Disruptionof Cell Polarity by Enteropathogenic Escherichia coli EnablesBasolateral Membrane Proteins To Migrate Apically and ToPotentiate PhysiologicalConsequences

Abstract

Enteropathogenic Escherichia coli (EPEC) disrupts the structure and barrier function of host intestinal epithelial tight junctions (TJs). The impact of EPEC on TJ "fence function," i.e., maintenance of cell polarity, has not been investigated. In polarized cells, proteins such as ␤ 1 -integrin and Na ؉ /K ؉ ATPase are restricted to basolateral (BL) membranes. The outer membrane EPEC protein intimin possesses binding sites for the EPEC translocated intimin receptor (Tir) and ␤ 1 -integrin. Restriction of ␤ 1 -integrin to BL domains, however, precludes opportunity for interaction. We hypothesize that EPEC perturbs TJ fence function and frees BL proteins such as ␤ 1 -integrin to migrate to apical (AP) membranes of host cells, thus allowing interactions with bacterial adhesins such as intimin. The aim of this study was to determine whether EPEC alters the polar distribution of BL proteins, in particular ␤ 1 -integrin, and if such redistribution contributes to pathogenesis. Human intestinal epithelial T84 cells and EPEC strain E2348/69 were used. Selective biotinylation of AP or BL membrane proteins and confocal microscopy showed the presence of ␤ 1 -integrin and Na ؉ /K ؉ ATPase on the AP membrane following infection. ␤ 1 -Integrin antibody afforded no protection against the initial EPECinduced decrease in transepithelial electrical resistance (TER) but halted the progressive decrease at later time points. While the effects of EPEC on TJ barrier and fence function were Tir dependent, disruption of cell polarity by calcium chelation allowed a tir mutant to be nearly as effective as wild-type EPEC. In contrast, deletion of espD, which renders the type III secretory system ineffective, had no effect on TER even after calcium chelation, suggesting that the putative ␤ 1 -integrin-intimin interaction serves to provide intimate contact, like that of Tir and intimin, making translocation of effector molecules more efficient. We conclude that the initial alterations of TJ barrier and fence function by EPEC are Tir dependent but that later disruption of cell polarity and accessibility of EPEC to BL membrane proteins, such as ␤ 1 -integrin, potentiates the physiological perturbations.

Polyethylene glycol diminishes pathological effects ofCitrobacter rodentiuminfection by blocking bacterial attachment to the colonic epithelia

“…17,18 During infection, β 1 -integrin relocalizes from the basolateral to the apical membrane to interact with bacterial intimin, which is essential for disruption of intestinal barrier function. 19 This study demonstrates that PEG decreases the amount of β 1 -integrin in intestinal epithelia and that pretreatment with PEG is sufficient to reduce attachment of C. rodentium. It has been demonstrated that high-molecular weight PEG inhibits the attachment of Pseudomonas to host cells by forming a physical barrier between the bacteria and host cells; 32 however, this type of PEG is not for systemic oral use.…”

“…We showed that low-molecular weight PEG downregulates β 1 -integrin, a host cell surface receptor critical for A/E pathogen attachment. [16][17][18][19] Pretreatment of colonic epithelia with PEG, concurrent presence of PEG during infection, or PEG treatment after the start of infection decreases the attachment of C. rodentium to colonic epithelia. We also presented evidence that PEG attenuates EGFR signaling and improves clinical signs of disease in mouse colon infected with C. rodentium.…”

“…18 These interactions with integrin generate signals in host cells 18 that disrupt the intestinal barrier. 18,19 Inhibiting interaction with integrin may be an efficient strategy for preventing enteric infections. infection in the mouse colon.…”

The role of epithelial malfunction in the pathogenesis of enteropathogenic E. coli-induced diarrhea

“…Upon EPEC infection, basolateral b 1 -integrin moves to the apical side of enterocytes where it can interact with intimin. 50 Although Tir remains essential for bacterial attachment, cytoskeletal rearrangement, and barrier defects, the interaction of b 1 -integrin with intimin further enhances the attachment of EPEC to IECs and is required for maximal decreases in TER. 50 It should be noted that certain gastrointestinal pathogens, such as the parasite Giardia lambia, are known to modulate epithelial permeability through the induction of apoptosis.…”

“…50 Although Tir remains essential for bacterial attachment, cytoskeletal rearrangement, and barrier defects, the interaction of b 1 -integrin with intimin further enhances the attachment of EPEC to IECs and is required for maximal decreases in TER. 50 It should be noted that certain gastrointestinal pathogens, such as the parasite Giardia lambia, are known to modulate epithelial permeability through the induction of apoptosis. 51,52 As mentioned before, EPEC induces apoptosis in infected IECs in a caspase-dependent manner.…”