V. parahaemolyticus is a prevalent Gram-negative halophilic foodborne pathogen that is ubiquitously distributed in freshwaters, seawaters and aquatic products (Fan et al., 2022). V. parahaemolyticus infections are mostly related to consumption of undercooked or raw contaminated seafood, such as oysters, shrimp, fish and mussels (Baker-Austin et al., 2018). V. parahaemolyticus was first found in Japan and it has been thought to be the cause of occasional infections associated with seafood in some Asian countries (Paranjpye et al., 2012). Over the past 20 years, there have been multiple large-scale foodborne outbreaks of V. parahaemolyticus recorded in the Pacific Northwest region (Turner et al., 2013; Zhang et al., 2020). V. parahaemolyticus infections are steadily rising in the US (Abanto et al., 2020). Furthermore, outbreaks of V. parahaemolyticus infections are frequent in China. V. parahaemolyticus has been considered as the most frequent cause of bacterial diarrhea infection and food poisoning in China (Cao et al., 2021). Type III secretion systems (T3SS), hemolysin, motility, type VI secretion systems (T6SS), adhesins (Type I pilus), protease synthesis, biofilm formation and iron uptake systems have been identified as virulence factors of V. parahaemolyticus infections (Ashrafudoulla et al., 2019; Gu et al., 2019).

Biofilm is an architecturally complex assembly of microorganisms that forms on abiotic or biotic interfaces (Han et al., 2016). The primary constituents of biofilm include exopolysaccharides, extracellular proteins, lipids and extracellular DNA (eDNA) (Li et al., 2020a). Biofilm is usually produced by bacteria in the presence of microbial inhibitors, nutrient deficiencies and other adverse environments (Liu et al., 2021a). Biofilm formation of V. parahaemolyticus involves numerous regulatory systems and specific structures, including cyclic-di-GMP (c-di-GMP), quorum sensing (QS), type IV pili, and flagella. The strong ability of V. parahaemolyticus to form biofilm on various food contact surfaces has been reported as a major food safety concern in the seafood industry (Ashrafudoulla et al., 2020). Approximately 80 % of bacteria exist in the form of biofilm (Zheng et al., 2020), but it is challenging to eradicate biofilm. Biofilm cells demonstrated enhanced resistance to antibiotics and host immunological responses. Biofilm has been reported to be the root cause of many chronic and persistent bacterial infections (Chen et al., 2020). Hence, comprehending the molecular mechanisms of biofilm formation and virulence in V. parahaemolyticus is essential to alleviating infections and contamination in food industry.

The colonial morphology is known to reflect the different expressions of bacterial surface components within the colonies (Haase et al., 1999). The rough-colony protein, RcpA was first discovered in the outer membrane fraction of the adherent rough-colony-forming Actinobacillus actinomycetemcomitans cells (Clock et al., 2008; Saito et al., 2010). RcpA is a transcriptional regulator and that regulates the whole secretin gene superfamily. Its closely related homologs comprise a distinct and novel subfamily (Pourhajibagher et al., 2017). It has been demonstrated to regulate the expression of fimbria-associated gene in Aggregatibacter actinomycetemcomitans (Pourhajibagher et al., 2017). Nevertheless, no research has been conducted about the role of rcpA in V. parahaemolyticus.

In this study, we constructed the rcpA mutant to investigate the impact of rcpA gene on multiple factors involved in V. parahaemolyticus biofilm formation and virulence in vivo. Additionally, RNA-seq was used to unravel the regulons of rcpA and explore the potential mechanisms of rcpA in biofilm formation and virulence of V. parahaemolyticus.