EspC Promotes Epithelial Cell Detachment by Enteropathogenic Escherichia coli via Sequential Cleavages of a Cytoskeletal Protein and then Focal Adhesion Proteins

Abstract

EspC is a non-locus of enterocyte effacement (LEE)-encoded autotransporter produced by enteropathogenic Escherichia coli (EPEC) that is secreted to the extracellular milieu by a type V secretion system and then translocated into epithelial cells by the type III secretion system. Here, we show that this efficient EspC delivery into the cell leads to a cytopathic effect characterized by cell rounding and cell detachment. Thus, EspC is the main protein involved in epithelial cell cytotoxicity detected during EPEC adhesion and pedestal formation assays. The cell detachment phenotype is triggered by cytoskeletal and focal adhesion disruption. EspC-producing EPEC is able to cleave fodrin, paxillin, and focal adhesion kinase (FAK), but these effects are not observed when cells are infected with an espC isogenic mutant. Recovery of these phenotypes by complementing the mutant with the espC gene but not with the espC gene mutated in the serine protease motif highlights the role of the protease activity of EspC in the cell detachment phenotype. In vitro assays using purified proteins showed that EspC, but not EspC with an S256I substitution [EspC S256I ], directly cleaves these cytoskeletal and focal adhesion proteins. Kinetics of protein degradation indicated that EspCproducing EPEC first cleaves fodrin (within the 11th and 9th repetitive units at the Q1219 and D938 residues, respectively), and this event sequentially triggers paxillin degradation, FAK dephosphorylation, and FAK degradation. Thus, cytoskeletal and focal adhesion protein cleavage leads to the cell rounding and cell detachment promoted by EspC-producing EPEC.

A Novel Mechanism for Protein Delivery by the Type 3 Secretion System for Extracellularly Secreted Proteins

“…EspC protein has to get inside the cells in order to cleave their intracellular targets such as fodrin and focal adhesion proteins such as FAK and paxillin (27) as well as proteins related to the apoptosis cascade such as pro-caspase 3 (28). Thus, a key step for the cell damage induced by EspC is its internalization process.…”

“…EspC is able to exert cytotoxic effects on epithelial cells, and these effects depend on its serine protease motif (26). EspC protein has to get inside the cells in order to cleave intracellular targets such as fodrin and focal adhesion proteins such as focal adhesion kinase (FAK) and paxillin (27) as well as proteins related to the apoptosis cascade such as pro-caspase 3 (28). The cleavage of these intracellular targets by EspC leads to cell rounding and detachment followed by cell death through apoptosis and necrosis (28).…”

Diarrheagenic Escherichia coli

“…One such protein, EspC, which is secreted by the type V secretion system and is injected by the T3SS in epithelial cells, has an IgA protease-like activity and, once in the host cytoplasm, has various cytopathic effects, including cytoskeletal damage, 68 enhanced lysozyme resistance, 70 hemoglobin degradation, 69 hydrolysis of pepsin, factor V, and spectrin, 70 and fodrin and focal adhesion protein degradation 71 . In addition, oligomerization of EspC gives rise to rope-like structures that serve as a substratum for adherence and biofilm formation as well as protecting bacteria from antimicrobial compounds 72 …”

Serine Protease Autotransporters of the Enterobacteriaceae (SPATEs): Out and About and Chopping It Up

“…EspC cleavage of fodrin occurred outside of the calmodulin binding domain [55]. Once inside the cells, kinetics of protein degradation indicate that purified EspC cleaves fodrin at two sites (within the 11th repetitive unit between Q 1219 and L1 220 and within the 9th repetitive unit between D 938 and L 939 ) which then results in disruption of focal adhesion including dephosphorylation and degradation of paxillin and FAK; leading to cell rounding and detachment [58]. However, entry to a target cell (cytosol) is critical for EspC cytotoxicity.…”