Polysaccharide Capsule

Capsules are produced by organisms in a variety of habitats, many of which are found in the marine environment (245). Expression is considered a basic cellular function, as judged by its early evolution and development (245). Pathogenic bacteria are often classified on the basis of the complex polysaccharides found on the surface, usually capsular polysaccharides or lipopolysaccharides. It is common in the clinical microbiology laboratory to use reactivity with antisera that specifically recognize these various cell surface carbohydrates for identification purposes.

Early studies reported by Kreger et al. (183,184) and others (186,246) supported the concept that the virulence of V. vulnificus was associated with its ability to resist phagocytosis, as well as its resistance to the bactericidal action of human serum (185-187,247) and its inability to activate complement (186). These results taken together suggest that V. vulnificus expressed an antiphago-cytic surface antigen, such as a capsule. Amako et al. (180) and Kreger et al. (184) both demonstrated in 1984 that V. vulnificus expressed such a surface structure. Amako and colleagues examined two clinical strains for the presence of a polysaccharide capsule by using a ruthenium red staining procedure. The proportion of ruthenium red-stained cells correlated well with mouse virulence and with the susceptibility of the organisms to the bactericidal activity of normal human serum. Amako's observations on the ultrastructure of the capsule were confirmed by several other studies (201,248-

FIGURE 3 Transmission electron photomicrograph showing encapsulated (A) and unencapsulated (B) phase variant Vibrio vulnificus cells stained with Alcian blue. Bar makers represent 0.1 |lm.

250). Figure 3 shows the ultrastructure of the capsule stained with Alcian blue and lysine in both encapsulated and unencapsulated phase variants. These studies further demonstrated that encapsulation correlated with colonial opacity and that variation in the opacity of colonies formed by the organism was accompanied by variation of capsular formation. Furthermore, encapsulated strains were also more resistant to the bactericidal action of human serum, possessed greater antiphagocytic activity, and were highly lethal for mice.

Multiple capsule types have been described for V. vulnificus (182,251), but virulence does not appear to correlate with any one particular capsule type (251,252). Reversible phase variation for opaque and translucent colony morphologies has been described, and both biotype 1 and 2 strains exhibit these properties (181,190,248,249). Capsule expression in biotype 3 has not been characterized. Phase variation or the spontaneous conversion of opaque or encapsulated strains to translucent colonies (and vice versa) was found to occur at similar rates (~10~4) (248-250), and Kaysner et al. (253) showed that phase conversion can occur in vivo.

Capsular polysaccharide was purified from a virulent strain of V. vulnificus by Reddy et al. (254). Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer is composed of a repeating structure with four sugar residues per subunit: a residue of 2-acetamido-2,6-dideoxy-hexo pyranose in the alpha-gluco configuration (QuiNAc), a residue of 2-acetamido-2,6-dideoxy hexopyranose in the alpha-galacto configuration (FucNAc), a residue of 2-acetamido-2,6-dideoxy hexopyranose in the alpha-manno configuration (RhaNAc), and a residue of 2-acetamido-2,6-dideoxy hexouronate in the alpha-galacto configuration (GalNAcA). This was the first reported occurrence of RhaNAc in a bacterial capsular polysaccharide.

Currently there are over 15 capsular serotypes (251). Capsular polysaccharide (CPS) from three opaque V. vulnificus strains was purified and characterized by Simonson and Siebeling (182). The purified acidic capsule contained considerable amounts of hexosamine, and given alone by injection, the purified capsule was poorly immunogenic for rabbits and mice; repeated injections produced little detectable anticapsular antibody. To improve immunogenicity, they conjugated the capsule to keyhole limpet hemocyanin. They then armed S. aureus cells with each of the three anticapsular antibodies and showed that each antibody could only co-agglutinate the homologous opaque strain. These findings suggested the existence of at least three capsular types and are reminiscent of earlier work performed with E. coli, which is known to produce over 70 capsular polysaccharides (255). Bush et al. (252) attempted to improve upon these results with a chemotyping method for bacterial capsular polysaccharides based on carbohydrate analysis of an acid hydrolysate of the capsule. Using a high-performance anion-exchange chromatography procedure coupled to an electrochemical detection method, they were able to semi-quantitatively produce a capsule "fingerprint," which was used to discriminate among isolates of V. vulnificus that expressed different capsular polysaccharide structures. The procedure was applied to a collection of 120 isolates of V. vulnificus from both clinical cases of septicemia and from such environmental sources such as seawater, sediments, and shellfish.

TABLE 6 Population Dynamics of Biofilm Formation by Encapsulated and Unencapsulated Phase Variants of Vibrio vulnificus on Glass Coverslips

Phase variant

Time (h)

CFU/ml, CS

CFU/ml, Sup.

Incubation/ colonizing

Unencapsulated

0

7 x

103

9x

< 105

0.7

Unencapsulated

3

9.3 x

104

6.9 >

< 106

1.3

Unencapsulated

18

1.1 x

107

1.2 x

< 1010

0.1

Encapsulated

0

2x

102

8.5 >

< 105

0.02

Encapsulated

3

2.2 x

103

2.2 x

< 106

0.1

Encapsulated

18

1.3 x

107

1.4 >

< 1010

0.1

CS, coverslip. Source: Ref. 201.

CS, coverslip. Source: Ref. 201.

They found a number of unusual sugars, including many amino sugars and a wide variety of capsular carbotypes.

Apart from its involvement in protection against host bacterial defense mechanisms, the capsule may also play a role in adherence and biofilm formation (245). Tall et al. (201,256), in two separate studies, present evidence that unencapsulated V. vulnificus strains may be more adept at colonizing cultured tissue cells (256) and sterile surfaces, such as glass coverslips (201), than were counterpart encapsulated phase variants (Table 6).

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