.org/10.1128/iai.01289-07.
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ABSTRACT Capsular polysaccharide (CPS) is a major virulence factor in Vibrio vulnificus, and encapsulated strains have an opaque, smooth (OpS) colony morphology, while nonencapsulated strains have a translucent, smooth (TrS) colony morphology. Previously, we showed that OpS and TrS parental strains can yield a third colony type, rugose (R), and that the resulting strains, with the OpR and TrR phenotypes, respectively, form copious biofilms. Here we show that while OpR and TrR strains both produce three-dimensional biofilm structures that are indicative of rugose extracellular polysaccharide (rEPS) production, OpR strains also retain expression of CPS and are virulent in an iron-supplemented mouse model, while TrR strains lack CPS and are avirulent. Chlorine resistance assays further distinguished OpR and TrR isolates as exposure to 3 μg/ml NaOCl eradicated both OpS and OpR strains, while both TrS and TrR strains survived, but at rates which were significantly different from one another. Taken together, these results further emphasize the importance of CPS for virulence of V. vulnificus and establish a correlation between CPS expression and chlorine sensitivity in this organism. Using reverse transcriptase PCR, we also identified a nine-gene cluster associated with both CPS and rEPS expression in V. vulnificus, designated the wcr (capsular and rugose polysaccharide) locus, with expression occurring primarily in R variants. The latter results set the stage for characterization of functional determinants which individually or collectively contribute to expression of multiple EPS forms in this pathogen.
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Choi, Eun Hwa, Fan Zhang, Ying-Jie Lu, and Richard Malley. "Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies." Clinical and Vaccine Immunology 23, no. 2 (December 16, 2015): 162–67. http://dx.doi.org/10.1128/cvi.00591-15.
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ABSTRACTThe efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes iswzydependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. Thein vitro-released CPS concentrations per 107CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum fromStreptococcus pneumoniaeST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant fromS. pneumoniaeST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary.
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Cuccui, Jon, Timothy S. Milne, Nicholas Harmer, Alison J. George, Sarah V. Harding, Rachel E. Dean, Andrew E. Scott, et al. "Characterization of the Burkholderia pseudomallei K96243 Capsular Polysaccharide I Coding Region." Infection and Immunity 80, no. 3 (January 17, 2012): 1209–21. http://dx.doi.org/10.1128/iai.05805-11.
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Burkholderia pseudomalleiis the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule inB. pseudomalleiis essential for virulence.B. pseudomalleicapsular polysaccharide (CPS) I comprises unbranchedmanno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivatinggmhA,wcbJ, andwcbNinB. pseudomalleiK96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with theB. pseudomalleiK96243 mutants lacking a functional copy of eithergmhAorwcbJwere afforded significant levels of protection against a wild-typeB. pseudomalleiK96243 challenge.
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Nesper, Jutta, Stefan Schild, Crystal M. Lauriano, Anita Kraiss, Karl E. Klose, and Joachim Reidl. "Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization." Infection and Immunity 70, no. 11 (November 2002): 5990–96. http://dx.doi.org/10.1128/iai.70.11.5990-5996.2002.
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ABSTRACT Since the first occurrence of O139 Vibrio cholerae as a cause of cholera epidemics, this serogroup has been investigated intensively, and it has been found that its pathogenicity is comparable to that of O1 El Tor strains. O139 isolates express a thin capsule, composed of a polymer of repeating units structurally identical to the lipopolysaccharide (LPS) O side chain. In this study, we investigated the role of LPS O side chain and capsular polysaccharide (CPS) in intestinal colonization by with genetically engineered mutants. We constructed CPS-negative, CPS/LPS O side chain-negative, and CPS-positive/LPS O side chain-negative mutants. Furthermore, we constructed two mutants with defects in LPS core oligosaccharide (OS) assembly. Loss of LPS O side chain or CPS resulted in a ≈30-fold reduction in colonization of the infant mouse small intestine, indicating that the presence of both LPS O side chain and CPS is important during the colonization process. The strain lacking both CPS and LPS O side chain and a CPS-positive, LPS O side chain-negative core OS mutant were both essentially unable to colonize. To characterize the role of surface polysaccharides in survival in the host intestine, resistance to several antimicrobial substances was investigated in vitro. These investigations revealed that the presence of CPS protects the cell against attack of the complement system and that an intact core OS is necessary for survival in the presence of bile.
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Hua, Kuo-Feng, Feng-Ling Yang, Hsiao-Wen Chiu, Ju-Ching Chou, Wei-Chih Dong, Chien-Nan Lin, Chai-Yi Lin, et al. "Capsular Polysaccharide Is Involved in NLRP3 Inflammasome Activation by Klebsiella pneumoniae Serotype K1." Infection and Immunity 83, no. 9 (June 15, 2015): 3396–409. http://dx.doi.org/10.1128/iai.00125-15.
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Klebsiella pneumoniae(strain 43816, K2 serotype) induces interleukin-1β (IL-1β) secretion, but neither the bacterial factor triggering the activation of these inflammasome-dependent responses nor whether they are mediated by NLRP3 or NLRC4 is known. In this study, we identified a capsular polysaccharide (K1-CPS) inK. pneumoniae(NTUH-K2044, K1 serotype), isolated from a primary pyogenic liver abscess (PLAK. pneumoniae), as theKlebsiellafactor that induces IL-1β secretion in an NLRP3-, ASC-, and caspase-1-dependent manner in macrophages. K1-CPS induced NLRP3 inflammasome activation through reactive oxygen species (ROS) generation, mitogen-activated protein kinase phosphorylation, and NF-κB activation. Inhibition of both the mitochondrial membrane permeability transition and mitochondrial ROS generation inhibited K1-CPS-mediated NLRP3 inflammasome activation. Furthermore, IL-1β secretion in macrophages infected with PLAK. pneumoniaewas shown to depend on NLRP3 but also on NLRC4 and TLR4. In macrophages infected with a K1-CPS deficiency mutant, an lipopolysaccharide (LPS) deficiency mutant, or K1-CPS and LPS double mutants, IL-1β secretion levels were lower than those in cells infected with wild-type PLAK. pneumoniae. Our findings indicate that K1-CPS is one of theKlebsiellafactors of PLAK. pneumoniaethat induce IL-1β secretion through the NLRP3 inflammasome.
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Smith, Amy B., and Ronald J. Siebeling. "Identification of Genetic Loci Required for Capsular Expression in Vibrio vulnificus." Infection and Immunity 71, no. 3 (March 2003): 1091–97. http://dx.doi.org/10.1128/iai.71.3.1091-1097.2003.
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ABSTRACT Transposon mutagenesis of an encapsulated, virulent strain of Vibrio vulnificus 1003(O) led to the identification of four genetic regions that are essential to capsular polysaccharide (CPS) expression and virulence. Of the four regions, three are believed to be part of a capsule gene locus comprised of biosynthesis, polymerization, and transport genes clustered on a single chromosomal fragment. Genes indicating a Wzy-dependent system of polymerization and transmembrane export are present, suggesting that the CPS of V. vulnificus is lipid linked. The fourth region, while it contains a gene essential for CPS expression, is characteristic of an integron-gene cassette region, similar to the super integron of V. cholerae. It is not believed to be part of a CPS gene locus and is located in a region of the chromosome separate from the putative CPS loci. It is comprised of open reading frames (ORFs) carrying genes of unknown function surrounded by direct repeats. This region also contains IS492, an insertion sequence located numerous times throughout a region of the genome, demonstrating a restriction fragment length polymorphism among an encapsulated and nonencapsulated morphotype of V. vulnificus. Collectively, 22 ORFs were recognized: 13 capsule synthesis genes, 4 insertion sequences, 1 truncated biosynthesis gene, and 4 genes of unknown function. This study has led to the identification of previously unrecognized genetic loci that may help to increase the understanding of capsular genetics and antigenic diversity among V. vulnificus strains.
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Llull, Daniel, Patrick Veiga, Josselyne Tremblay, and Saulius Kulakauskas. "Immobilization-based isolation of capsule-negative mutants of Streptococcus pneumoniae." Microbiology 151, no. 6 (June 1, 2005): 1911–17. http://dx.doi.org/10.1099/mic.0.27862-0.
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The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococcus pneumoniae, a human pathogen of the upper respiratory tract. One limitation in studies of S. pneumoniae surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0·04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical S. pneumoniae type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS− mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS− phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned tts gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the tts gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS− mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.
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Scott, Andrew E., Mary N. Burtnick, Margaret G. M. Stokes, Adam O. Whelan, E. Diane Williamson, Timothy P. Atkins, Joann L. Prior, and Paul J. Brett. "Burkholderia pseudomallei Capsular Polysaccharide Conjugates Provide Protection against Acute Melioidosis." Infection and Immunity 82, no. 8 (May 27, 2014): 3206–13. http://dx.doi.org/10.1128/iai.01847-14.
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ABSTRACTBurkholderia pseudomallei, the etiologic agent of melioidosis, is a CDC tier 1 select agent that causes severe disease in both humans and animals. Diagnosis and treatment of melioidosis can be challenging, and in the absence of optimal chemotherapeutic intervention, acute disease is frequently fatal. Melioidosis is an emerging infectious disease for which there are currently no licensed vaccines. Due to the potential malicious use ofB. pseudomalleias well as its impact on public health in regions where the disease is endemic, there is significant interest in developing vaccines for immunization against this disease. In the present study, type A O-polysaccharide (OPS) andmanno-heptose capsular polysaccharide (CPS) antigens were isolated from nonpathogenic, select-agent-excluded strains ofB. pseudomalleiand covalently linked to carrier proteins. By using these conjugates (OPS2B1 and CPS2B1, respectively), it was shown that although high-titer IgG responses against the OPS or CPS component of the glycoconjugates could be raised in BALB/c mice, only those animals immunized with CPS2B1 were protected against intraperitoneal challenge withB. pseudomallei. Extending upon these studies, it was also demonstrated that when the mice were immunized with a combination of CPS2B1 and recombinantB. pseudomalleiLolC, rather than with CPS2B1 or LolC individually, they exhibited higher survival rates when challenged with a lethal dose ofB. pseudomallei. Collectively, these results suggest that CPS-based glycoconjugates are promising candidates for the development of subunit vaccines for immunization against melioidosis.
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Goyette-Desjardins, Guillaume, Cynthia Calzas, Tze Chieh Shiao, Axel Neubauer, Jennifer Kempker, René Roy, Marcelo Gottschalk, and Mariela Segura. "Protection against Streptococcus suis Serotype 2 Infection Using a Capsular Polysaccharide Glycoconjugate Vaccine." Infection and Immunity 84, no. 7 (April 25, 2016): 2059–75. http://dx.doi.org/10.1128/iai.00139-16.
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Streptococcus suisserotype 2 is an encapsulated bacterium and one of the most important bacterial pathogens in the porcine industry. Despite decades of research for an efficient vaccine, none is currently available. Based on the success achieved with other encapsulated pathogens, a glycoconjugate vaccine strategy was selected to elicit opsonizing anti-capsular polysaccharide (anti-CPS) IgG antibodies. In this work, glycoconjugate prototypes were prepared by couplingS. suistype 2 CPS to tetanus toxoid, and the immunological features of the postconjugation preparations were evaluatedin vivo. In mice, experiments evaluating three different adjuvants showed that CpG oligodeoxyribonucleotide (ODN) induces very low levels of anti-CPS IgM antibodies, while the emulsifying adjuvants Stimune and TiterMax Gold both induced high levels of IgGs and IgM. Dose-response trials comparing free CPS with the conjugate vaccine showed that free CPS is nonimmunogenic independently of the dose used, while 25 μg of the conjugate preparation was optimal in inducing high levels of anti-CPS IgGs postboost. With an opsonophagocytosis assay using murine whole blood, sera from immunized mice showed functional activity. Finally, the conjugate vaccine showed immunogenicity and induced protection in a swine challenge model. When conjugated and administered with emulsifying adjuvants,S. suistype 2 CPS is able to induce potent IgM and isotype-switched IgGs in mice and pigs, yielding functional activityin vitroand protection against a lethal challengein vivo, all features of a T cell-dependent response. This study represents a proof of concept for the potential of glycoconjugate vaccines in veterinary medicine applications against invasive bacterial infections.
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Nakhamchik, Alina, Caroline Wilde, Henry Chong, and Dean A. Rowe-Magnus. "Evidence for the Horizontal Transfer of an Unusual Capsular Polysaccharide Biosynthesis Locus in Marine Bacteria." Infection and Immunity 78, no. 12 (October 4, 2010): 5214–22. http://dx.doi.org/10.1128/iai.00653-10.
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ABSTRACT The most intensely studied of the Vibrio vulnificus virulence factors is the capsular polysaccharide (CPS). All virulent strains produce copious amounts of CPS. Acapsular strains are avirulent. The structure of the CPS from the clinical isolate ATCC 27562 is unusual. It is serine modified and contains, surprisingly, N-acetylmuramic acid. We identified the complete 25-kb CPS biosynthesis locus from ATCC 27562. It contained 21 open reading frames and was allelic to O-antigen biosynthesis loci. Two of the genes, murA CPS and murB CPS, were paralogs of the murA PG and murB PG genes of the peptidoglycan biosynthesis pathway; only a single copy of these genes is present in the strain CMCP6 and YJ016 genomes. Although MurACPS and MurBCPS were functional when expressed in Escherichia coli, lesions in either gene had no effect on CPS production, virulence, or growth in V. vulnificus; disruption of 8 other genes within the locus resulted in an acapsular phenotype and attenuated virulence. Thus, murA CPS and murB CPS were functional but redundant. Comparative genomic analysis revealed that while completely different CPS biosynthesis loci were found in the same chromosomal region in other V. vulnificus strains, most of the CPS locus of ATCC 27562 was conserved in another marine bacterium, Shewanella putrefaciens strain 200. However, the average GC content of the CPS locus was significantly lower than the average GC content of either genome. Furthermore, several of the encoded proteins appeared to be of Gram-positive and archaebacterial origin. These data indicate that the horizontal transfer of intact and partial CPS loci drives CPS diversity in marine bacteria.
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Chaffin, D. O., L. M. Mentele, and C. E. Rubens. "Sialylation of Group B Streptococcal Capsular Polysaccharide Is Mediated by cpsK and Is Required for Optimal Capsule Polymerization and Expression." Journal of Bacteriology 187, no. 13 (July 1, 2005): 4615–26. http://dx.doi.org/10.1128/jb.187.13.4615-4626.2005.
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ABSTRACT Several bacterial pathogens have evolved the means to escape immune detection by mimicking host cell surface carbohydrates that are crucial for self/non-self recognition. Sialic acid, a terminal residue on these carbohydrates, inhibits activation of the alternate pathway of complement by recruiting the immune modulating molecule factors H, I, and iC3b. Sialylation of capsular polysaccharide (CPS) is important for virulence of group B streptococci (GBS), a significant human pathogen. We previously reported that cpsK, a gene within the cps locus of type III GBS, could complement a sialyltransferase deficient lst mutant of Haemophilus ducreyi, implicating its role in sialylation of the GBS capsule. To explore the function of cpsK in GBS capsule production, we created a mutant in cpsK. Immunoblot analysis and enzyme-linked immunosorbent assay using anti-type III CPS antisera demonstrated that the mutant CPS did not contain sialic acid. This was confirmed by high-performance liquid chromatography after mild acid hydrolysis of the CPS. Although increased CPS chain length was seen for this strain, CPS production was <20% of the parental isolate. An episomal cpsK copy restored synthesis of sialo-CPS to wild-type levels. These data support our hypothesis that cpsK encodes the GBS CPS sialyltransferase and provide further evidence that lack of CPS oligosaccharide sialylation reduces the amount of CPS expressed on the cell surface. These observations also imply that one or more of the components involved in synthesis or transport of oligosaccharide repeating units requires a sialo-oligosaccharide for complete activity.
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Laus, Marc C., Trudy J. Logman, Anton A. N. van Brussel, Russell W. Carlson, Parastoo Azadi, Mu-Yun Gao, and Jan W. Kijne. "Involvement of exo5 in Production of Surface Polysaccharides in Rhizobium leguminosarum and Its Role in Nodulation of Vicia sativa subsp. nigra." Journal of Bacteriology 186, no. 19 (October 1, 2004): 6617–25. http://dx.doi.org/10.1128/jb.186.19.6617-6625.2004.
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ABSTRACT Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum, RBL5808 and RBL5812, revealed independent Tn5 transposon integrations in a single gene, designated exo5. As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid. A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides. Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid. Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures. Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS. The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure. Nodulation studies with Vicia sativa subsp. nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization. This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl. Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS. However, in this case colonization of the nodule tissue was impaired.