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Enteropathogenic Escherichia coli (EPEC) infections among infants and young children are common in many developing countries (24); however, the pathophysiology of resultant diarrhea remains uncertain. EPEC produces no recognized enterotoxins (22, 25, 28); rather, diarrhea results from direct interaction of bacteria with the small intestinal epithelium. EPEC adheres to enterocytes and transduces signals (13) which produce an attaching and effacing (A/E) lesion in the brush border membrane characterized by a loss of microvilli and intimate adhesion of bacteria to the apical cell membrane, beneath which host cell cytoskeletal elements accumulate (18, 19, 34).

To study the effects of EPEC interaction on host cell electrolyte transport, we developed an in vitro model of EPEC infection in which monolayers of the human intestinal cell line Caco-2 are rapidly infected with EPEC before being mounted into Ussing chambers (2). Using this technique, we demonstrated a rapid stimulation of short-circuit current (I_sc) across Caco-2 cell monolayers caused by EPEC which indicated modulation of transepithelial electrolyte transport (2). Moreover, the peak of this I_sc response coincided with development of A/E lesions and was partially (~35%) chloride dependent, consistent with EPEC-induced stimulation of chloride secretion.

The A/E phenotype is determined by a 35-kbp region of the EPEC chromosome designated the locus of enterocyte effacement (LEE) (7, 26). At the right end of this pathogenicity island are genes encoding three EPEC secreted proteins, EspA, EspD, and EspB, which are essential for signal transduction to host cells and A/E activity (4, 8, 14, 21); these proteins are exported via a type III secretion apparatus encoded by esc and sep genes at the left end of the LEE (7, 11). EPEC-induced signal transduction results in a number of intracellular changes, including a cascade of inositol phosphates, phosphorylation of host cell proteins, and most significantly, tyrosine phosphorylation of a large protein (formerly Hp90) in the host cell membrane (6, 9, 29). This protein, recently shown to be bacterial in origin and renamed Tir (15), acts as the receptor for intimin, a 94-kDa outer membrane protein product of the LEE eae gene (12). Intimin binding is essential for intimate adhesion of EPEC to host cells and promotes accretion of cytoskeletal elements beneath attached bacteria to produce the mature A/E lesion (30).

The present study was designed to investigate the roles of specific EPEC virulence proteins in the generation of I_sc in Caco-2 cell monolayers; we utilized isogenic EPEC mutants deficient in expression of EspA, EspB, or EspD, in functional type III secretion (escN mutant), or in the expression of intimin (eae mutant). The origins and characteristics of EPEC strains used in this study are listed in Table 1. Bacteria were grown in bicarbonate-buffered Dulbecco's modified Eagle's medium (DMEM; Sigma Chemical Co., Poole, United Kingdom) in an atmosphere of 5% CO₂ in air at 37°C for 4 to 5 h to stimulate expression of LEE-encoded virulence proteins (activated EPEC) (10, 17). As described elsewhere (2), bacteria (10⁸ to ~10⁹ CFU) were centrifuged onto Caco-2 cell monolayers, which had been grown on Transwell polycarbonate microporous cell culture inserts, to facilitate synchronous initial attachment. Infected monolayers were immediately mounted into a modified Ussing chamber apparatus and maintained at 37°C in DMEM (gassed with 5% CO₂-95% O₂) for 60 min; during this time I_sc and transepithelial electrical resistance (TEER) of monolayers were measured continuously (2). Uninfected Caco-2 cell monolayers, subject to all the procedures involved in the infection method except exposure to bacteria, maintained both a constant I_sc and a stable TEER throughout the 60-min study period.

To investigate the respective roles of Esp proteins, functional type III secretion, and intimin-mediated adhesion in EPEC-induced electrical responses, Caco-2 cell monolayers were infected with EPEC wild-type strain E2348/69; with espA, espB, espD, escN, or eae mutants of E2348/69; or with their complementary transformants in which the deficient gene was reintroduced on a plasmid. Light microscopy studies revealed that for all strains, initial infection consistently produced adhesion of discrete bacterial colonies to at least 75% of cells (data not shown).

EPEC stimulates I_sc in Caco-2 cell monolayers. Consistent with our previous observations (2), infection of Caco-2 cells with the wild-type EPEC strain, E2348/69, stimulated a rapid increase in I_sc, which peaked 10 to 15 min after initial infection (mean change in I_sc ± standard error of the mean [SEM] at 10 min, 26.4 [2.6] µA/cm² above the basal I_sc of uninfected monolayers; n = 8; Student's t test, P < 0.001) before falling gradually (Fig. 1A). The peak of this I_sc response was not associated with any significant change in TEER (TEER) of monolayers; however, between 10 and 60 min after initial infection, E2348/69 induced a 31% loss in TEER (mean TEER [SEM] between 10 and 60 min postinfection, 81 [8]  · cm²; P < 0.001; n = 8) similar to that described previously (2).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Isc change (Isc; µA/cm2) in Caco-2 cell monolayers during infection with wild-type EPEC E2348/69 (A); signal transduction-defective mutant UMD872 (espA), UMD864 (espB), UMD870 (espD), or CVD452 (escN) (B); complementary plasmid transformant UMD872pMSD2 (EspA+), UMD864pMSD3 (EspB+), or UMD870pLCL123 (EspD+) (C); and intimin-deficient mutant CVD206 (eae) or its plasmid transformant CVD206pCVD438 (intimin positive) (D). Each point represent the mean change in Isc ± SEM (error bars) of 5 to 10 monolayers compared with the basal Isc of uninfected cells (n = 25).

EspA, EspB, and EspD are required for stimulation of Caco-2 cell I_sc. Changes in I_sc following infection of Caco-2 cell monolayers with E2348/69 gene deletion mutants or their plasmid transformants are shown in Fig. 1. The most striking finding was an absence of the characteristic EPEC-induced I_sc response (Fig. 1A) when Caco-2 cell monolayers were infected with signal transduction-defective mutant UMD872 (espA), UMD864 (espB), UMD870 (espD), or CVD452 (escN) (Fig. 1B). Moreover, the I_sc response was qualitatively restored, albeit at a reduced peak magnitude, when cells were infected with complementary plasmid transformants UMD872pMSD2 (EspA⁺) or UMD864pMSD3 (EspB⁺) (Fig. 1C). Infection with the EspD⁺ transformant strain UMD870pLCL123 induced a more gradual increase in I_sc than that stimulated by the parental strain; this response peaked after ~30 min (Fig. 1C). Therefore, the EPEC genes required for expression and functional type III secretion of three separate signalling proteins (i.e., EspA, EspB, and EspD) all appear to be prerequisite for eliciting an increase in I_sc in Caco-2 cell monolayers. In contrast, infection with the intimin-deficient strain CVD206 (eae) or its transformant CVD206pCVD438 (intimin positive) induced I_sc responses which were qualitatively similar to, although somewhat attenuated compared to, that seen with E2348/69 infection (Fig. 1D), thereby revealing that modulation of transepithelial electrolyte transport is independent of intimate EPEC adherence to host cells.

EPEC signal transduction and intimin expression are necessary for inducing loss of TEER. The reduction in TEER of Caco-2 cell monolayers, seen during infection with strain E2348/69, was not apparent during infection with signal transduction-deficient strain UMD872 (espA), UMD864 (espB), UMD870 (espD), or CVD452 (escN) (Fig. 2A). However, marked reductions in TEER were induced by transformant strains UMD864pMSD3 (EspB⁺) and UMD870pLCL123 (EspD⁺) (Fig. 2B); infection with strain UMD872pMSD2 (EspA⁺) did not significantly reduce TEER. In contrast, infection with the intimin-deficient strain CVD206 (eae) produced a significant increase in TEER over 60 min (Fig. 2A), while infection with its intimin-positive transformant CVD206pCVD438 caused a reduction in TEER comparable to that induced by E2348/69 (Fig. 2B). A detrimental effect of EPEC infection on TEER has been described for several cultured epithelial cell lines, and although this effect was initially attributed to a transcellular defect (1), it is now considered to result primarily from the disruption of intercellular tight junctions, which reduces barrier function of the cell monolayer (27, 31). In Caco-2 cells, we found that both EPEC signal transduction and intimin expression were prerequisite for inducing loss of TEER. A similar observation was previously made by Canil et al. (1), who also noted an increase in TEER during infection with the intimin-deficient strain CVD206. Recently, specific signalling events induced in host cells by EPEC have been found to be dependent on intimin binding (16). Therefore, the detrimental effect of EPEC on the TEER of Caco-2 cell monolayers may occur after intimin binding as a result of late signalling events which alter the integrity of tight junctions. The increase in TEER observed during infection with the intimin-deficient strain CVD206 requires further investigation.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Change in TEER (TEER;  · cm2) in uninfected Caco-2 cell monolayers between 10 and 60 min and after infection with wild-type EPEC E2348/69 or gene-deficient mutant UMD872 (espA), UMD864 (espB), UMD870 (espD), CVD452 (escN), or CVD206 (eae) (A) or complementary plasmid transformants UMD872pMSD2 (EspA+), UMD864pMSD3 (EspB+), UMD870pLCL123 (EspD+), or CVD206pCVD438 (intimin positive) (B). Each bar represents the mean ± SEM (error bar) for 5 to 10 infected monolayers. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 versus uninfected monolayers (n = 25).