Multiple aspartic proteases process PE and PPE proteins on the mycobacterial surface of pathogenic mycobacteria

Mycobacterium tuberculosis is the world’s leading bacterial infectious agent. The absence of an effective vaccine and the rise of multidrug-resistant M. tuberculosis strains emphasize the need to identify novel targets for prevention and intervention strategies. Previously, the Mycobacterium marinum orthologue of the aspartic protease PecA (PE_PGRS35) was found on the bacterial cell surface, after secretion by the specialized Type VII secretion system. PecA was shown to be responsible for the processing of secreted PE_PGRS proteins, including itself. Both M. marinum and M. tuberculosis encode two additional predicted aspartic proteases with similar secretion domains (PE_PGRS16 and PE26). The roles of the M. marinum PecA, PecB, and PecC were studied using frameshift mutants generated by CRISPR-Cas9 technology. Numerous PecA substrates were identified using analysis of semi-tryptic peptides detected by proteomics of secreted, surface-associated protein fractions. The most abundant substrates are members of the PE and PPE protein families. In addition, analysis of semi-tryptic peptides revealed consensus cleavage sites. Interestingly, these cleavage sites were also situated within the Type VII secretion motif (YxxxD/E), thereby effectively removing the entire PE domain, further strengthening the notion that these domains function as secretion peptides. PecB and PecC were also predicted to process PE and PPE proteins, although to a lesser extent than PecA. Follow-up experiments revealed that PecC is involved in the processing of a small subset of the PPE protein family. Finally, our evidence indicates that PecA functions similarly in M. tuberculosis, as PecA is also responsible for cleavage of PE_PGRS proteins in this species.