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Identification of a second Arcanobacterium pyogenes neuraminidase and involvement of neuraminidase activity in host cell adhesion.

ABSTRACT: Arcanobacterium pyogenes, a common inhabitant of the upper respiratory and urogenital tracts of economically important animals, such as cattle and swine, is also an opportunistic pathogen associated with suppurative infections in these animals. A. pyogenes expresses neuraminidase activity encoded by the nanH gene, and previously, construction of a nanH mutant of A. pyogenes BBR1 indicated that a second neuraminidase is present in this strain. A 5,112-bp gene, nanP, was cloned and sequenced, and this gene conferred neuraminidase activity on an Escherichia coli host strain. The predicted 186.8-kDa NanP protein exhibited 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. As expected, insertional inactivation of the nanP gene in A. pyogenes BBR1 resulted in a mutant with reduced neuraminidase activity. However, insertional inactivation of the nanP gene in the nanH mutant strain resulted in a complete lack of neuraminidase activity. Like NanH, NanP was localized to the A. pyogenes cell wall. However, unlike the nanH gene, which was present in 100% of the strains examined, nanP was present in only 64.2% of the isolates (n = 53). A. pyogenes adheres to HeLa cells, and a nanP mutant displayed a wild-type adhesion phenotype with these cells. In contrast, the ability of a nanH nanP double mutant to bind to HeLa cells was reduced by 53%. The wild-type adhesion phenotype was restored by providing nanP in trans. These data indicate that the neuraminidases of A. pyogenes play a role in adhesion of this organism to host epithelial cells.

SUBMITTER: Jost BH

PROVIDER: S-EPMC127772 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The Arcanobacterium pyogenes collagen-binding protein, CbpA, promotes adhesion to host cells.

Project description:Arcanobacterium pyogenes is an opportunistic pathogen associated with suppurative diseases in economically important food animals such as cattle, pigs, and turkeys. A. pyogenes adheres to host epithelial cells, and adhesion is promoted by the action of neuraminidase, which is expressed by this organism. However, a neuraminidase-deficient mutant of A. pyogenes only had a reduced ability to adhere to host epithelial cells, indicating that other factors are involved in adhesion. Far Western blotting revealed the presence of an approximately 120-kDa A. pyogenes cell wall protein that binds collagen type I. The 3.5-kb gene that encodes the 124.7-kDa CbpA protein was cloned, and sequence analysis indicated that CbpA contains a typical MSCRAMM protein domain structure. Recombinant, six-His-tagged CbpA (HIS-CbpA) was capable of binding collagen types I, II, and IV but not fibronectin. In addition, CbpA was involved in the ability of A. pyogenes to adhere to HeLa and 3T6 cells, as a cbpA knockout strain had 38.2 and 57.0% of wild-type adhesion, respectively. This defect could be complemented by providing cbpA on a multicopy plasmid. Furthermore, HIS-CbpA blocked A. pyogenes adhesion to HeLa or 3T6 cells in a dose-dependent manner. cbpA was only present in 48% of the A. pyogenes strains tested (n = 75), and introduction of plasmid-encoded cbpA into a naturally cbpA-deficient strain increased the ability of this strain to bind to HeLa and 3T6 cells 2.9- and 5.7-fold, respectively. These data indicate that CbpA, a collagen-binding protein of A. pyogenes, plays a role in the adhesion of this organism to host cells.

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