The Enteropathogenic Escherichia coli Effector Cif Induces Delayed Apoptosis in Epithelial Cells

Abstract

The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins, the cyclomodulins, which modulate the host cell cycle. Upon injection into the host cell by the type III secretion system of enteropathogenic Escherichia coli (EPEC), Cif induces both G 2 and G 1 cell cycle arrests. The cell cycle arrests correlate with the accumulation of p21 waf1 and p27 kip1 proteins that inhibit CDK-cyclin complexes, whose activation is required for G 1 /S and G 2 /M transitions. Increases of p21 and p27 levels are independent of p53 transcriptional induction and result from protein stabilization through inhibition of the ubiquitin/proteasome degradation pathway. In this study, we show that Cif not only induces cell cycle arrest but also eventually provokes a delayed cell death. Indeed, 48 h after infection with EPEC expressing Cif, cultured IEC-6 intestinal cells were positive for extracellular binding of annexin V and exhibited high levels of cleaved caspase-3 and lactate dehydrogenase release, indicating evidence of apoptosis. Cif was necessary and sufficient for inducing this late apoptosis, and the cysteine residue of the catalytic site was required for Cif activity. These results highlight a more complex role of Cif than previously thought, as a cyclomodulin but also as an apoptosis inducer.Enteropathogenic Escherichia coli (EPEC) constitutes a major cause of severe infant diarrhea in developing countries (25). Infection of intestinal epithelial cells with EPEC produces a characteristic histopathological feature known as an "attaching and effacing" (A/E) lesion. This lesion is characterized by intimate bacterial attachment, formation of an actin-rich pedestal structure, and localized destruction of the brush border microvilli (15,16). This bacterial attachment is detected in vitro through the use of the fluorescent actin staining test (15). The genes required for the formation of A/E lesions are clustered on the pathogenicity island named the locus of enterocyte effacement (LEE), which codes for a type III secretion system (T3SS), a molecular syringe that allows translocation into the host cell of up to 40 effector proteins that subvert eukaryotic cellular pathways for the pathogen's benefit (20, 21). The LEE does not carry all genes necessary for EPEC virulence. Indeed, the cycle inhibiting factor (Cif) belongs to a repertoire of proteins that use the T3SS to be injected into the host cell (19). The cif gene is located on a lambdoid prophage found in the chromosomes of some EPEC and enterohemorrhagic E. coli strains (18,19). The Cif protein is composed of an exchangeable N-terminal domain that is necessary for its secretion and translocation through the T3SS (2). Cif displays substantial structural and functional homology with four putative proteins found in pathogenic and symbiotic bacteria. The crystal structures of different homologs of Cif and the presence of a conserved catalytic triad (Cys109-His165-Gln185) suggest that Cif belongs to a superfamily of cysteine proteases, transglutaminases, and acetyltrans...

Delivery of antibodies to the cytosol

“…Another commonly used readout for protein delivery is cell death as an indicator for the delivery of toxic proteins. 41,[48][49][50] However, the specificity of this readout has to be critically inspected considering the large variety of factors that may influence cell viability in addition to the toxicity that is associated with most transfection reagents.…”

Gut Inflammation: Current Update on Pathophysiology, Molecular Mechanism and Pharmacological Treatment Modalities

“…Colitogenic bacteria can damage the epithelial barrier functions either directly or via producing toxins. For instance AIEC strain LF82 disrupts the tight junction protein zonula occludens-1 [165] and through binding to the cell adhesion molecule CEACAM 6 can lead to abnormal expression of claudin 2 [166], while enteropathogenic Escherichia coli (EPEC) induces epithelial cell apoptosis by producing a bacterial toxin called cycle inhibiting factor [167]. Both results in an increased intestinal permeability, which permits the penetration of luminal antigens and microbes, that can stimulate pro-inflammatory responses.…”

The Cyclomodulin Cycle Inhibiting Factor (CIF) Alters Cullin Neddylation Dynamics

“…CIF induces cytopathic effects such as cell cycle arrest, focal adhesion formation, and stress fiber accumulation and delays apoptosis (21)(22)(23)(24). These correlate to inactivation of a subset of CRLs and have been attributed to CIF-mediated conversion of glutamine 40 of NEDD8 to glutamic acid (Q40E) (25,26).…”