Abstract
Tuberculosis, one of the world's most severe infectious diseases, is caused by Mycobacterium tuberculosis A major weapon of this pathogen is a unique cell wall that protects the pathogen from eradication by the immune system. Mycobacteria have specialized secretion systems, e.g., type VII secretion or ESX systems, to transport substrates across this cell wall. The largest group of proteins that are secreted by these ESX systems are the PE proteins. Previously, it was shown that the N-terminal PE domain of about 100 amino acids is required for secretion. Here, we describe the identification of an aspartic protease, designated PecA, that removes (part of) this PE domain at the cell surface. Nearly all of the observed PE_PGRS proteins are processed by PecA. Interestingly, the protease itself is also a secreted PE protein and subject to self-cleavage. Furthermore, a defect in surface processing has no effect on the activity of the PE lipase protein LipY but does seem to affect the functioning of other virulence factors, as a pecA mutant strain of Mycobacterium marinum shows moderate attenuation in zebrafish larvae. In conclusion, our results reveal the presence of a functional aspartic acid protease in M. marinum that cleaves LipY, itself as well as other members of the PE_PGRS family. Finally, mutants lacking PecA show growth attenuation in vivo, suggesting that PecA plays a role during infection.IMPORTANCE Aspartic proteases are common in eukaryotes and retroviruses but are relatively rare among bacteria (N. D. Rawlings and A. Bateman, BMC Genomics 10:437, 2009, https://doi.org/10.1186/1471-2164-10-437). In contrast to eukaryotic aspartic proteases, bacterial aspartic proteases are generally located in the cytoplasm. We have identified a surface-associated mycobacterial aspartic protease, PecA, which cleaves itself and many other type VII secretion substrates of the PE_PGRS family. PecA is present in most pathogenic mycobacterial species, including M. tuberculosis In addition, pathogenicity of M. marinum is reduced in the ΔpecA mutant, indicating that PecA contributes to virulence.
Highlights
Tuberculosis, one of the world’s most severe infectious diseases, is caused by Mycobacterium tuberculosis
To identify proteins involved in PE protein secretion, we generated and screened a transposon mutant library of M. marinum expressing LipYOVA2
We expected to find mutants that were blocked in secretion because they contained a mutation in the gene coding for the LipY partner protein, but we did not identify these
Summary
Tuberculosis, one of the world’s most severe infectious diseases, is caused by Mycobacterium tuberculosis. Its causative agent is Mycobacterium tuberculosis, a facultative intracellular pathogen with a highly unique diderm cell envelope This unique cell envelope is highly impermeable and requires specialized secretion systems for protein transport to the cell surface [2, 3]. PE/PPE proteins are characterized by a conserved N-terminal domain of 100 and 180 amino acids (aa), respectively, which includes the Pro-Glu or Pro-Pro-Glu motifs close to the N terminus after which they were named [20] Both these protein families lack a classical signal peptide, the PE proteins do have a conserved ESX secretion motif, consisting of a helix-turn-helix domain directly followed by YxxxD/E [17]
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