| Abstract |
V. parahaemolyticus 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 V. parahaemolyticus. 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.
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