Characterization of Molecular Chaperone GroEL as a Potential Virulence Factor in Cronobacter sakazakii

The molecular chaperone GroEL of C. sakazakii, a highly conserved protein encoded by the gene grol, has the basic function of responding to heat shock, thus enhancing the bacterium's adaptation to dry and high-temperature environments, which poses a threat to food safety and human health. Our previous study demonstrated that GroEL was found in the bacterial membrane fraction and caused a strong immune response in C. sakazakii. In this study, we tried to elucidate the subcellular location and virulent effects of GroEL. In live C. sakazakii cells, GroEL existed in both the soluble and insoluble fractions. To study the secretory mechanism of GroEL protein, a non-reduced Western immunoblot was used to analyze the form of the protein, and the result showed that the exported GroEL protein was mainly in monomeric form. The exported GroEL could also be located on bacterial surface. To further research the virulent effect of C. sakazakii GroEL, an indirect immunofluorescence assay was used to detect the adhesion of recombinant GroEL protein to HCT-8 cells. The results indicated that the recombinant GroEL protein could adhere to HCT-8 cells in a short period of time. The recombinant GroEL protein could activate the NF-κB signaling pathway to release more pro-inflammatory cytokines (TNF-α, IL-6 and IL-8), downregulating the expression of tight-junction proteins (claudin-1, occluding, ZO-1 and ZO-2), which collectively resulted in dose-dependent virulent effects on host cells. Inhibition of the grol gene expression resulted in a significant decrease in bacterial adhesion to and invasion of HCT-8 cells. Moreover, the deficient GroEL also caused slow growth, decreased biofilm formation, defective motility and abnormal filamentation of the bacteria. In brief, C. sakazakii GroEL was an important virulence factor. This protein was not only crucial for the physiological activity of C. sakazakii but could also be secreted to enhance the bacterium's adhesion and invasion capabilities.