# Targeted Regulation of Protein Expression in Vibrio parahaemolyticus

## TL;DR

Researchers developed tools to control protein levels in Vibrio parahaemolyticus, enhancing cytotoxicity studies and enabling new ways to explore bacterial physiology.

## Contribution

A novel arabinose-regulated expression and targeted degradation system for proteins in Vibrio parahaemolyticus was developed.

## Key findings

- VP1680 expression increases cytotoxicity to mammalian cells in a dose-dependent manner.
- Targeted degradation of ssrA-tagged proteins achieved over 50% degradation within 20 minutes.
- An enhanced green fluorescent protein variant was developed for visualizing intracellular proteins in V. parahaemolyticus.

## Abstract

Cellular phenotypes arise from quantitative and qualitative changes in proteins. Here, we developed an arabinose-regulated effector protein expression system in Vibrio parahaemolyticus and showed that VP1680 expression enhances cytotoxicity to mammalian cells in a dose-dependent manner. We also established a targeted protein degradation system using VP0917 (ClpP), VP0918 (ClpX), and VP1014 (ClpA) to degrade ssrA-tagged proteins, achieving over 50% degradation within 20 min. As a byproduct, we obtained an enhanced green fluorescent protein (EGFP) variant with strong fluorescence in V. parahaemolyticus. This variant serves as a useful tool for visualizing intracellular proteins. These tools enable precise manipulation of intracellular protein levels, offering new ways to explore and redirect bacterial physiology.

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.