A novel mutation alters the stability of PapA2 resulting in the complete abrogation of sulfolipids in clinical mycobacterial strains

Abstract

The analysis of whole genomes has revealed specific geographical distribution of Mycobacterium tuberculosis (Mtb) strains across the globe suggestive of unique niche dependent adaptive mechanisms. We provide an important correlation of a genome-based mutation to a molecular phenotype across two predominant clinical Mtb lineages of the Indian subcontinent. We have identified a distinct lineage specific mutation-G247C, translating into an alanine-proline conversion in the papA2 gene of Indo-oceanic lineage 1 (L1) Mtb strains, and restoration of cell wall sulfolipids by simple genetic complementation of papA2 from lineage 3 (L3) or from H37Rv (lineage 4-L4) attributed the loss of this glycolipid to this specific mutation in Indo-Oceanic L1 Mtb. The investigation of structure of Mtb PapA2 revealed a distinct nonribosomal peptide synthetase (NRPS) C domain conformation with an unconventional presence of a zinc binding motif. Surprisingly, the A83P mutation did not map to either the catalytic center in the N-terminal subdomain or any of the substrate-binding region of the protein. On the contrary, the inherent ability of mutant PapA2 to form insoluble aggregates and molecular simulations with the wild-type/ mutant (Wt/mut) PapA2 purports an important role for the surface associated 83rd residue in protein conformation. This study demonstrates the importance of a critical structural residue in the papA2 protein of Mtb and helps establish a link between observed genomic alteration and its molecular consequence in the successful human pathogen Mtb. SignificanceWe demonstrate the effect of a unique SNP in PapA2 gene of Indo-oceanic Mycobacterium tuberculosis (Mtb) strains leading to the loss of sulfolipid from these strains. By X-ray crystallographic analysis and molecular dynamics (MD) simulations, we show the importance of this residue in the global PapA2 structure. The presence of a Zn atom has not been reported before for this class of proteins. Here, we provide an important link between genomic alteration and its molecular consequence in Mtb highlighting one of the many adaptive mechanisms that Abbreviations: Mtb, Mycobacterium tuberculosis; SNP, sinlge nucleotide polymorphism; PapA2, polyketide synthase associated protein A2; Mut, mutation; Wt, wild type; SL-1, sulfolipids-1.

Modern clinical Mycobacterium tuberculosis strains leverage type I IFN pathway for a pro-inflammatory response in the host

“…We have previously demonstrated that L1 strains harbor a mutation in papA2, rendering them incapable of sulfolipid synthesis ( 28 ). A dominant role for the Mtb sulfolipids has also been demonstrated in modulation of the cellular phagosome maturation and acidification ( 29-31 ).…”

“…While sulfolipids have been demonstrated in promoting phagosomal acidification and L1 strains lack sulfolipids, our results do not purport an active role for this lipid in the type I IFN response. Despite the presence of mature sulfolipids in the L1 subtype Mtb strains T83 ( 28 ), induction of type I IFN was relatively low in THP1 macrophages. Moreover, while differences in sulfolipid expression between H 37 Ra and H 37 Rv is well established ( 47-48 ), there was no discernable alteration of IFN expression between the two Mtb strains.…”

Robust lysosomal rewiring in Mtb infected macrophages mediated by Mtb lipids restricts the intracellular bacterial survival

“…Clinical isolates of Mtb exhibit clade specific virulence patterns with strong correlations of their phylogenetic relationships with gene expression profiles and host inflammatory responses (Portevin et al, 2011; Reiling et al, 2013; Shankaran et al, 2019). Some strains of the ‘ancestral’ Clade 2 show reduced expression of genes in the SL-1 biosynthetic pathway (Homolka et al, 2010), while a recent report shows an Mtb strain belonging to the ancestral lineage L1 having a point mutation in the pap A2 gene, which confers it a loss of SL-1 phenotype (Panchal et al, 2019). The contribution of the lysosomal alterations and their differential sub-cellular localization to the distinct inflammatory responses elicited by these phylogenetically distant strains will be interesting to explore.…”