Virulence of Vtbrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/0) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the a-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the a-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 'H and "3C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is:The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/0.Vibio vulnificus is a halophilic bacterium that can cause severe wound infections or a syndrome of primary septicemia that is frequently fatal (mortality, >50%) in persons with underlying liver disease or hemochromatosis or who are immunocompromised (7,23,29). The organism is common in the estuarine environment (20,31). Wound infections are associated with seawater exposure, whereas primary septicemia apparently results from ingestion of V. vulnificus in raw oysters or other shellfish (29). In 1984, Kreger et al. (25) first described a major protective antigen of V. vulnificus that appeared, by electron microscopy, to be a ruthenium red-staining acidic polysaccharide layer or capsule located on the bacterial surface. The degree of encapsulation (estimated by electron microscopy) was subsequently correlated with colony morphology: encapsulated cells produced opaque colonies, whereas bacteria with a reduced ruthenium red-staining layer produced translucent colonies (36, 47). Opaque, encapsulated strains were found to shift to a translucent morphology at a rate of ca. 10-4 (36, 44, 47); shifts from translucent to opaque morphologies have been demonstrated at comparable frequencies (44). The opaque morphology has been correlated with increased virulence in mice, resistance to serum bactericidal activity, decreased hydrophobicity, and ability to utilize iron bound to saturated transferrin (36, 44). V. vulnificus produces a number of extracellular products that have been * Corresponding author. implicated as possible virulence factors, including cytolysin (15), elastolytic protease (24),...
Vibrio vulnificus produces a capsular polysaccharide (CPS) that is essential for virulence. CPS from V. vulnificus clinical strain MO6-24 has been purified and the structure determined. In preliminary screening with antisera raised to MO6-24 CPS, 4 (19%) of 21 clinical isolates (including MO6-24), but none of 67 environmental V. vulnificus isolates, agglutinated with anti-MO6-24 antisera (P = .003). CPS was isolated from a subset of 12 clinical and 7 environmental isolates and analyzed by high-performance anion-exchange chromatography and one-dimensional nuclear magnetic resonance. MO6-24 and 1 other serologically positive strain had identical CPS structures; the other 2 serologically positive strains had substitutions in two of four sugar residues. Thirteen other capsular types were identified among the remaining 15 strains from which CPS was extracted.
Vibrio vulnificus causes septicemia and wound infections in immunocompromised humans. The capsular polysaccharide of Vibrio vulnificus (VvPS) is critical for virulence. We synthesized conjugate vaccines of carbotype 1 VvPS under conditions and in formulations suitable for human use. Purified VvPS was conjugated to tetanus toxoid (TT) or to inactivated V. vulnificus cytolysin or elastase by two different schemes. All conjugates elicited elevated anticapsular immunoglobulin G (IgG) and IgM and antiprotein IgG responses in mice compared with saline placebo. The conjugates prepared through carboxyl activation of VvPS (VvPS-TTa, VvPS-cytolysin, and VvPS-elastase) were more immunogenic than the one prepared through hydroxyl activation (VvPS-TTb). The protective efficacy of conjugated and unconjugated formulations of VvPS and that of protein carriers were evaluated in a mouse septicemia model. Eighty percent of mice actively immunized with VvPS-TTa vaccine survived challenge with carbotype 1 V. vulnificus, while VvPS-cytolysin and VvPS-elastase conjugates conferred 44 and 40% protection, respectively. Control mice immunized with VvPS, cytolysin, or elastase alone, or saline only, showed 70 to 100% mortality. VvPS-TTa vaccine is nontoxic, immunogenic, and protective in mice.
Vibrio vulnificus is a pathogenic gram-negative bacterium, endemic to brackish waters, which is often isolated from sediments, from the water column or from shellfish. It is associated with wound infections and septicemia in humans and the virulence of V. vulnificus has been strongly associated with encapsulation. The capsular polysaccharide purified from a virulent strain of V. vulnificus 6353 did not show cross reactivity with antibodies to the capsular polysaccharide of a related pathogenic strain of V. vulnificus (MO6-24) the structure of which was recently reported. NMR spectroscopic analysis of the purified polysaccharide from strain 6353 showed that the polymer is composed of four sugar residues per repeating subunit including 2,6-dideoxyThe 1 H-and 13 C-NMR spectra were completely assigned by homonuclear and heteronuclear NMR spectroscopy. Sugar types and anomeric configurations were determined from proton homonuclear coupling constants and glycosidic linkages were determined from 1 HϪ 13 C heteronuclear multiple bond correlation spectra. Sugar identities were confirmed by high performance anion-exchange chromatography and absolute configurations were determined by gas chromatography in combination with molecular modeling and NMR spectroscopy. The structure of the polysaccharide repeating unit is:While there are some common features shared among the structures of the capsular polysaccharides of pathogenic strains of V. vulnificus, there are distinct differences in the detailed structures.Keywords : bacteria ; polysaccharide ; structure ; Vibrio; NMR. Vibrio vulnificus is a gram-negative halophilic bacterium thatV. vulnificus produces a capsular polysaccharide which is is capable of causing severe wound infections and septicemia in essential for virulence [6]. This capsule provides the bacterium humans [1, 2]. While wound infections can occur in healthy with resistance to serum bactericidal activity and phagocytosis. individuals, septicemia primarily affects compromised hosts The capsular material has also been shown to directly stimulate with underlying conditions such as hemachromatosis, cirrhosis, the release of tumor necrosis factor A and other cytokines from and alcoholism. Over 50% of persons with septicemia die, and peripheral blood mononuclear cells [7]. Capsular polysacchathe mortality rate among patients who are hypotensive within ride-protein conjugate vaccines provide protection against lethal 24 h of hospital admission exceeds 90% [3]. The organism is infection in mouse models. Antibodies raised to the purified capcommon in the estuarine environment [4,5]. Infection generally sular material are also protective against strains of the homolodevelops following ingestion of raw oysters or other shellfish or gous capsular type [8,9]. These observations underscore the exposure of a skin break to sea water containing the bacterium need to carefully catalog V. vulnificus capsular types, both for [2]. studies of the role of the capsular material in pathogenesis and in exploring development of antisera...
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