Enteropathogenic Escherichia coli (EPEC) causes severe diarrheal disease and is present globally. EPEC virulence requires a bacterial
type III secretion system to inject >20 effector
proteins into human intestinal cells. Three effectors travel to mitochondria and modulate apoptosis; however, the mechanisms by which effectors control apoptosis from within mitochondria are unknown. To identify and quantify global changes in mitochondrial proteolysis during
infection, we applied the mitochondrial terminal proteomics technique mitochondrial stable isotope labeling by
amino acids in cell culture-terminal
amine isotopic labeling of substrates (MS-TAILS). MS-TAILS identified 1,695 amino N-terminal
peptides from 1,060 unique
proteins and 390 N-terminal
peptides from 215
mitochondrial proteins at a false discovery rate of 0.01.
Infection modified 230 cellular and 40
mitochondrial proteins, generating 27 cleaved mitochondrial neo-N termini, demonstrating altered proteolytic processing within mitochondria. To distinguish proteolytic events specific to EPEC from those of canonical apoptosis, we compared mitochondrial changes during
infection with those reported from chemically induced apoptosis. During
infection, fewer than half of all mitochondrial cleavages were previously described for canonical apoptosis, and we identified nine mitochondrial proteolytic sites not previously reported, including several in
proteins with an annotated role in apoptosis, although none occurred at canonical
Asp-Glu-Val-Asp (DEVD) sites associated with
caspase cleavage. The identification and quantification of novel neo-N termini evidences the involvement of noncaspase human or EPEC
protease(s) resulting from mitochondrial-targeting effectors that modulate cell death upon
infection. All proteomics data are available via ProteomeXchange with identifier PXD016994IMPORTANCE To our knowledge, this is the first study of the mitochondrial
proteome or N-terminome during
bacterial infection. Identified cleavage sites that had not been previously reported in the mitochondrial N-terminome and that were not generated in canonical apoptosis revealed a pathogen-specific strategy to control human cell apoptosis. These data inform new mechanisms of
virulence factors targeting mitochondria and apoptosis during
infection and highlight how enteropathogenic Escherichia coli (EPEC) manipulates human cell death pathways during
infection, including candidate substrates of an EPEC
protease within mitochondria. This understanding informs the development of new antivirulence strategies against the many human pathogens that target mitochondria during
infection. Therefore, mitochondrial stable isotope labeling by
amino acids in cell culture-terminal
amine isotopic labeling of substrates (MS-TAILS) is useful for studying other pathogens targeting human cell compartments.