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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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Phenotype of lysX strains.Panel A: The growth and viability of the Mtb lysX strains in 7H9 broth in the absence of antibiotics -i. A-ii: The growth and viability of cultures grown in the presence of 1.0 µg/ml Van. At the indicated times, growth was measured. After 3 days of growth in broth, viability was determined by plating cells on Middlebrook 7H11 agar and determining the CFU. Symbols: Filled diamonds – Rv-03; grey squares – Rv81-ami; white triangles – Rv-80lys; crosses – Rv-82med. The inset shows the viable cell count. Black bars - Rv-03, grey bars - Rv-81ami, white bars - Rv-80lys and dashed bars - Rv82-med. A-iii: The growth and viability of cultures grown in the presence of 100 units/ml PMB. * Represents a P value<0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method); bars represent means±standard error. Panel B: The growth and viability of Mtb strains in the presence of 1 mg/ml lysozyme -i- or HNP-1 -ii. There was no significant reduction in viability compared to cultures grown without TFA (data not shown). The stars represent P<0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method). The bars represent the mean±standard error.
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ppat-1000534-g003: Phenotype of lysX strains.Panel A: The growth and viability of the Mtb lysX strains in 7H9 broth in the absence of antibiotics -i. A-ii: The growth and viability of cultures grown in the presence of 1.0 µg/ml Van. At the indicated times, growth was measured. After 3 days of growth in broth, viability was determined by plating cells on Middlebrook 7H11 agar and determining the CFU. Symbols: Filled diamonds – Rv-03; grey squares – Rv81-ami; white triangles – Rv-80lys; crosses – Rv-82med. The inset shows the viable cell count. Black bars - Rv-03, grey bars - Rv-81ami, white bars - Rv-80lys and dashed bars - Rv82-med. A-iii: The growth and viability of cultures grown in the presence of 100 units/ml PMB. * Represents a P value<0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method); bars represent means±standard error. Panel B: The growth and viability of Mtb strains in the presence of 1 mg/ml lysozyme -i- or HNP-1 -ii. There was no significant reduction in viability compared to cultures grown without TFA (data not shown). The stars represent P<0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method). The bars represent the mean±standard error.

Mentions: Gram-positive organisms such as S. aureus and B. subtilis are sensitive to cationic antimicrobial antibiotics (CAMAs) such as vancomycin (Van) and polymyxin-B (PMB) and to cationic antimicrobial peptides (CAMPs) such as human neutrophil peptide (HNP-1) and lysozyme. On the other hand, Mtb is generally tolerant to these compounds. HNP-1 and lysozyme are produced in neutrophils and macrophages, respectively. It is generally believed that CAMPs induce cell death by interfering with the integrity of the negatively charged membrane. Furthermore, the ability of intracellular pathogens to resist the action of CAMPs produced by the host is, in part, responsible for pathogen proliferation upon infection [13]. The presence of lysine groups on the acidic PG would impart a net positive charge and, therefore, could affect the net ratio of positively charged to negatively charged PL species. Thus, the absence of L-PG could make the Mtb membrane relatively acidic, thereby sensitizing the bacterium to the action of CAMAs. To test this possibility, actively growing Rv-03, Rv-80lys, Rv-80ami and Rv-82med were exposed to Van and PMB, and growth and viability were measured (Fig. 3A). Van and PMB interfered with the growth and viability of Rv-80lys and Rv-82med (see Fig. 3A-ii and 3A-iii), inset showing viability after 3 days of exposure; and Figure S1 showing viability after 6 days of exposure). Comparisons of growth, measured as the change in optical density (OD), and viability, measured as the change in CFU, revealed that while the lysX mutant was relatively more sensitive to Van and PMB than Rv-03, it was able to recover when grown in the absence of antibiotics, indicating that Van and PMB do not exert potent bactericidal activity. All of the strains grew well in the absence of antibiotics, although the lysX mutant showed a small reduction in growth rate in the absence of antibiotics (Fig. 3A-i), inset shows an approximately 0.3 log reduction in viability). Visualization of Rv-80lys cells following nucleoid staining and bright field or fluorescence microscopy did not reveal any significant differences in cell morphology or nucleoid organization (data not shown).


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)

Phenotype of lysX strains.Panel A: The growth and viability of the Mtb lysX strains in 7H9 broth in the absence of antibiotics -i. A-ii: The growth and viability of cultures grown in the presence of 1.0 µg/ml Van. At the indicated times, growth was measured. After 3 days of growth in broth, viability was determined by plating cells on Middlebrook 7H11 agar and determining the CFU. Symbols: Filled diamonds – Rv-03; grey squares – Rv81-ami; white triangles – Rv-80lys; crosses – Rv-82med. The inset shows the viable cell count. Black bars - Rv-03, grey bars - Rv-81ami, white bars - Rv-80lys and dashed bars - Rv82-med. A-iii: The growth and viability of cultures grown in the presence of 100 units/ml PMB. * Represents a P value<0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method); bars represent means±standard error. Panel B: The growth and viability of Mtb strains in the presence of 1 mg/ml lysozyme -i- or HNP-1 -ii. There was no significant reduction in viability compared to cultures grown without TFA (data not shown). The stars represent P<0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method). The bars represent the mean±standard error.
© Copyright Policy
Related In: Results  -  Collection

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ppat-1000534-g003: Phenotype of lysX strains.Panel A: The growth and viability of the Mtb lysX strains in 7H9 broth in the absence of antibiotics -i. A-ii: The growth and viability of cultures grown in the presence of 1.0 µg/ml Van. At the indicated times, growth was measured. After 3 days of growth in broth, viability was determined by plating cells on Middlebrook 7H11 agar and determining the CFU. Symbols: Filled diamonds – Rv-03; grey squares – Rv81-ami; white triangles – Rv-80lys; crosses – Rv-82med. The inset shows the viable cell count. Black bars - Rv-03, grey bars - Rv-81ami, white bars - Rv-80lys and dashed bars - Rv82-med. A-iii: The growth and viability of cultures grown in the presence of 100 units/ml PMB. * Represents a P value<0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method); bars represent means±standard error. Panel B: The growth and viability of Mtb strains in the presence of 1 mg/ml lysozyme -i- or HNP-1 -ii. There was no significant reduction in viability compared to cultures grown without TFA (data not shown). The stars represent P<0.001 versus Rv-03 and Rv-81ami (Student-Newman-Keuls Method). The bars represent the mean±standard error.
Mentions: Gram-positive organisms such as S. aureus and B. subtilis are sensitive to cationic antimicrobial antibiotics (CAMAs) such as vancomycin (Van) and polymyxin-B (PMB) and to cationic antimicrobial peptides (CAMPs) such as human neutrophil peptide (HNP-1) and lysozyme. On the other hand, Mtb is generally tolerant to these compounds. HNP-1 and lysozyme are produced in neutrophils and macrophages, respectively. It is generally believed that CAMPs induce cell death by interfering with the integrity of the negatively charged membrane. Furthermore, the ability of intracellular pathogens to resist the action of CAMPs produced by the host is, in part, responsible for pathogen proliferation upon infection [13]. The presence of lysine groups on the acidic PG would impart a net positive charge and, therefore, could affect the net ratio of positively charged to negatively charged PL species. Thus, the absence of L-PG could make the Mtb membrane relatively acidic, thereby sensitizing the bacterium to the action of CAMAs. To test this possibility, actively growing Rv-03, Rv-80lys, Rv-80ami and Rv-82med were exposed to Van and PMB, and growth and viability were measured (Fig. 3A). Van and PMB interfered with the growth and viability of Rv-80lys and Rv-82med (see Fig. 3A-ii and 3A-iii), inset showing viability after 3 days of exposure; and Figure S1 showing viability after 6 days of exposure). Comparisons of growth, measured as the change in optical density (OD), and viability, measured as the change in CFU, revealed that while the lysX mutant was relatively more sensitive to Van and PMB than Rv-03, it was able to recover when grown in the absence of antibiotics, indicating that Van and PMB do not exert potent bactericidal activity. All of the strains grew well in the absence of antibiotics, although the lysX mutant showed a small reduction in growth rate in the absence of antibiotics (Fig. 3A-i), inset shows an approximately 0.3 log reduction in viability). Visualization of Rv-80lys cells following nucleoid staining and bright field or fluorescence microscopy did not reveal any significant differences in cell morphology or nucleoid organization (data not shown).

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