Role of the Chaperone ClpB in Mycoplasma bovis Virulence: Implications for stress response, adhesion, biofilm development, and secreted nuclease activity

ClpB, an ATP-dependent molecular chaperone belonging to the Hsp100/Clp subfamily of AAA+ ATPases, plays a crucial role in protein disaggregation, thereby enhancing bacterial survival under stress conditions. Despite its well-conserved function in prokaryotes, the specific contributions of ClpB to the pathogenesis of the ruminant pathogen Mycoplasma bovis remain largely unexplored. In this study, we identified and functionally characterized a ClpB homolog in M. bovis. Biochemical assays confirmed that the recombinant ClpB protein exhibits intrinsic ATPase activity and, in cooperation with the DnaK chaperone system, efficiently mediates protein disaggregation in vitro. Through genome-wide transposon mutagenesis of the M. bovis HB0801 strain, we generated ClpB-deficient mutants that maintained normal growth kinetics and morphology at 37°C but exhibited significant growth defects under thermal (42°C) and osmotic stress conditions. Phenotypic characterization revealed that ClpB disruption attenuated key virulence attributes, including: impaired host cell adhesion capacity, significant reduction in biofilm formation (p