Although several groups have explored the effect of EPEC on host cell survival, the role of apoptosis in the pathogenesis of this organism is not entirely clear. There is considerable evidence that the secreted protein EspF localizes to the mitochondria of EPEC-infected cells and promotes permeabilization of the mitochondrial membrane, release of cytochrome c
, and cleavage of caspase-3, implicating a role for this protein in host cell death (19
). Even so, EPEC itself is a relatively weak inducer of apoptosis. This could be because EPEC stimulates the EGF receptor, NF-κB, and PKC pathways, all of which promote survival (10
). Moreover, in a rabbit model of EPEC infection, apoptosis was unchanged or possibly less than baseline levels (14
Since EPEC EspF is substantially responsible for infection-induced disruption of epithelial TJs and consequent loss of barrier function, we investigated whether the effects of EspF on host cell death had a role in alteration of paracellular permeability. Two independent lines of investigation failed to demonstrate a causal link between apoptosis and TJ disruption in infected monolayers. First, a site-directed mutant of EspF impaired for causing host cell death promoted TJ disruption and altered barrier function. Second, the pan-caspase inhibitor Q-VD-OPH did not interfere with EPEC-induced barrier disruption.
These results are consistent with what is presently known about these two phenotypes in infected intestinal epithelial cells. First, EPEC-induced effects on tight junctions are observed as early as 2 h postinfection, at a point of time when very little host cell death is evident (18
). Second, one of the limitations of studies demonstrating mitochondrial targeting and apoptosis related to EPEC infection is the need to utilize a high MOI to see such effects (21
) (data not shown). Third, it has been suggested that the EPEC mitochondria-associated protein (Map) and EspF work in concert to effect changes in TJs in a manner that is independent of Map mitochondrial targeting (11
). Finally, there is some evidence that the COOH-terminal region of EspF, lacking the mitochondrial localization signals, can disrupt the epithelial barrier (25
). In this context, our findings suggest that EspF features independent of its mitochondrial localization, such as the interaction of its COOH-terminal domains with N-WASP, contributes to epithelial barrier function disruption. Although our studies rule out apoptosis as a primary cause for EPEC-mediated TJ alterations, we cannot preclude the possibility that cell death may augment barrier disruption at later points of infection. Also, apoptosis may contribute to small conductive leaks, as has been observed in response to TNF-α-induced single cell apoptotic events (13
Apoptosis-dependent barrier function alterations, however, have been observed in other systems. TNF-α-treated HT-29/B6 cells display a progressive decrease in TER, which was attenuated by the caspase inhibitor, Z-VAD-FMK (3
). Caspase-dependent cleavage of occludin was considered to be at least partially responsible for this phenotype. Agonists of the G protein-coupled receptor proteinase-activated receptor (PAR1
) similarly induced apoptosis and concomitantly increased epithelial permeability in a caspase-3-dependent manner in the nontumorigenic SCBN cell line, as well as in mouse colonic epithelium (7
). Caspase-3 inhibitors, as well as myosin light chain kinase inhibitors, abolished PAR1
-dependent TJ alterations. Some strains of the intestinal pathogen Giardia lamblia
) induce apoptosis, which in turn results in increased paracellular permeability (6
). Pharmacological inhibition of caspase-3 blocked G. lamblia
-induced barrier disruption, suggesting a causal role for epithelial apoptosis in barrier disruption. Similarly, it has been shown that single-cell apoptosis in TNF-α-treated monolayers leads to transient small conductive leaks (13
). On the other hand, the pan-caspase inhibitor Z-VAD-FMK failed to block cytokine (IFN-γ and TNF-α)-mediated epithelial barrier function alteration (4
), possibly suggesting the existence of compensatory mechanisms that maintain monolayer integrity.
Studies from various laboratories suggest a complex multifunctional role for EspF. Aside from its role in apoptosis and barrier disruption, EspF inhibits phosphatidylinositol-3 kinase-dependent uptake of EPEC into macrophages by a process independent of its localization to the mitochondria (25
). The ability of this protein to orchestrate such diverse host cell functions and the role of these changes in causing disease remain to be understood.