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

Area of Science:

• Microbiology
• Molecular Biology
• Proteomics

Background:

• Mycobacterium tuberculosis is a major global pathogen, necessitating new intervention targets due to drug resistance and lack of vaccines.
• Secreted PE and PPE proteins are abundant in mycobacteria, but their processing and roles remain incompletely understood.
• The aspartic protease PecA (PE_PGRS35) in M. marinum is known to process PE_PGRS proteins via the Type VII secretion system.

Purpose of the Study:

• To elucidate the roles of aspartic proteases PecA, PecB, and PecC in processing secreted proteins in M. marinum.
• To identify substrates of these proteases, particularly PE and PPE family members.
• To determine if PecA functions similarly in Mycobacterium tuberculosis.

Main Methods:

• CRISPR-Cas9 technology was used to generate frameshift mutants of M. marinum PecA, PecB, and PecC.
• Proteomics analysis of secreted and surface-associated protein fractions was performed to identify protease substrates via semi-tryptic peptides.
• Consensus cleavage sites were analyzed to understand protease specificity and the function of PE domains.

Main Results:

• PecA was identified as the primary protease processing numerous PE and PPE proteins, including itself, in M. marinum.
• Cleavage sites were found within the Type VII secretion motif, suggesting PE domains act as secretion peptides.
• PecB and PecC also process PE and PPE proteins, with PecC specifically involved in a subset of PPE processing. PecA's function in M. tuberculosis was confirmed.

Conclusions:

• The aspartic protease PecA plays a critical role in processing PE and PPE proteins in M. marinum and M. tuberculosis.
• The PE domains function as secretion peptides, removed by PecA during the secretion process.
• These findings highlight the importance of PecA and related proteases in mycobacterial protein secretion and virulence.