Global Profiling of Proteolysis from the Mitochondrial Amino Terminome during Early Intrinsic Apoptosis Prior to Caspase-3 Activation.

The human genome encodes ∼20 mitochondrial proteases, yet we know little of how they sculpt the mitochondrial proteome, particularly during important mitochondrial events such as the initiation of apoptosis. To characterize global mitochondrial proteolysis we refined our technique, terminal amine isotopic labeling of substrates, for mitochondrial SILAC (MS-TAILS) to identify proteolysis across mitochondria and parent cells in parallel. Our MS-TAILS analyses identified 45% of the mitochondrial proteome and identified protein amino (N)-termini from 26% of mitochondrial proteins, the highest reported coverage of the human mitochondrial N-terminome. MS-TAILS revealed 97 previously unknown proteolytic sites. MS-TAILS also identified mitochondrial targeting sequence (MTS) removal by proteolysis during protein import, confirming 101 MTS sites and identifying 135 new MTS sites, revealing a wobbly requirement for the MTS cleavage motif. To examine the relatively unknown initial cleavage events occurring before the well-studied activation of caspase-3 in intrinsic apoptosis, we quantitatively compared N-terminomes of mitochondria and their parent cells before and after initiation of apoptosis at very early time points. By identifying altered levels of >400 N-termini, MS-TAILS analyses implicated specific mitochondrial pathways including protein import, fission, and iron homeostasis in apoptosis initiation. Notably, both staurosporine and Bax activator molecule-7 triggered in common 7 mitochondrial and 85 cellular cleavage events that are potentially part of an essential core of apoptosis-initiating events. All mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD009054.

Universal Genotyping for Tuberculosis Prevention Programs: a 5-Year Comparison with On-Request Genotyping.

Prospective universal genotyping of tuberculosis (TB) isolates is used by many laboratories to detect clusters of cases and inform contact investigations. Prior to universal genotyping, most TB prevention programs genotyped isolates on request only, relying on requests from public health professionals whose knowledge of a patient's clinical, demographic, and epidemiological characteristics suggested potential transmission. To justify the switch from on-request to universal genotyping-particularly in the public health domain, with its limited resources and competing priorities-it is important to demonstrate the additional benefit provided by a universal genotyping program. We compared the clustering patterns revealed by retrospective 24-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat genotyping of all culture-positive isolates over a 5-year period to the patterns previously established by our genotyping-on-request program in the low-incidence setting of British Columbia, Canada. We found that 23.8% of isolates were requested during the study period, and while requested isolates had increased odds of belonging to a genotype cluster (adjusted odds ratio, 2.3; 95% confidence interval, 1.5 to 3.3), only 54.6% clustered with the requested comparator strain. Universal genotyping revealed 94 clusters ranging in size from 2 to 53 isolates (mean = 5) and involving 432 individuals. On-request genotyping missed 54 (57.4%) of these clusters and 130 (30.1%) clustered individuals. Our results underscore that TB patient networks are complex, with unrecognized linkages between patients, and a prospective province-wide universal genotyping program provides an informative, bias-free tool to explore transmission to a degree not possible with on-request genotyping.

Master Sculptor at Work: Enteropathogenic Escherichia coli Infection Uniquely Modifies Mitochondrial Proteolysis during Its Control of Human Cell Death.

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.