Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions

Abstract

SummaryEnteropathogenic Escherichia coli (EPEC) increases tight junction permeability in part by phosphorylating the 20 kDa myosin light chain (MLC 20 ) that induces cytoskeletal contraction. The impact of this enteric pathogen on specific tight junction (TJ) proteins has not been investigated. We examined the effect of EPEC infection on occludin localization and phosphorylation in intestinal epithelial cells. After infection by EPEC, a progressive shift of occludin from a primarily TJ-associated domain to an intracellular compartment occurred, as demonstrated by immunofluorescent staining. A reverse in the ratio of phosphorylated to dephosphorylated occludin accompanied this morphological change. Eradication of EPEC with gentamicin resulted in the normalization of occludin localization and phosphorylation. The serine/threonine phosphatase inhibitor, calyculin A, prevented these events. The EPEC-associated decrease in transepithelial electrical resistance, a measure of TJ barrier function, returned to baseline after gentamicin treatment. Non-pathogenic E. coli, K-12, did not induce these changes. Transformation of K-12 with the pathogenicity island of EPEC, however, conferred the phenotype of wild-type EPEC. Deletion of specific EPEC genes encoding proteins involved in EPEC type III secretion markedly attenuated these effects. These findings suggest that EPEC-induced alterations in occludin contribute to the pathophysiology associated with this infection.

Comparative Analysis of EspF from Enteropathogenic and Enterohemorrhagic Escherichia coli in Alteration of Epithelial Barrier Function

“…EPEC and EHEC similarly disrupt occludin from TJ. EPEC has been shown to redistribute the transmembrane TJ protein occludin, an event that correlates with both occludin dephosphorylation and a decrease in TER (34). Occludin contributes to formation of the TJ barrier, consistent with its uniform and peripheral distribution in uninfected cells, as seen in Fig.…”

“…Occludin migrates as two or more bands, based on its phosphorylation status; P refers to phosphorylated occludin (33). The amount of lesser-phosphorylated occludin increases as EHEC infection progresses, as previously reported for EPEC (33,34 teins by EPEC and EHEC. EPEC EspF has previously been shown to be expressed at low levels in LB and highly upregulated in DMEM in a CesF-dependent manner (9).…”

“…Like EPEC, EHEC also redistributed occludin, as shown by a loss of protein from the membrane and increased intracellular localization ( Fig. 2A) (34). When analyzed by Western blotting, occludin resolves according to its phosphorylation status, with phosphorylated forms migrating as a higher-molecularweight band than the lesser or nonphosphorylated forms.…”

“…T84 monolayers on Transwell inserts (Costar) were infected with mid-log growth phase bacteria, and the transepithelial electrical resistance (TER) was measured at 2, 4, and 6 h postinfection as previously described (34). Monolayers infected with EPEC or EHEC at an MOI of 100 (unless otherwise indicated) for 1 h were washed to remove nonadherent bacteria and incubated for additional time (1, 3, or 5 h) in the presence of fresh DMEM.…”

“…Transfer of the cloned EPEC LEE into E. coli K-12 confers the ability to induce A/E lesions and inject effector proteins into host cells (22). This strain was also able to displace occludin from the tight junctions (TJ) and disrupt the barrier function of host cells (34). In contrast, E. coli K-12 transformed with a clone containing the EHEC LEE was unable to induce A/E lesions or inject effector proteins into host cells, even when cotransformed with fragments from EPEC LEE (12).…”