Although proteolytic activity has been shown to be critical for virulence in mycoplasmas, the role of this activity in the pathogenesis of infections with Mycoplasma bovis is yet to be fully elucidated. Two putative peptidase genes (MBOVPG45_0176 and MBOVPG45_0685) of M. bovis were previously shown to be required for the survival of the organism in association with host cells. To investigate the metabolic functions of the two genes, proteolytic screening of M. bovis cell-associated and secreted proteins, together with metabolomic profiling by gas chromatography-mass spectrometry (GC-MS), were used in this study to compare mutants in which the genes were disrupted by a transposon with their parent strain. Cloning and expression of the two genes were also attempted to study their enzymatic activity. Zymography using different peptidase substrates detected secreted proteins of M. bovis with proteolytic activity against gelatin and collagen. Although peptidase activity was reduced in the mutant in which the MBOVPG45_0176 gene was disrupted, the expressed glutathione S-transferase-tagged recombinant product of this gene had no detectable proteolytic activity, possibly due to the absence of propeptide cleavage required for peptidase maturation. Comparative metabolomic profiling revealed that mutants with either of these genes disrupted had significantly lower abundances of amino acids, nucleoside metabolites, and glycolytic intermediates. These results suggest the two putative peptidase genes have a role in metabolic and extracellular proteolytic activity of M. bovis, although further investigation is needed to determine the targets of the proteolytic activity of their products and their precise role in host-mycoplasma interactions.
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