Demonstration of lipopolysaccharide on sheathed flagella of Vibrio cholerae O:1 by protein A-gold immunoelectron microscopy

Abstract

Monoclonal antibodies with group and type specificity for lipopolysaccharide antigens were used in combination with protein A-colloidal gold labeling and transmission electron microscopy to demonstrate the presence of lipopolysaccharide antigens on both the sheathed flagellum and the cel surface of Inaba and Ogawa strains of Vibrio cholerae 0:1. Labeling was associated with the sheath of the flagellum rather than the core, and flagellar cores were not labeled. Flagellum and cell shared a common set of lipopolysaccharide antigens characteristic of the strain serotype.Recent research on the pathogenesis of cholera has concentrated on the factors responsible for adhesion of Vibrio cholerae cells to the intestinal mucosa and the nature of the antigens responsible for stimulating an immune response to infection (1,(4)(5)(6)20). Although the important protective antigens have yet to be unequivocally defined, purified lipopolysaccharide (LPS) has been reported to induce significant protection against cholera in humans and experimental animals (16). The flagellum of V. cholerae has been implicated in pathogenesis of cholera in two ways: with respect to its role as a motility organelle (12,18,25) and with respect to its possible role in adherence of V. cholerae to the host intestinal surface (1). The role of the flagellum as a carrier of the adhesins responsible for attachment to intestinal tissue may be of greater significance than its role in conferring motility (1).While the flagellum of V. cholerae is of possible significance in pathogenesis, its chemical and antigenic composition are poorly understood. The flagellum possesses a membranous sheath covering a core which is similar in structure to naked flagella of other bacteria (7,8). In Vibrio metchnikovii, continuity between the sheath and the outer membrane has been demonstrated (10). The question of whether the flagellar sheath of V. cholerae shares the macromolecular composition of the cell wall outer membrane has not been resolved, however. It has been proposed, on the basis of immunoferritin electron microscopy using polyclonal antibodies to LPS, that the sheath does not contain LPS and that the sheath is thus not a simple extension of the outer membrane (17). However, reports that polyclonal antibodies against somatic 0 antigen and monoclonal antibodies against LPS determinants immobilize motile V. cholerae cells are consistent with the occurrence of LPS in the flagellar sheath (1,14). One way to resolve this problem is to localize LPS directly, utilizing highly specific monoclonal antibodies against LPS antigenic determinants and an electron-dense protein A-gold marker for immunoelectron microscopy. LPS determinants distinguish two major serotypes of V. cholerae 0:1: Ogawa, with determinants A and B, and Inaba, with determinants A and C (13). The establishment of hybrid cell lines producing monoclonal antibodies against such groupand type-specific 0 antigens of V. cholerae 0:1 (13,15) Serotypes were checked routinely by slide agglutination with polytypic (an...

Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs

“…Monoclonal antibodies 1D1, 6E4, and 2E11 recognize C. fetus S-layer proteins and have been used previously (28); 1D1 recognizes the 97-kDa proteins of either serotype A or B strains, 6E4 strongly recognizes the 96-to 98-kDa proteins of serotype A only, and 2E11 recognizes 80-to 149-kDa proteins of serotype A. Immunoblotting was performed as described elsewhere (24). Immunogold electron microscopy was done essentially by a previously described procedure (9). After reacting with M442, the cells were labeled with an anti-mouse immunoglobulin G conjugate with 30-nm-diameter gold particles (Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada).…”

Polar Flagellar Motility of the Vibrionaceae

“…Little is known about the composition, formation, or function of flagellar sheaths, which are found in many bacteria, including marine Vibrio species, V. cholerae, B. bacteriovorus, and Helicobacter pylori (reviewed in reference 164). Evidence from these organisms suggests that the sheath contains both lipopolysaccharide and proteins and that it may exist as a stable membrane domain distinct from the outer membrane (42,51,58,69,144). The lipid content of the sheath of B. bacteriovorus is distinct from that of the outer membrane, and the sheath appears to be a highly fluid, symmetric bilayer (179).…”

The bacterial flagellin gene as a biomarker for detection, population genetics and epidemiological analysis

“…(Sjoblad et al, 1983) and Helicobacter pylori (Geis et al, 1989), where a sheath protein has been identified (Luke & Penn, 1995). In Vibrio cholerae (Fuerst & Perry, 1988) and H . pylori (Doig & Trust, 1994), there is evidence from the study of lipopolysaccharide antigens indicating that outer membrane components are present in the flagellar sheath.…”