Abstract

Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD+ and reduce O2 to H2O2. The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovisNOX–expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovisNOX– for cell adhesion and H2O2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.

Highlights

  • Mycoplasma bovis is a major bacterial pathogen, causing pneumonia, mastitis, and arthritis in cattle throughout the world[1,2,3,4]

  • When the putative M. bovis NADH oxidase (NOX) protein was compared with the crystal structure of S. pyogenes NOX using DNAMAN, M. bovis NOX was found to have one catalytic residue (Cys 42), two FAD-binding domains (Gly 7–Gly 12; Ile 272–Asp 283), and a NADH-binding domain (Gly 157 to Gly 162) (Fig. 1)

  • The amino acids in FAD-binding domain 1, the NADH-binding domain, and the active site are identical between the NOX proteins of S. pyogenes and M. bovis, indicating that they are highly conserved

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Summary

Introduction

Mycoplasma bovis is a major bacterial pathogen, causing pneumonia, mastitis, and arthritis in cattle throughout the world[1,2,3,4]. In M. bovis, several membrane proteins, including lipoprotein P269, a VpmaX-like lipoprotein[10], and lipoprotein P6811, are known to function as adhesins. Despite these preliminary findings, the adherence of M. bovis to its host cells remains to be clarified. Because a homologue of the nox gene exists in the M. bovis genome, we hypothesized that this homologue might function as both an active enzyme and an adhesin in M. bovis.

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