<b>Role of the Chaperone ClpB in</b><b> </b><b>Mycoplasma bovis</b><b> Virulence: Implications for </b><b>stress response, a</b><b>dhesion, </b><b>b</b><b>iofilm </b><b>d</b><b>evelopment, and </b><b>secreted n</b><b>uclease </b><b>activity</b>

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&lt;0.01), diminished induction of proinflammatory cytokines (IL-1β, IL-6, TNF-α) in infected BoMac cells, decreased extracellular nuclease activity. Furthermore, the reduced virulence of the ClpB mutant was investigated by DIA proteomic analyses, which revealed that the ClpB mutant strain altered distinct protein expression patterns related to protein degration, including serine-type peptidase activity, serine hydrolase activity, and chaperone-mediated protein folding that contribute to the stress response and virulence. These findings collectively demonstrate that ClpB serves as a multifunctional virulence determinant in M. bovis, orchestrating stress adaptation, host-pathogen interactions, and pathogenic potential through modulation of both protein quality control systems and virulence-associated pathways.