Characterization of the hippurate hydrolase enzyme of Campylobacter jejuni.

摘要

Campylobacter jejuni is isolated from cases of human gastroenteritis far more commonly than other Campylobacter species, suggesting that it may possess species-specific factors that enable it to preferentially cause disease in humans. The hippurate hydrolase enzyme of C. jejuni, a species-specific enzyme encoded for by the hipO gene, was examined to determine its potential contribution to virulence in C. jejuni. The hipO gene is located on a possible 10 to 15 kb pathogenicity island, in close proximity to the gene for a known adhesin. The usefulness of this enzyme as a target for a species-specific immunoassay was also evaluated.; The hippurate hydrolase was found to be a cytoplasmic, zinc metallocarboxypeptidase with a neutral pH optimum, a thermophilic temperature optimum, and a substrate preference for small, aliphatic N-acylated amino acids. Site-directed mutagenesis studies suggested that the Asp76, Asp104, Glu134 , Glu135, His161, and His356 amino acids contributed to enzyme catalysis and/or zinc-binding in this enzyme. Enzyme activity was enhanced under low-iron conditions, and the C. jejuni ferric uptake regulator (Fur) protein bound to hipO upstream DNA, suggesting that the hipO gene was Fur regulated, as are several bacterial genes expressed within a mammalian host. Campylobacter jejuni hipO- mutants, however, were not significantly reduced in their motility, or abilities to adhere to or invade INT407 cells, colonize BALB/c, and produce cytolethal distending toxin, characteristics associated with virulence in C. jejuni, suggesting that the enzyme did not contribute directly to bacterial virulence.; Eleven murine monoclonal antibodies recognizing the hippurate hydrolase of C. jejuni were generated. One of these monoclonal antibodies was used in a species-specific immunoassay for detection of C. jejuni that displayed 100% sensitivity and 100% specificity for C. jejuni when tested against 54 C. jejuni, 26 Campylobacter coli, 6 Campylobacter lari, 5 Campylobacter hyointestinalis, 6 Campylobacter upsaliensis , 1 Campylobacter mucosalis, and 1 Campylobacter fetus subsp. fetus strains. The assay also detected five out of eight naturally-occurring hippurate hydrolase negative C. jejuni variants, suggesting that this immunoassay might be more sensitive than the traditional assay for detection of atypical C. jejuni strains.