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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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Determination of the membrane potential of Mtb strains.The relative membrane potential was calculated using the average mean fluorescence intensity (the ratio between the average red fluorescence and the average green fluorescence). The graph shows the average of four experiments. Wild type and lysX mutant strains were incubated with 3 µM DiOC2(3) for 5 h in either the presence (+) or absence (−) of 100 µM CCCP. The bars represent the mean±standard error. * Represents P<0.002 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method), whereas # refers to P = 0.001 compared to untreated with CCCP.
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ppat-1000534-g004: Determination of the membrane potential of Mtb strains.The relative membrane potential was calculated using the average mean fluorescence intensity (the ratio between the average red fluorescence and the average green fluorescence). The graph shows the average of four experiments. Wild type and lysX mutant strains were incubated with 3 µM DiOC2(3) for 5 h in either the presence (+) or absence (−) of 100 µM CCCP. The bars represent the mean±standard error. * Represents P<0.002 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method), whereas # refers to P = 0.001 compared to untreated with CCCP.

Mentions: We wished to test whether the absence of L-PG production in Rv-80lys cells was associated with changes in the properties of the PL bilayer (e.g., membrane potential). The membrane potential of the Rv-80lys cells was determined using a slow-response membrane potential-sensitive dye, DiOC2(3), and comparing with Rv-03 cells. This cationic cyanine dye exhibits green fluorescence (Ex = 488 nm and Em = 520 nm) in the monomeric state and red fluorescence (Ex = 488 nm and Em = 620 nm) in the aggregated or oligomeric state. As a negative control, the membrane potential was measured following exposure of the cells to the proton ionophore m-chlorophenylhydrazone (CCCP), which is known to eliminate the proton gradient across the membrane. As seen in Figure 4, the membrane potentials (measured as the ratio of red to green fluorescence) of the lysX mutant Rv-80lys and Rv-82med were 21% and 17%, respectively, higher than that of the Rv-03 and complemented Rv-81ami (P<0.002). The increased ratio of red to green fluorescence observed in lysX mutants suggests accumulation of the positively charged lipophilic dye on the negatively charged membrane. The red to green fluorescence ratio in all strains was decreased to similar levels (∼41%) in the presence of CCCP (P = 0.001). Presumably, this reduction reflects the completely depolarized state of the membrane. Together, these results indicate that the membranes of Rv-80lys and Rv-82med are hyperpolarized relative to Rv-03 and Rv-81ami.


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)

Determination of the membrane potential of Mtb strains.The relative membrane potential was calculated using the average mean fluorescence intensity (the ratio between the average red fluorescence and the average green fluorescence). The graph shows the average of four experiments. Wild type and lysX mutant strains were incubated with 3 µM DiOC2(3) for 5 h in either the presence (+) or absence (−) of 100 µM CCCP. The bars represent the mean±standard error. * Represents P<0.002 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method), whereas # refers to P = 0.001 compared to untreated with CCCP.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713425&req=5

ppat-1000534-g004: Determination of the membrane potential of Mtb strains.The relative membrane potential was calculated using the average mean fluorescence intensity (the ratio between the average red fluorescence and the average green fluorescence). The graph shows the average of four experiments. Wild type and lysX mutant strains were incubated with 3 µM DiOC2(3) for 5 h in either the presence (+) or absence (−) of 100 µM CCCP. The bars represent the mean±standard error. * Represents P<0.002 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method), whereas # refers to P = 0.001 compared to untreated with CCCP.
Mentions: We wished to test whether the absence of L-PG production in Rv-80lys cells was associated with changes in the properties of the PL bilayer (e.g., membrane potential). The membrane potential of the Rv-80lys cells was determined using a slow-response membrane potential-sensitive dye, DiOC2(3), and comparing with Rv-03 cells. This cationic cyanine dye exhibits green fluorescence (Ex = 488 nm and Em = 520 nm) in the monomeric state and red fluorescence (Ex = 488 nm and Em = 620 nm) in the aggregated or oligomeric state. As a negative control, the membrane potential was measured following exposure of the cells to the proton ionophore m-chlorophenylhydrazone (CCCP), which is known to eliminate the proton gradient across the membrane. As seen in Figure 4, the membrane potentials (measured as the ratio of red to green fluorescence) of the lysX mutant Rv-80lys and Rv-82med were 21% and 17%, respectively, higher than that of the Rv-03 and complemented Rv-81ami (P<0.002). The increased ratio of red to green fluorescence observed in lysX mutants suggests accumulation of the positively charged lipophilic dye on the negatively charged membrane. The red to green fluorescence ratio in all strains was decreased to similar levels (∼41%) in the presence of CCCP (P = 0.001). Presumably, this reduction reflects the completely depolarized state of the membrane. Together, these results indicate that the membranes of Rv-80lys and Rv-82med are hyperpolarized relative to Rv-03 and Rv-81ami.

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