Abstract
ABSTRACTEnteropathogenic Escherichia coli (EPEC) has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS), but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. EspC produced by EPEC cleaves fodrin, paxillin, and focal adhesion kinase (FAK), which are also cleaved by caspases and calpains during apoptosis. Here we show the role of EspC in cell death induced by EPEC. EspC is involved in EPEC-mediated cell death and induces both apoptosis and necrosis in epithelial cells. EspC induces apoptosis through the mitochondrial apoptotic pathway by provoking (i) a decrease in the expression levels of antiapoptotic protein Bcl-2, (ii) translocation of the proapoptotic protein Bax from cytosol to mitochondria, (iii) cytochrome c release from mitochondria to the cytoplasm, (iv) loss of mitochondrial membrane potential, (v) caspase-9 activation, (vi) cleavage of procaspase-3 and (vii) an increase in caspase-3 activity, (viii) PARP proteolysis, and (ix) nuclear fragmentation and an increase in the sub-G1 population. Interestingly, EspC-induced apoptosis was triggered through a dual mechanism involving both independent and dependent functions of its EspC serine protease motif, the direct cleavage of procaspase-3 being dependent on this motif. This is the first report showing a shortcut for induction of apoptosis by the catalytic activity of an EPEC protein. Furthermore, this atypical intrinsic apoptosis appeared to induce necrosis through the activation of calpain and through the increase of intracellular calcium induced by EspC. Our data indicate that EspC plays a relevant role in cell death induced by EPEC.
Highlights
Enteropathogenic Escherichia coli (EPEC) has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS), but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood
To further understand the relationship between cytotoxicity and cell death caused by EspC, we sought the correlation between the cytotoxic effects and cell death caused by EspC by using EPEC wild-type (WT) strain E2348/69, an espC mutant (⌬espC) strain, and the complemented (⌬espC/pespC) strain
The complementation of the mutant with a site-directed mutation in the serine protease motif of EspC (⌬espC/pespCS256I) caused similar damage to the ⌬espC strain (Fig. 1E), suggesting an important role played by the enzymatic activity in the development of cell toxicity. (In this complemented strain, pespCS256I is a plasmid carrying the espC gene that encodes a change from serine to isoleucine at amino acid position 256.)
Summary
Enteropathogenic Escherichia coli (EPEC) has the ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the type III secretion system (T3SS), but the total number of these effectors and the overall functional relationships between these effectors during infection are poorly understood. We show for the first time that this serine protease motif is able to cleave procaspase-3, thereby reaching the terminal stages of caspase cascade activation leading to apoptosis This overlapped apoptosis appears to potentiate cell death through necrosis, where EspC induces calpain activation and increases intracellular calcium. Several EPEC T3SS effectors interfere with host apoptotic pathways, such as NleD and NleB1/NleB2 for the extrinsic pathway and NleH1/NleH2, EspZ, EspT, and EspM (the latter two by inhibiting EspH) for the intrinsic pathway, as well as NleF, which directly inhibits caspases involved in both apoptotic pathways [8, 27] All of these data suggest EPEC’s ability to antagonize host apoptosis during infection through promotion and inhibition of effectors injected by the T3SS; many other effectors could play a role in driving to a final outcome. We showed that during EPEC infection, EspC is secreted from the bacteria by T5SS and can be efficiently translocated into epithelial cells by the T3SS translocon [32]
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