Enteropathogenic E. coli (EPEC) infection is a leading cause of morbidity and mortality from diarrhea in developing countries. The mechanisms underlying its pathogenesis are not completely understood. Epithelial tight junctions (TJs) restrict the passage of water and ions across the epithelium. TJs are not static; proteins constantly recycle between the plasma membrane and the cytoplasm with contributions of newly synthesized TJ proteins. EPEC induce TJ disruption in a type III secretion system dependent manner. <P> The hypothesis of this proposal is that specific EPEC effector molecules block homeostatic movement of TJ proteins that maintain and restore TJ structure and barrier function. EPEC effectors EspG1/2 destroy microtubule (MT) networks. We hypothesize that these networks are essential for recycling TJ proteins back to the membrane. NleA interferes with anterograde transport through the ER and Golgi, potentially preventing newly synthesized TJ proteins from reaching the TJ. Together, these effectors likely contribute to disruption of TJ structure and function by impeding repair mechanisms. <P> The following specific aims will address this hypothesis. 1: To determine if EPEC infection delays recovery of TJ structure and barrier function following disruption. The impact of EPEC effectors on TJ recovery after calcium chelation will be determined. The duration of effector expression in infected intestinal epithelial cells (IEC) will be defined using myc-tagged effectors. It is anticipated that effector proteins will persist during TJ recovery and that recovery will be significantly delayed. <P> 2: To investigate the effect of EPEC- induced microtubule disruption on the homeostatic maintenance and recovery of TJs. Biotinylation assays will be used to determine the rate of TJ protein recycling after infection with EPEC WT or DespG1/2. IEC will be transfected to express EspG1, or treated with nocodazole to disrupt MTs. Effects on TJ recovery will be determined. In vivo studies will be performed using a transgenic EGFP-occludin mouse. The rates of intestinal epithelial TJ recovery following infection with wild-type (WT) EPEC versus DespG1/2 will be compared. It is anticipated that more TJ proteins will recycle and TJ recovery will be more rapid in the absence of EspG1/2. 3: To investigate the contribution of EPEC-induced interference of ER-Golgi trafficking of newly synthesized proteins on TJ maintenance and reconstruction and to determine the combined effect of EPEC effectors on TJ recovery. IEC will be transfected to express NleA, or treated with brefeldin A, and the effects on TJ recovery following calcium chelation will be measured. In vivo studies using EGFP-occludin transgenic mice will be performed to compare the rate of TJ recovery after infection with WT EPEC and DnleA. A triple DespG1/2/nleA mutant will be created and its effects on recovery of TJ structure and function will be determined. We hypothesize that NleA will significantly impact TJ recovery structurally and functionally, but that the triple mutant will recover the most rapidly. <P> This proposal is the first to investigate TJ recovery as a pathogenic target. It directly addresses a need for greater understanding of how EPEC affects the intestinal epithelial barrier.
Public Health Relevance: Morbidity in the developing world due to recurrent enteropathogenic E. coli (EPEC) infections contributes to developmental problems in infancy and early childhood due to malnutrition. Long lasting effects on intellectual ability and adult health have also been observed. Our proposal directly addresses a need for greater understanding of how EPEC affects the epithelial barrier, creating the potential for new therapeutics.