# Role of the TPR family protein VPA1365 in regulating type III secretion system 2 and virulence in Vibrio parahaemolyticus

## TL;DR

A new protein, VPA1365, was found to regulate a key system in Vibrio parahaemolyticus that helps it cause disease in the gut.

## Contribution

VPA1365, a TPR family protein, is a novel regulator of T3SS2 and virulence in Vibrio parahaemolyticus.

## Key findings

- VPA1365 positively regulates T3SS2 and its associated virulence genes.
- Deletion of VPA1365 reduces cytotoxicity, adhesion, biofilm formation, and hemolytic activity.
- VPA1365 binds to promoters of vtrA, scrG, pilA, and mshA to control virulence.

## Abstract

Vibrio parahaemolyticus is a notable seafood-borne pathogen capable of colonizing the intestines of hosts and inducing acute gastroenteritis. The intestinal colonization and enterotoxicity of V. parahaemolyticus are highly reliant on the type III secretion system 2 (T3SS2), encoded within the pathogenicity island (Vp-PAI). The expression of Vp-PAI is strictly regulated by bile acid signals and transcriptional regulators VtrA/VtrB. In this study, we identified a tetratricopeptide repeat (TPR) family protein named VPA1365, which regulates the expression of T3SS2 and is indispensable for the intestinal colonization of V. parahaemolyticus. The expression and secretion of the T3SS2-dependent protein VopD2 were significantly reduced in Δvpa1365 compared to that of the wild type (WT), suggesting that VPA1365 positively regulates the function of T3SS2. Further research indicated that VPA1365 directly binds to the promoters of vtrA, thereby increasing the expression levels of T3SS2-associated genes. Additionally, the deletion of vpa1365 markedly reduced the cytotoxicity, adhesion ability, biofilm formation, and hemolytic activity of V. parahaemolyticus. VPA1365 was found to control the expression levels of these virulence-associated genes by binding to the promoters of scrG, pilA, and mshA. In a zebrafish infection model, the Δvpa1365 infected groups demonstrated a higher survival rate compared to the zebrafish infected with WT. In conclusion, this study identified a TPR family protein VPA1365, which regulates the expression levels of T3SS2 and virulence-associated genes in V. parahaemolyticus, further broadening our understanding of its virulence factors.

The type III secretion system 2 (T3SS2) is of crucial significance for the pathogenicity of Vibrio parahaemolyticus; nevertheless, the biological functions of many genes within the T3SS2 gene cluster and the transcriptional regulatory network of T3SS2 remain ambiguous. In this study, we identified VPA1365, a tetratricopeptide repeat family regulator encoded in the T3SS2 gene cluster, which differs from other known T3SS2 regulatory factors, such as OmpR, ToxR, or LysR family proteins. VPA1365 not only positively regulated the expression and secretion of T3SS2-related proteins but also enhanced the virulence in infant rabbits and zebrafish. Moreover, we identified several novel functions of VPA1365, such as its contribution to hemolytic activity, biofilm formation, cytotoxicity, and adhesion ability, uncovering its global physiological role in V. parahaemolyticus. The putative VPA1365-binding site was predicted and identified through the MEME-Suite tool and electrophoretic mobility shift analysis. Collectively, these results broaden our understanding of the regulatory pathways of T3SS2 and virulence.