Exoproteomic Evidence for BcTSPO‐Mediated Regulation of Virulence Factors in <i>Bacillus cereus</i>

Bacillus cereus translocator protein (BcTSPO) is a transmembrane protein that plays a regulatory role in various Bacillus cereus phenotypes, potentially including its virulence. Given that the exoproteome is a major determinant of B. cereus virulence, particularly during the late stages of active growth, its analysis is crucial for understanding the regulatory functions of BcTSPO. In this study, we conducted a comparative analysis of the exoproteomes and cellular proteomes of a Δtspo mutant and the wild-type ATCC 14579 strain during the late exponential and early stationary growth phases. Shotgun proteomics revealed that the absence of BcTSPO significantly depleted the exoproteome of secreted virulence factors, including the HBL, NHE, and CytK enterotoxins. Interestingly, the cholesterol-dependent cereolysin O (CLO) was more abundant in the Δtspo exoproteome, indicating a distinct regulatory mechanism. The cellular proteome analysis further confirmed that BcTSPO positively regulates, likely indirectly, the synthesis of enterotoxins and negatively regulates the production of CLO. Additionally, the Δtspo exoproteome was enriched in moonlighting proteins, reflecting the impact of BcTSPO absence on cellular metabolism and stress responses. Overall, these findings highlight the critical role of BcTSPO in shaping the composition of both the exoproteome and cellular proteome, with significant implications for the virulence of B. cereus.