THE ESTABLISHED INTIMIN-RECEPTOR TIR AND THE PUTATIVE EUCARYOTIC INTIMIN-RECEPTORS NUCLEOLIN AND BETA1 INTEGRIN LOCALIZE AT OR NEAR THE SITE OF ENTEROHEMORRHAGIC ESCHERICHIA COLI O157:H7 ADHERENCE TO ENTEROCYTES IN VIVO

Authors: SINCLAIR, JAMES - UNIFORMED SRVCS UNIV HLTH; Nystrom, Evelyn; O'BRIEN, ALISON - UNIFORMED SRVCS UNIV HLTH

Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/26/2005
Publication Date: 2/1/2006
Citation: Sinclair, J.F., Nystrom, E.A., O'Brien, A.D. 2006. The established intimin receptor Tir and the putative eucaryotic intimin receptors nucleolin and beta1 integrin localize at or near the site of enterohemorrhagic Escherichia coli O157:H7 adherence to enterocytes in vivo. Infection and Immunity. 74:(2):1255-1265.

Interpretive Summary: Cattle are important reservoirs of Shiga-like toxin-producing Escherichia coli (STEC) O157:H7 foodborne pathogens that cause severe diarrhea and sometimes kidney failure and death in humans. STEC O157:H7 must express an adhesin called intimin and a receptor for intimin (Tir), which it inserts into the surface of the host intestinal epithelial cells. Intimin can bind to eukaryotic proteins called nucleolin and beta1 integrin in cultured tissue cells. Our objective was to evaluate the potential biological relevance of these eucaryotic proteins in the infection process in vivo. We used immunofluorescent staining to compare the proximity of Tir, nucleolin, and beta1 integrin to regions where STEC O157:H7 bacteria were attached in formalin-fixed sections of intestinal tissues obtained from neonatal calves, piglets, and mice inoculated with STEC O157:H7. We noted immunostained Tir and nucleolin closely associated with adherent O157-positive bacteria in intestinal sections from all three animal species. We also observed immunostained beta1 integrin clustered at locations of bacterial adherence in porcine and bovine tissues. These findings indicate that nucleolin and beta1 integrin are present on surface of intestinal epithelial cells and are potentially accessible as receptors for intimin during STEC O157:H7 infection. This new information increases our understanding of host factors involved in STEC O157:H7 infections and will facilitate development of strategies to reduce STEC infections in food animals.

Technical Abstract: For Enterohemorrhagic E. coli (EHEC) O157:H7 to adhere tightly to the intestinal epithelium and produce attach and efface (A/E) lesions, the organism must express the adhesion intimin and insert the bacterially-encoded translocated intimin receptor TIR into the plasma membrane of the host enterocyte. Additionally, some reports based on tissue culture experiments indicated that intimin has affinity for the eucaryotic proteins nucleolin and beta1 integrin. To address the potential biological relevance of these eucaryotic proteins in the infection process in vivo, we sought to compare the proximity of Tir, nucleolin, and beta1 integrin to regions of EHEC O157:H7 attachment in intestinal sections from three different inoculated animals: piglets, neonatal calves, and mice. Piglets and neonatal calves were chosen because intimin-mediated adherence of EHEC O157:H7 and subsequent A/E lesion formation occurs at high levels in these animals. Mice were selected because of their ease of manipulation but only after we first demonstrated that in competition with the normal mouse gut flora, an EHEC O157:H7 strain with a non-polar deletion in the intimin gene was cleared faster than strains that produced wild-type or hybrid intimin. In all three animal species, we noted immunostained Tir beneath, and stained nucleolin closely associated with adherent bacteria in intestinal sections. We also observed immunostained beta1 integrin clustered at locations of bacterial adherence in porcine and bovine tissue. These fines indicate that nucleolin and beta1 integrin are present on the luminal surface of intestinal epithelia and are potentially accessible as receptors for intimin during EHEC O157:H7 infection.