ClpB protein regulates biofilm formation in Methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) biofilms contribute significantly to chronic infections and treatment failure. The chaperone protein ClpB has been implicated in bacterial stress responses, but its role in MRSA biofilm regulation and toxicity remains unclear. We constructed a clpB knockout strain (ΔclpB) in MRSA USA300 and quantitatively compared its biofilm phenotype to the wild type (WT) using crystal violet staining, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and extracellular matrix (eDNA, polysaccharide, protein). A mouse skin infection model was used to evaluate wound healing, histopathology (H&E staining), and host inflammatory response (TNF-α/IL-6 ELISA and RT-PCR). ΔclpB showed significantly reduced biofilm biomass (p < 0.0001) and impaired three-dimensional structure with reduced extracellular matrix composition. In vivo, mice infected with ΔclpB showed accelerated wound closure, less severe tissue damage, and reduced levels of TNF-α and IL-6 at protein and transcription levels compared to controls infected with WT. ClpB is a key regulator of MRSA biofilm formation and toxicity, possibly by regulating extracellular matrix and host inflammation. Targeting ClpB could represent a novel anti-biofilm strategy against MRSA infections.