Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems. The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus. However, the role of these proteins remains largely elusive . Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising>80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion . Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the 'modern' Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution.

Unexpected Genomic and Phenotypic Diversity of Mycobacterium africanum Lineage 5 Affects Drug Resistance, Protein Secretion, and Immunogenicity

“…3). It should be noted that three L5.2 isolates (76, NRC2, and NRC4), reproducibly secreted residual amounts of PE_PGRS, which is unlike the phenotype of M. tuberculosis ppe38 -deleted strains (Ates, Dippenaar et al 2018). The pattern of both expressed (whole-cell lysate) and secreted PE_PGRS proteins of L6 strains were clearly distinguishable from the other strains (fig.…”

“…We previously showed that the ESX-5 substrate PPE38 is required for the secretion of two large subgroups of other ESX-5 substrates called the PE_PGRS and PPE-MPTR proteins (Ates, Dippenaar et al 2018). Deletions of the ppe38 -locus and concurring secretion defects increase virulence of M. tuberculosis during late stage infection.…”

“…Interestingly, all other L5 strains were found to have only one copy of ppe38 , while the L6 strains and H37Rv possessed the more common four-gene ppe38-71 locus (McEvoy et al. 2009; Ates, Dippenaar et al 2018; Ates, Sayes et al. 2018).…”

“…Source: http://www.kazusa.or.jp/codon/). To test if reduced ppe38 transcription or translation was a possible explanation for the PE_PGRS secretion defect, we introduced an integrative plasmid that constitutively expresses the ppe38-71 locus ( mt2419-22 ) and successfully complements PPE38-dependent secretion in L2, L4, or BCG isolates, in two L5.2 strains, that is, isolate 68, with functional EsxN secretion and in the RD5-deleted isolate 77 (Ates, Dippenaar et al 2018; Ates, Sayes et al. 2018).…”

“…2016) can be found in supplementary table 4, Supplementary Material online. The pMV:: mt2419-22 ( ppe38-71 ) plasmid was previously published (Ates, Dippenaar et al 2018). …”