Heterogeneity of environmental, retail, and clinical isolates of Vibrio vulnificus as determined by lipopolysaccharide-specific monoclonal antibodies.

The opportunistic pathogen Vibrio vulnificus expresses lipopolysaccharide (LPS) antigens on its outer membrane surface. A serological typing system was developed for these antigens, utilizing the discriminatory recognition of monoclonal antibodies (MAb) by ELISA. MAb were used to recognize five unique types of LPS-associated antigens for examination of clinical. environmental, and retail isolates of V. vulnificus. The overall serotype profile of the clinical isolates was significantly different (P < 0.05) from that of the environmental and retail isolates. A higher percentage of clinical isolates were typable (61%) compared to the environmental isolates (41%) and retail isolates (44%). In particular, the percentage of serotype 1/5 among clinical isolates (33%), compared to that of environmental (9%) and retail (4%), was highly significant (P < 0.0001). Among the environmental Gulf Coast isolates, there were differences in the prevalence of serotypes 2 and 3 (P < 0.05), depending on whether isolates were obtained from Louisiana or Alabama harvest sites. There were no statistically significant differences between the serotype profiles of Gulf and Atlantic Coast retail isolates despite the absence of serotype 1/5 from the Atlantic Coast. While some serotype diversity was detected in V. vulnificus isolated during different seasons, from different geographic locations, and at retail versus at harvest, there was no apparent concordance between any of the serotype distributions obtained from oysters versus that isolated clinically. The heterogeneity of environmental isolates and relative homogeneity among clinical isolates suggest that human risk may not be predicted on quantitative exposure alone.

MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium.

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli chi7122 conferred mannose-resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain chi7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low-copy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature- and RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.

An epimerase gene essential for capsule synthesis in Vibrio vulnificus.

The extracellular capsule polysaccharide (CPS) of Vibrio vulnificus is a primary virulence factor which allows survival of the bacteria in the human host. To study the genes involved in expression of the capsule, we generated mutants that lost the ability to produce CPS following the insertion of a minitransposon into the genome of an encapsulated, clinical strain of V. vulnificus. A genomic region, from one nonencapsulated mutant, containing the transposon and flanking V. vulnificus DNA was cloned, and a probe complementary to the chromosomal DNA immediately adjacent to the transposon was used to locate this fragment in the genome of the encapsulated parent strain. The fragment, which contained a putative capsule gene, was cloned and, when supplied in trans, complemented the mutation in the nonencapsulated mutant to restore capsule production. In addition, virulence studies, using the 50% lethal dose assay, showed that the restoration of capsule production also restored the virulence of the organism. Sequence analysis of the gene disrupted by the transposon revealed that it matched a nucleotide-sugar epimerase of Vibrio cholerae O139, with 75 and 85% identities at the nucleotide and amino acid levels, respectively. In addition, computer analysis recognized epimerases of various organisms as highly similar to the putative epimerase of V. vulnificus. Finally, a combination of PCR amplification and Southern blotting showed that this epimerase is common to at least 10 strains of V. vulnificus that each express a serologically distinct CPS. Our results indicate that the epimerase gene is essential for capsule expression in V. vulnificus.