Cloning, Expression, and Characterization of a Neuraminidase Gene fromArcanobacterium pyogenes

Abstract

Arcanobacterium pyogenes is an opportunistic pathogen, associated with suppurative infections in domestic animals. In addition to pyolysin, a pore-forming, 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 neuraminidase activity on an Escherichia coli host strain. The predicted 107-kDa NanH protein displayed similarity to a number of bacterial neuraminidases and contained 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°C, respectively. Insertional deletion of the nanH gene resulted in the reduction, but not absence, of neuraminidase activity, indicating the presence of a second neuraminidase 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 cells. The role of NanH in host epithelial cell adherence may be masked by the presence of a second neuraminidase in A. pyogenes.Arcanobacterium pyogenes is a common inhabitant of the upper respiratory, urogenital (12, 54), and gastrointestinal tracts (35; B. H. Jost, K. W. Post, and S. J. Billington, unpublished data) of many domestic animal species. However, a physical or microbial insult to the host can lead to a variety of suppurative A. pyogenes infections, such as mastitis in dairy cows (27) and goats (2), liver abscesses in feedlot cattle (31, 34), and pneumonia in pigs (26) and various species of wildlife (17,42,57). A. pyogenes can also infect avian species (10) and humans (5,16,19), although infections in humans are rare.A. pyogenes elaborates a number of extracellular proteins, including the hemolytic exotoxin pyolysin (PLO) (8), several proteases (48, 51), a DNase (30), and at least one neuraminidase (47). While all these proteins are putative virulence factors, only for PLO is there definitive evidence of involvement in the pathogenesis of infections by A. pyogenes (29).Recently, there has been interest in the neuraminidases of bacterial pathogens and the potential role they play in pathogenesis. Neuraminidase (N-acetylneuraminyl hydrolase; EC 3.2.1.18) removes sialic acid from glycolipids, glycoproteins, and poly-and oligosaccharides. Bacterial neuraminidases have only 20 to 30% amino acid sequence identity, but they contain two conserved motifs, the RIP/ RLP motif (Arg-Ile/Leu-Pro) and the Asp box motif (Ser-X-Asp-X-Gly-X-Thr-Trp), which occurs four or five times in the enzyme (14,21,43).Neuraminidases are virulence factors, especially in bacteria that inhabit mucosal surfaces (20, 22, 52, 55), and they may play several roles in virulence. This enzyme can make sialic acid available as a carbon source to promote growth in a nutrient-limited environment (11, 23). The action of neuraminidase can decrease m...

Properties and antimicrobial susceptibility of Trueperella pyogenes isolated from bovine mastitis in China

“…In addition, two neuraminidases, H (nanH: 40%) and P (nanP: 38%) were found in this study. Previous studies (Jost et al, 2001) showed that all investigated T. pyogenes isolates recovered from different types of infection were positive for nanH activity, and 64.2% of these isolates harboured the nanP gene. Both the nanH and nanP genes also occurred in all isolates recovered from the uterus of dairy cows (Silva and Lobato, 1999).…”

Identification of a SecondArcanobacterium pyogenesNeuraminidase and Involvement of Neuraminidase Activity in Host Cell Adhesion

“…An A. pyogenes BBR1 nanH insertion deletion mutant exhibited neuraminidase activity (16), suggesting that a second neuraminidase gene was present in this strain. Plaques from a GEM-12 library of A. pyogenes BBR1 genomic DNA (16) were overlaid with MUAN-containing top agar and were visualized under UV light.…”

“…A. pyogenes strain BBR1 was isolated from a bovine abscess. A. pyogenes strain NANH-1, in which the entire nanH gene was deleted and replaced with an erythromycin resistance cassette, was described previously (16). Other A. pyogenes strains used in this study were obtained from veterinary diagnostic laboratories or personal collections.…”

“…pyogenes expresses neuraminidase activity, and this activity is in part due to NanH, a cell wall-bound neuraminidase found in all isolates (16). Construction of a nanH insertion deletion mutant in A. pyogenes resulted in a strain with reduced neuraminidase activity, but neuraminidase activity was still present, suggesting that a second enzyme was present (16).…”

“…pyogenes expresses neuraminidase activity, and this activity is in part due to NanH, a cell wall-bound neuraminidase found in all isolates (16). Construction of a nanH insertion deletion mutant in A. pyogenes resulted in a strain with reduced neuraminidase activity, but neuraminidase activity was still present, suggesting that a second enzyme was present (16). The nanH mutant was not defective for adhesion to epithelial cells (16), but the role of NanH in adhesion of A. pyogenes to host cells may have been masked by the presence of the second neuraminidase.…”