Roles and mechanisms of gapA and gap-encoded proteins in cronobacter sakazakii adhesion to and invasion of intestinal cells and neonatal rats

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen whose initial process involves intestinal cell adhesion mediated by numerous virulence factors encoded in various genes. The key metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, also known as GapA), is encoded by gapA in the glycolysis pathway and acts as an adhesin in some bacteria. In C. sakazakii, there is also a key enzyme in the gluconeogenesis pathway, Gap, with type I GAPDH and erythrose-4-phosphate dehydrogenase activities and encoded by gap. This study aimed to investigate the virulence properties of GapA and Gap of C. sakazakii in adhesion to and invasion of HCT-8 and HIEC-6 cells and damage to the colon and brain of neonatal rats, by gene silencing. In addition, the role of both recombinant proteins in intestinal cell adhesion and invasion was investigated, and their role in inducting inflammatory cytokine expression was assessed by ELISA and Western blot. Silencing gapA or gap decreased the viability and swimming motility of bacterial cells and reduced bacterial adhesion to and invasion of both types of intestinal cells. Both recombinant proteins contributed to C. sakazakii adhesion in both cells, enhanced protein phosphorylation of NF-κB, and induced inflammatory cytokine expression. Finally, silenced expression of GapA and Gap also weakened bacterial damage to the brain and colon of neonatal rats. In conclusion, we demonstrated for the first time the virulence properties of GapA and Gap in C. sakazakii adhesion to and invasion of intestinal cells and neonatal rats and induction of inflammatory responses.