The Two-Domain LysX Protein of Mycobacterium tuberculosis Is Required for Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides

Abstract

The well-recognized phospholipids (PLs) of Mycobacterium tuberculosis (Mtb) include several acidic species such as phosphatidylglycerol (PG), cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG), is a component of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF)-lysyl-tRNA synthetase (lysU) protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows increased association with lysosome-associated membrane protein–positive vesicles, and it exhibits altered membrane potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and for survival of the pathogen upon infection.

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…The LysRS domain of LysX is required for synthesis of lysylated phosphatidylglycerol in M. tuberculosis , suggesting that free Lys-tRNA Lys produced by the translation machinery is not used by LysX for lipid modification. 29 …”

“…The effects of aa-PG on antibiotic resistance have been investigated in several important human pathogens such as S. aureus (for review see [4]), L. monocytogenes , 53 B. anthracis , 58 P. aeruginosa , 24,59 M. tuberculosis , 29,60 Enterococcus species, 35,36,61 and B. subtilis 62,63 (for review see [1]). aa-PGs in these organisms primarily enhance bacterial resistance to positively charged compounds targeting the membrane (such as CAMPs) and last resort lipopetides like daptomycin.…”

“…59 In M. tuberculosis , Lys-PG was shown to increase resistance to vancomycin. 29 β-lactams and vancomycin are antibiotics that do not interact with the cytoplasmic membrane; both compounds target distinct components of the peptidoglycan synthesis machinery inside the periplasm (i.e., penicillin binding proteins and peptidoglycan connecting pentapeptides, respectively). Increased resistance to these antibiotics may be due to indirect effects of the aaPGS lipid modification process on the homeostasis and general properties of the cell wall.…”

“…53 In M. tuberculosis , deletion of LysPGS (i.e., lysX ) decreases bacterial growth in the lungs of guinea pigs relative to the wild-type strain. 29 …”

“…Despite its low abundance, Lys-PG in this species is important for maintaining membrane potential, virulence, CAMP resistance, and survival in mononuclear phagocytes. 29,71,90 It was determined that the phenotypes observed in mutants deprived of Lys-PG may, in fact, be due to indirect effects of the modified lipids on phospholipid catabolism and cell division. 90 These findings indicate that PG-lysylation in M. tuberculosis plays a broader role outside of the typical electrostatic effects associated with aa-PGs, which might be relevant to many bacterial species.…”