Cloning, expression, and characterization of a neuraminidase gene from Arcanobacterium pyogenes

Arcanobacterium pyogenes is an opportunistic pathogen, associated with supp urative infections in domestic animals. In addition to pyolysin, a pore-for ming, cholesterol-binding toxin, A. pyogenes expresses a number of putative virulence factors, including several proteases and neuraminidase activity. A 3,009-bp gene, nanH, was cloned and sequenced and conferred neuraminidas e activity on an Escherichia coli host strain. The predicted 107-kDa NanH p rotein displayed similarity to a number of bacterial neuraminidases and con tained the RIP/RLP motif and five copies of the Asp box motif found in all bacterial neuraminidases. Recombinant His-tagged NanH was found to have pH and temperature optima of 5.5 to 6.0 and 55 degreesC, respectively. Inserti onal deletion of the nanH gene resulted in the reduction, but not absence, of neuraminidase activity, indicating the presence of a second neuraminidas e gene in A, pyogenes, NanH was localized to the A. pyogenes cell wall. A. pyogenes adhered to HeLa, CHO, and MDBK cells in a washing-resistant manner . However, the nanH mutant was not defective for adherence to epithelial ce lls. The role of NanH in host epithelial cell adherence may be masked by th e presence of a second neuraminidase in A. pyogenes.