Extensive surface diversity of a commensal microorganism by multiple DNA inversions.

The dynamic interactions between a host and its intestinal microflora that lead to commensalism are unclear. Bacteria that colonize the intestinal tract do so despite the development of a specific immune response by the host. The mechanisms used by commensal organisms to circumvent this immune response have yet to be established. Here we demonstrate that the human colonic microorganism, Bacteroides fragilis, is able to modulate its surface antigenicity by producing at least eight distinct capsular polysaccharides-a number greater than any previously reported for a bacterium-and is able to regulate their expression in an on-off manner by the reversible inversion of DNA segments containing the promoters for their expression. This means of generating surface diversity allows the organism to exhibit a wide array of distinct surface polysaccharide combinations, and may have broad implications for how the predominant human colonic microorganisms, the Bacteroides species, maintain an ecological niche in the intestinal tract.

Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis.

Bacteroides fragilis, though only a minor component of the human intestinal commensal flora, is the anaerobe most frequently isolated from intra-abdominal abscesses. B. fragilis 9343 expresses at least three capsular polysaccharides-polysaccharide A (PS A), PS B, and PS C. Purified PS A and PS B have been tested in animal models and are both able to induce the formation of intra-abdominal abscesses. Mutants unable to synthesize PS B or PS C still facilitate abscess formation at levels comparable to those of wild-type 9343. To determine the contribution of PS A to abscess formation in the context of the intact organism, the PS A biosynthesis region was cloned, sequenced, and deleted from 9343 to produce a PS A-negative mutant. Animal experiments demonstrate that the abscess-inducing capability of 9343 is severely attenuated when the organism cannot synthesize PS A, despite continued synthesis of the other capsular polysaccharides. The PS A of 9343 contains an unusual free amino sugar that is essential for abscess formation by this polymer. PCR analysis of the PS A biosynthesis loci of 50 B. fragilis isolates indicates that regions flanking each side of this locus are conserved in all strains. The downstream conserved region includes two terminal PS A biosynthesis genes that homology-based analyses predict are involved in the synthesis and transfer of the free amino sugar of PS A. Conservation of these genes suggests that this sugar is present in the PS A of all serotypes and may explain the abscessogenic nature of B. fragilis.

Immunochemical and biological characterization of three capsular polysaccharides from a single Bacteroides fragilis strain.

Although Bacteroides fragilis accounts for only 0.5% of the normal human colonic flora, it is the anaerobic species most frequently isolated from intra-abdominal and other infections with an intestinal source. The capsular polysaccharides of B. fragilis are part of a complex of surface polysaccharides and are the organism's most important virulence factors in the formation of intra-abdominal abscesses. Two capsular polysaccharides from strain NCTC 9343, PS A1 and PS B1, have been characterized structurally. Their most striking feature is a zwitterionic charge motif consisting of both positively and negatively charged substituent groups on each repeating unit. This zwitterionic motif is essential for abscess formation. In this study, we sought to elucidate structural features of the capsular polysaccharide complex of a commonly studied B. fragilis strain, 638R, that is distinct from strain 9343. We sought a more general picture of the species to establish basic structure-activity and structure-biosynthesis relationships among abscess-inducing polysaccharides. Strain 638R was found to have a capsular polysaccharide complex from which three distinct carbohydrates could be isolated by a complex purification procedure. Compositional and immunochemical studies demonstrated a zwitterionic charge motif common to all of the capsular polysaccharides that correlated with their ability to induce experimental intra-abdominal abscesses. Of interest is the range of net charges of the isolated polysaccharides-from positive (PS C2) to balanced (PS A2) to negative (PS 3). Relationships among structural components of the zwitterionic polysaccharides and their molecular biosynthesis loci were identified.

Bacteroides fragilis NCTC9343 produces at least three distinct capsular polysaccharides: cloning, characterization, and reassignment of polysaccharide B and C biosynthesis loci.

Bacteroides fragilis produces a capsular polysaccharide complex (CPC) that is directly involved in its ability to induce abscesses. Two distinct capsular polysaccharides, polysaccharide A (PS A) and PS B, have been shown to be synthesized by the prototype strain for the study of abscesses, NCTC9343. Both of these polysaccharides in purified form induce abscesses in animal models. In this study, we demonstrate that the CPC of NCTC9343 is composed of at least three distinct capsular polysaccharides: PS A, PS B, and PS C. A previously described locus contains genes whose products are involved in the biosynthesis of PS C rather than PS B as was originally suggested. The actual PS B biosynthesis locus was cloned, sequenced, and found to contain 22 genes in an operon-type structure. A mutant with a large chromosomal deletion of the PS B biosynthesis locus was created so that the contribution of PS B to the formation of abscesses could be assessed in a rodent model. Although purified PS B can induce abscesses, removal of this polysaccharide does not attenuate the organism's ability to induce abscesses.

Genetic diversity of the capsular polysaccharide C biosynthesis region of Bacteroides fragilis.

A genetic approach was used to assess the heterogeneity of the capsular polysaccharide C (PS C) biosynthesis locus of Bacteroides fragilis and to determine whether distinct loci contain genes whose products are likely to be involved in conferring charged groups that enable the B. fragilis capsular polysaccharides to induce abscesses. A collection of 50 B. fragilis strains was examined. PCR analysis demonstrated that the genes flanking the PS C biosynthesis region are conserved, whereas the genes within the loci are heterogeneous. Only cfiA(+) B. fragilis strains, which represent 3% of the clinical isolates of B. fragilis, displayed heterogeneity in the regions flanking the polysaccharide biosynthesis genes. Primers were designed in the conserved regions upstream and downstream of the PS C locus and were used to amplify the region from 45 of the 50 B. fragilis strains studied. Fourteen PS C genetic loci could be differentiated by a combination of PCR and extended PCR. These loci ranged in size from 14 to 26 kb. Hybridization analysis with genes from the PS C loci of strains 9343 and 638R revealed that the majority of strains contain homologs of wcgC (N-acetylmannosamine dehydrogenase), wcfF (putative dehydrogenase), and wcgP (putative aminotransferase). The data suggest that the synthesis of polysaccharides that have zwitterionic characteristics rendering them able to induce abscesses is common in B. fragilis.

Interstrain variation of the polysaccharide B biosynthesis locus of Bacteroides fragilis: characterization of the region from strain 638R.

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of Bacteroides fragilis 638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of B. fragilis NCTC 9343. Two genes of the region, wcgD and wcgC, are shown by complementation to encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.

Analysis of a capsular polysaccharide biosynthesis locus of Bacteroides fragilis.

A major clinical manifestation of infection with Bacteroides fragilis is the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designated wcf) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar to O-acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that the wcf locus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides of B. fragilis 9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.

Vibriocidal antibody responses in North American volunteers exposed to wild-type or vaccine Vibrio cholerae O139: specificity and relevance to immunity.

The emergence of a new agent of cholera, Vibrio cholerae O139, has prompted a reevaluation of the vibriocidal antibody assay. This assay, primarily directed to lipopolysaccharide, is an important correlate of O1 immunity. V. cholerae O139 strains are encapsulated, rendering them relatively resistant to killing by serum. Recent reports suggest that there is strain-to-strain variability in the sensitivity of the vibriocidal assay to fully encapsulated O139 strains. We have assessed a modified vibriocidal assay for fully encapsulated O139 strain AI-1837 and its unencapsulated mutant 2L in sera from 53 volunteers given wild-type AI-1837 or its attenuated derivative CVD 112 and from 48 controls challenged with V. cholerae O1 or strains of the family Enterobacteriaceae. Vibriocidal responses to the AI-1837 and 2L strains were seen in 67 and 89% of volunteers, respectively, following a single exposure to the wild-type strain. However, >50% of all controls had low-level vibriocidal responses to both strains. These nonspecific responses were transient and of the immunoglobulin G isotype. No binding activity against purified O139 lipopolysaccharide (LPS) by immunoblotting was seen in control sera. In contrast, vibriocidal assay and strain 2L LPS responses by immunoblotting were detectable in 91% of tested volunteers following a single exposure to O139. The presence of vibriocidal antibody to AI-1837 or 2L was not associated with protection in rechallenge studies with O139 strain AI-1837. The vibriocidal assay with unencapsulated strain 2L may be used to detect exposure to O139 strain AI-1837 in controlled research trials. However, its lack of specificity does not make it useful for determining exposure to V. cholerae O139 in the field.

Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1.

Vibrio cholerae serogroup O139 Bengal is the first documented serogroup other than O1 to cause epidemic cholera. The O139 Bengal strains are very similar to V. cholerae serogroup O1 biotype El Tor strains. The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule. We previously described three TnphoA mutants of O139 strain AI1837 which abolish both O antigen and capsule production. These TnphoA insertions were mapped to a 21.5 kb EcoRI fragment of the O139 chromosome. We describe here the cloning and mapping of this 21.5 kb EcoRI fragment and it was shown to complement each of the mutants in trans to produce O antigen and capsule. The EcoRI fragment contains 13 kb of DNA that is specific to O139 and 8.5 kb of DNA that is common to O1 and O139. Sequence analysis of the 13 kb of O139-specific DNA revealed that it contains 11 open reading frames all of which are transcribed in the same direction. Eight of the 11 open reading frames are homologous to sugar biosynthesis genes from other organisms. Using extended polymerase chain reactions, we show that the extent of the DNA region in O139 that is not present in O1 is approximately 35 kb. The site of insertion of this O139-specific DNA in the O1 chromosome was mapped to the rfbO1 region. We also demonstrate that O139 Bengal strain AI1837 contains a deletion of 22 kb that in serogroup O1 strains contains the rfb region. Therefore, O139 Bengal probably arose from an O1 strain that had undergone genetic rearrangements including deletion of the O1 rfb region and acquisition of a 35 kb region of DNA which encodes O139 surface polysaccharide.

The capsule and O antigen in Vibrio cholerae O139 Bengal are associated with a genetic region not present in Vibrio cholerae O1.

Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.

A monoclonal antibody to OspA inhibits association of Borrelia burgdorferi with human endothelial cells.

Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B. burgdorferi.

Characterization of Borrelia burgdorferi invasion of cultured endothelial cells.

Borrelia burgdorferi can adhere to cultured endothelial cells and penetrate through cell monolayers by passing through intercellular tight junctions and through the host cell cytoplasm. Borrelia burgdorferi strains which were isolated from different sources and areas of the U.S. all demonstrated similar invasive capabilities. Bacterial penetration from the apical to the basal surface of the monolayer was 20 times more efficient than from the basal to the apical surface. Borreliae which were non-viable as a result of either heat treatment or ultraviolet (UV) irradiation showed reduced association with the endothelial cell monolayer and loss of invasive capabilities. Borreliae were able to invade when protein synthesis was inhibited with streptomycin or chloramphenicol. When assays were conducted at 4 degrees C, bacterial penetration of the monolayer was completely inhibited. Treatment of borreliae with proteases affecting outer surface proteins greatly reduced cell association and bacterial invasion.

Penetration of endothelial cell monolayers by Borrelia burgdorferi.

The ability of Borrelia burgdorferi, the agent of Lyme disease, to penetrate cultured human umbilical vein endothelial cell monolayers was investigated. After 4 h of coincubation, approximately 7.7% of added bacteria passed through the host cell monolayers. Electron microscopy revealed that the borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing through them.

Interaction of Lyme disease spirochetes with cultured eucaryotic cells.

The association of the Lyme disease spirochete, Borrelia burgdorferi, with cultured human endothelial cells was investigated. Attachment was time and temperature dependent, with optimal adherence occurring after 4 h of incubation at 37 degrees C. Pretreatment of borreliae with heat, immune human serum, or monoclonal antibodies directed against outer surface protein B (OspB) reduced the attachment of organisms to host cell monolayers. These results suggest that the adherence of B. burgdorferi may be mediated, at least in part, by borrelial surface proteins.