Enteropathogenic Escherichia coli EspH-Mediated Rho GTPase Inhibition Results in Desmosomal Perturbations

Abstract

Background & AimsThe diarrheagenic pathogen, enteropathogenic Escherichia coli (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss.MethodsDSG2 abundance and localization was monitored via immunoblotting and immunofluorescence, respectively. Junctional perturbations were visualized by electron microscopy, and cell–cell adhesion was assessed using dispase assays. EspH alanine-scan mutants as well as pharmacologic agents were used to evaluate impacts on desmosomal alterations. EPEC-mediated DSG2 loss, and its impact on bacterial colonization in vivo, was assessed using a murine model.ResultsThe secreted virulence protein EspH mediates EPEC-induced DSG2 degradation, and contributes to desmosomal perturbation, loss of cell junction integrity, and barrier disruption in infected IECs. EspH sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatase signaling; EspH mutants impaired for Rho guanine nucleotide exchange factor interaction failed to inhibit RhoA or deplete DSG2. Cytotoxic necrotizing factor 1, which locks Rho guanosine triphosphatase in the active state, jasplakinolide, a molecule that promotes actin polymerization, and the lysosomal inhibitor bafilomycin A, respectively, rescued infected cells from EPEC-induced DSG2 loss. Wild-type EPEC, but not an espH-deficient strain, colonizes mouse intestines robustly, widens paracellular junctions, and induces DSG2 re-localization in vivo.ConclusionsOur studies define the mechanism and consequences of EPEC-induced desmosomal alterations in IECs. These perturbations contribute to the colonization and virulence of EPEC, and likely related pathogens.

Epithelial maturity influences EPEC-induced desmosomal alterations

“…15 We recently reported that enteropathogenic Escherichia coli (EPEC) perturbs desmosomes. 16 EPEC effector protein EspH, via its Rho GTPase inhibitory function, induces keratin IF retraction and desmosomal disruption. Beyond EPEC pathogenesis, our studies provide insights on how desmosomes are maintained, particularly in the intestines, and expand our knowledge of the role of Rho GTPases in desmosomal assembly and stability.…”

“…Consistent with a role for Rho-mediated actin polymerization in the maintenance of keratin IFs in intestinal epithelial cells, we observed that the actin-stabilizing compound jasplakinolide rescues infected cells from EPEC-induced keratin retraction. 16 But how does keratin retraction promote desmosomal plaque protein loss? Is the half-desmosome pulled into the cytoplasm while still tethered to the keratin IF, or does keratin dissociate from the desmosome prior to internalization and degradation of the desmosomal plaque components?…”

“…In our recent study, EPEC-induced keratin retraction occurred simultaneously with cytoplasmic redistribution of DSG2, though some DSG2 remained at the cell junctions of C2 BBe cells. 16 In those experiments, cells were grown until 3-days post-confluence, and were infected at a multiplicity of infection (MOI) of 100.…”

“…It should be noted, however, that there was a near-complete loss of DSG2 in most cells by 3-4 hours post-EPEC infection, irrespective of monolayer maturity. 16 To gain insight into the underlying mechanistic processes, we specifically focused on the few cells in which DSG2 was still present [ Figure 2, and in the immunofluorescence images in our previous study. 16 ] Collectively, our studies underscore the importance of keratin IF tethering in desmosome plaque stability in the intestinal epithelium, and the potency of RhoGTPase-inactivating compounds such as EPEC EspH in inducing desmosomal damage.…”

Desmosome Disruption by Enteropathogenic E coli

“…In this issue, Roxas et al 4 investigated the impact of EPEC on desmosome disruption in the intestinal epithelia. They examined the contribution of EspH and Rho GTPase signaling to the loss of intestinal epithelial integrity.…”