Studies On Interactions Between Enterotoxigenic Escherichia Coli, Intestinal Epithelial Cells And Lactic Acid Bacteria

Enterotoxigenic Escherichia coli (ETEC) are often associated with the outbreaks of diarrhea in animals and human beings worldwide. The adhesins of ETEC mediate bacterial adherence to the intestine, after binding of the fimbriae to enterocytes, ETEC proliferate rapidly to attain massive numbers and produce one or more types of enterotoxin, which stimulate fluid and electrolyte secretion by intestinal cells, thus leading to diarrhea. Some lactic acid bacteria were identified as probiotics, which can help the host with the improvement of gut microbial ecology and confer health benefits to animals and humans. Previous report showed that ETEC induced cell death of several intestinal epithelial cells in vitro, and lactic acid bacteria may protect the intestinal epithelial cells through the interactions with ETEC. However, the mechanisms of the actions are largely undefined. Therefore, understanding the ETEC pathogenesis of infecting host cells, and the potential protecting mechanisms of lactic acid bacteria would provide new insights into the study of how lactic acid bacteria protect the host.In this study, we incubated pig intestinal IPEC-J2 cells with two concentrations of ETEC K88 strain JG280. We observed that 108 CFU/ml of ETEC K88 strain JG280 caused more death of IPEC-J2 cells than did 109 CFU/ml, suggesting that ETEC-induced cell death was cell density-dependent and that quorum sensing (QS) may play a role in pathogenesis. Subsequent investigations demonstrated a positive correlation between autoinducer 2 (AI-2) activity of ETEC K88 strain JG280 and death of IPEC-J2 cells during the infection up to 3 hours. However, there was a negative correlation between AI-2 activity and expression of the ETEC K88 strain JG280 enterotoxin genes estA and estB when IPEC-J2 cells were exposed to the pathogen at 108 CFU/ml. To further understand the QS mechanisms of ETEC, we therefore cloned the luxS gene (responsible for AI-2 production) from ETEC K88 strain JG280, and overexpressed it in E. coli DH5?. Addition of culture fluid from E. coli DH5?with the overexpressed luxS reduced cell death of IPEC-J2 by 108 CFU/ml JG280. The addition also reduced the estA expression of ETEC K88 strain JG280. These results suggest the involvement of AI-2-mediated quorum sensing in K88+ ETEC pathogenesis, possibly through a negative regulation of STa production.In this study, we have also investigated possible protection of thirteen previously selected lactic acid bacteria (LAB) strains from swine, against intestinal cell damage by ETEC K88 strain JG280 infection and the underlying mechanisms. In previous study, we observed that the non-pathogenic K88+ E. coli strain JFF4 (toxins-negative) caused no cytotoxicity to IPEC-J2 cells at both concentrations of 108 CFU/ml and 109 CFU/ml, implying the effect of enterotoxins on cell damage/death. Five out of thirteen LAB strains were able to prevent the ETEC-induced cytotoxicity to IPEC-J2 cells. Further investigations on six LAB strains CL9, CL11, CL12, K67, S33 and S64 showed that IL-8 (interleukin-8, pro-inflammatory cytokine) production was decreased by these six strains, and IL-10 (interleukin-10, anti-inflammatory cytokine) production was increased by five strains, except K67. Quantitative PCR analysis showed that expression of two enterotoxin genes estA and estB of ETEC were reduced by CL9 (an effective LAB strain) during incubation. Subsequent investigations showed that LAB strain S8 significantly increased the AI-2 activity produced by ETEC, and meanwhile inhibited the ETEC cytotoxicity to IPEC-J2 cells. Together, our results indicate that some LAB strains inhibit the intestinal cell damage caused by ETEC through inhibiting the enterotoxin gene expression of ETEC, and some LAB might secrete a molecule(s) that interfere with ETEC quorum sensing signaling. Meanwhile inflammatory cytokines IL-8 and IL-10 may involve in the lactic acid bacteria-reduced inflammation of IPEC-J2. To determine the molecular mechanism of the protection offered by the LAB strains, further studies are required.