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The two-domain LysX protein of Mycobacterium tuberculosis is required for production of lysinylated phosphatidylglycerol and resistance to cationic antimicrobial peptides.

Maloney E, Stankowska D, Zhang J, Fol M, Cheng QJ, Lun S, Bishai WR, Rajagopalan M, Chatterjee D, Madiraju MV - PLoS Pathog. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, TX, USA.

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.

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Co-localization of Mtb with LAMP-1-expressing phagosomes.Panel A: THP-1-derived macrophages were infected with GFP-expressing Mtb strains (Rv-03, Rv-80lys, Rv-81ami and Rv-82med). Bacteria (green spots) inside LAMP-1-positive phagosomes (red) produce a yellow signal indicating co-localization (merged). Panel B: The percent co-localization was determined by visual scoring of yellow spots after 72 h of infection. We analyzed 126 macrophages for Rv-80lys, 164 for wild type, 168 for complemented, and 127 for Rv-82med and scored 534 bacterial cells for Rv-80lys, 1,307 for wild type, 848 for Rv-81ami, and 402 for Rv-82med. Mtb Rv-03 (black bar; 21.2±1.6%), Rv-80lys (white bar; 52.0±3.0%), Rv-81ami (grey bar; 26.4±2.2%) and Rv-82med (dashed bar; 52.6±2.8%). * P<0.001 versus Rv-03 and Rv-81ami using the Student-Newman-Keuls Method; bars represent mean±standard error.
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ppat-1000534-g006: Co-localization of Mtb with LAMP-1-expressing phagosomes.Panel A: THP-1-derived macrophages were infected with GFP-expressing Mtb strains (Rv-03, Rv-80lys, Rv-81ami and Rv-82med). Bacteria (green spots) inside LAMP-1-positive phagosomes (red) produce a yellow signal indicating co-localization (merged). Panel B: The percent co-localization was determined by visual scoring of yellow spots after 72 h of infection. We analyzed 126 macrophages for Rv-80lys, 164 for wild type, 168 for complemented, and 127 for Rv-82med and scored 534 bacterial cells for Rv-80lys, 1,307 for wild type, 848 for Rv-81ami, and 402 for Rv-82med. Mtb Rv-03 (black bar; 21.2±1.6%), Rv-80lys (white bar; 52.0±3.0%), Rv-81ami (grey bar; 26.4±2.2%) and Rv-82med (dashed bar; 52.6±2.8%). * P<0.001 versus Rv-03 and Rv-81ami using the Student-Newman-Keuls Method; bars represent mean±standard error.

Mentions: Intracellular replication of Mtb is, in part, due to its ability to resist the delivery of its phagosomes to lysosomes [15]. This process can be visualized by examining the co-localization of Mtb with the lysosome-associated-membrane protein (LAMP-1). In order to address whether the lysX mutant had a phagosome-lysosome fusion defect, we infected macrophages with Mtb strains expressing green-fluorescent protein and visualized co-localization with LAMP-1. Increased association of phagosomes containing Rv-80lys with lysosomes was evident compared to Rv-03 and complemented Rv-81ami (Fig. 6, p<0.001). Rv-82med behaved like Rv-80lys, indicating that the full-length lysX gene is required for functional activity. These results are consistent with the hypothesis that the lysX mutant is not as proficient as the Rv-03 strain in preventing fusion of phagosomes with lysosomes, which could contribute to defects in intramacrophage replication.


The two-domain LysX protein of Mycobacterium tuberculosis is required for production of lysinylated phosphatidylglycerol and resistance to cationic antimicrobial peptides.

Maloney E, Stankowska D, Zhang J, Fol M, Cheng QJ, Lun S, Bishai WR, Rajagopalan M, Chatterjee D, Madiraju MV - PLoS Pathog. (2009)

Co-localization of Mtb with LAMP-1-expressing phagosomes.Panel A: THP-1-derived macrophages were infected with GFP-expressing Mtb strains (Rv-03, Rv-80lys, Rv-81ami and Rv-82med). Bacteria (green spots) inside LAMP-1-positive phagosomes (red) produce a yellow signal indicating co-localization (merged). Panel B: The percent co-localization was determined by visual scoring of yellow spots after 72 h of infection. We analyzed 126 macrophages for Rv-80lys, 164 for wild type, 168 for complemented, and 127 for Rv-82med and scored 534 bacterial cells for Rv-80lys, 1,307 for wild type, 848 for Rv-81ami, and 402 for Rv-82med. Mtb Rv-03 (black bar; 21.2±1.6%), Rv-80lys (white bar; 52.0±3.0%), Rv-81ami (grey bar; 26.4±2.2%) and Rv-82med (dashed bar; 52.6±2.8%). * P<0.001 versus Rv-03 and Rv-81ami using the Student-Newman-Keuls Method; bars represent mean±standard error.
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ppat-1000534-g006: Co-localization of Mtb with LAMP-1-expressing phagosomes.Panel A: THP-1-derived macrophages were infected with GFP-expressing Mtb strains (Rv-03, Rv-80lys, Rv-81ami and Rv-82med). Bacteria (green spots) inside LAMP-1-positive phagosomes (red) produce a yellow signal indicating co-localization (merged). Panel B: The percent co-localization was determined by visual scoring of yellow spots after 72 h of infection. We analyzed 126 macrophages for Rv-80lys, 164 for wild type, 168 for complemented, and 127 for Rv-82med and scored 534 bacterial cells for Rv-80lys, 1,307 for wild type, 848 for Rv-81ami, and 402 for Rv-82med. Mtb Rv-03 (black bar; 21.2±1.6%), Rv-80lys (white bar; 52.0±3.0%), Rv-81ami (grey bar; 26.4±2.2%) and Rv-82med (dashed bar; 52.6±2.8%). * P<0.001 versus Rv-03 and Rv-81ami using the Student-Newman-Keuls Method; bars represent mean±standard error.
Mentions: Intracellular replication of Mtb is, in part, due to its ability to resist the delivery of its phagosomes to lysosomes [15]. This process can be visualized by examining the co-localization of Mtb with the lysosome-associated-membrane protein (LAMP-1). In order to address whether the lysX mutant had a phagosome-lysosome fusion defect, we infected macrophages with Mtb strains expressing green-fluorescent protein and visualized co-localization with LAMP-1. Increased association of phagosomes containing Rv-80lys with lysosomes was evident compared to Rv-03 and complemented Rv-81ami (Fig. 6, p<0.001). Rv-82med behaved like Rv-80lys, indicating that the full-length lysX gene is required for functional activity. These results are consistent with the hypothesis that the lysX mutant is not as proficient as the Rv-03 strain in preventing fusion of phagosomes with lysosomes, which could contribute to defects in intramacrophage replication.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, TX, USA.

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.

Show MeSH
Related in: MedlinePlus