Targeted Regulation of Protein Expression in Vibrio parahaemolyticus

<i>V. parahaemolyticus</i> has several virulence factors, including thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), and two separate type III secretion systems (T3SSs), T3SS1 and T3SS2. T3SS1 is responsible for cytotoxicity, primarily through the activity of its effector VP1680. To gain a detailed understanding of the relationship between the amount of effector, its expression timing, and cytotoxicity, a system is required to regulate protein expression levels and timing. In the present study, we developed an effector protein expression system controlled by an arabinose-dependent transcription factor and found that cytotoxicity toward mammalian cells increased in a VP1680-dependent manner. To ensure specific protein degradation, we also established a targeted protein degradation system, including VP0917 (ClpP) and VP0918 (ClpX)-, or VP0917 and VP1014 (ClpA)-mediated degradation of ssrA-tagged proteins (proteins bearing the C-terminal degradation tag encoded by tmRNA). By combining these systems, more than 50% of the targeted protein could be degraded within 20 min. As a byproduct of creating the systems, we obtained an enhanced green fluorescent protein variant that emits strong fluorescence in <i>V. parahaemolyticus</i>. The protein degradation system developed in this study has demonstrated the potential to control intracellular protein levels to a certain extent. Moreover, experimentally controlling intracellular protein levels will allow for a more detailed examination of the relationship between protein quantity and cellular phenotype, potentially overcoming the limitations of the "all-or-nothing" model.