Screening of genes involved in interactions with intestinal epithelial cells in Cronobacter sakazakii

Xin-jun Du, Xia Zhang, Ping Li, Rui Xue, Shuo Wang

Cronobacter sakazakii possesses a significant ability to adhere to and invade epithelial cells in its host. However, the molecular mechanisms underlying this process are poorly understood. In the current study, the adhesive and invasive capabilities of 56 C. sakazakii strains against human epithelial cells were evaluated, and one of them was selected for construction of a mutant library using the Tn5 transposon. In a systematic analysis of the adhesive and invasive capabilities of 1084 mutants, 10 mutants that showed more than a 50 % reduction in adhesion or invasion were obtained. Tail-PCR was used to sequence the flanking regions of the inserted transposon and 8 different genes (in 10 different mutants) were identified that encoded an exonuclease subunit, a sugar transporter, a transcriptional regulator, two flagellar biosynthesis proteins, and three hypothetical proteins. Raman spectroscopy was used to analyze variations in the biochemical components of the mutants, and the results showed that there were fewer amide III proteins, protein -CH deformations, nucleic acids and tyrosines and more phenylalanine, carotenes, and fatty acids in the mutants than in the wild type strain. Real-time PCR was used to further confirm the involvement of the genes in the adhesive and invasive abilities of C. sakazakii, and the results indicated that the expression levels of the 8 identified genes were upregulated 1.2- to 11.2-fold. The results of this study provide us with insight into the mechanism by which C. sakazakii infects host cells at molecular level.