Effects of ESA_00986 Gene on Adhesion/Invasion and Virulence of Cronobacter sakazakii and Its Molecular Mechanism

Cronobacter sakazakii is an opportunistic Gram-negative pathogen that has been identified as a causative agent of severe foodborne infections with a higher risk of mortality in neonates, premature infants, the elderly, and immunocompromised populations. The specific pathogenesis mechanisms of C. sakazakii, such as adhesion and colonization, remain unclear. Previously, we conducted comparative proteomic studies on the two strains with the stronger and weaker infection ability, respectively, and found an interesting protein, ESA_00986, which was more highly expressed in the strain with the stronger ability. This unknown protein, predicted to be a type of invasitin related to invasion, may be a critical factor contributing to its virulence. This study aimed to elucidate the precise roles of the ESA_00986 gene in C. sakazakii by generating gene knockout mutants and complementary strains. The mutant and complementary strains were assessed for their biofilm formation, mobility, cell adhesion and invasion, and virulence in a rat model. Compared with the wild-type strain, the mutant strain exhibited a decrease in motility, whereas the complementary strain showed comparable motility to the wild-type. The biofilm-forming ability of the mutant was weakened, and the mutant also exhibited attenuated adhesion to/invasion of intestinal epithelial cells (HCT-8, HICE-6) and virulence in a rat model. This indicated that ESA_00986 plays a positive role in adhesion/invasion and virulence. This study proves that the ESA_00986 gene encodes a novel virulence factor and advances our understanding of the pathogenic mechanism of C. sakazakii.