Vibrio parahaemolyticus is a species of gram-negative, halophilic bacteria ubiquitous in the marine environment worldwide and prevalent in fish, shellfish, shrimp, and other aquatic products [1]. It is a typical pathogenic bacterium found in aquatic animals [2]. Consumption of contaminated water or undercooked seafood may lead to V. parahaemolyticus infections in humans, resulting in acute gastroenteritis [3]. The significant increase in fresh seafood consumption in recent years has led to a high number of cases of food poisoning caused by V. parahaemolyticus [4], and this bacterium has become a key food pathogen worldwide.

A flagellum is the motility organelle in bacterial cells, which not only helps bacteria invade host cells but also plays a toxic role in the process of bacterial disease. Flagellin is a subunit protein of the flagellar filament in bacteria, and flagellins of different bacterial species vary in structure, size, and function [5,6]. Moreover, different flagellar proteins in a particular bacterial species have distinct immune functions [7,8]. Owing to its strong immune activation effect, flagellin has been widely used in vaccine research and development [9]. Previous studies have investigated the structure and immune function of flagellin and its role in signaling pathways, gradually enhancing the understanding of the function of flagellin. Nevertheless, the flagellin of V. parahaemolyticus remains underexplored. Gene knockout technology provides an effective approach to study the functional mechanism of V. parahaemolyticus flagellin by constructing a flagellin deletion mutant strain.

Bacterial gene knockout is a type of genetic modification technology [10]. Using this technology, a gene of interest can be precisely inactivated or deleted by altering or deleting the gene sequence; the resultant loss of gene function facilitates the investigation of changes in phenotypic traits or the related physical and chemical characteristics of the gene deletion mutant strain and thus the biological function of the gene [11]. To date, several gene knockout strategies have been used in bacteria, but no unified and effective knockout strategy has been identified for a specific bacterial species [[12], [13], [14]]. Although gene knockout technology has been widely used to explore gene function, it has limitations. For example, knockout of target genes in bacteria may lead to cell death; the same plasmid vector may play a significantly different role in the bacterial genetic system in different bacterial species; and different plasmids and bacterial species require diverse transformation conditions.

Bacterial species exhibit significant differences in their morphological structure, gene regulation system, and physical and chemical characteristics. Many bacteria exhibit thick cell walls and have poor permeability, leading to low gene knockout efficiency. The gene knockout strategies currently used in bacteria have several limitations such as low transformation efficiency, homologous arm length, and cumbersome protocol, which limit the study of bacterial gene function [15,16]. An effective gene knockout strategy has not yet been reported for V. parahaemolyticus, and the combined frequencies of plasmid transfer and recombination are low; therefore, several conjugations in parallel are necessary to obtain the desired strain. Therefore, in this study, we aimed to circumvent this problem by developing a gene knockout method suitable for V. parahaemolyticus.

In a previous study, we investigated the pro-inflammatory role of various flagellins of V. parahaemolyticus (flaA, flaB, flaC, flaD, flaE, and flaF); flaF exhibited the strongest immunostimulatory effect [17]. Therefore, in the present study, we aimed to further analyze the function of flaF in V. parahaemolyticus infection. We used overlapping PCR and two-step homologous recombination to construct a flaF-deficient mutant of V. parahaemolyticus and used the intestinal mucosal epithelial cells of silver pomfret (Pampus argenteus) as an in vitro model of the intestine to compare and analyze the virulence of wild-type (WT) and mutant strains. Our findings not only reveal the functional mechanism of flagellin but also offer a basis for the improvement and development of gene knockout approaches applicable to bacteria.