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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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Related in: MedlinePlus

Polar lipid and Southern blot analysis of the lysX mutant strain.A: Mtb strains were grown in the presence and absence of 14C-lysine. Total lipids were extracted in chloroform∶methanol (2∶1 v/v) and resolved by TLC on Silcia Gel 60 (EMD Chemicals, New Jersey) in a solvent system of chloroform∶methanol∶water (65∶25∶4 v/v/v). TLC plates were either visualized by autoradiography (lanes i, iv, vii and x), exposed to iodine vapors (lanes ii, v, viii and xi), or stained with ninhydrin (lanes iii, vi, ix and xii). B: Southern blot analysis of Mtb lysX mutant strains. B-i: The ClaI fragment bearing the wild type lysX gene (3.5 kb) with the locations of the mprF and lysU regions marked. The dark box designated as “probe 1” is an approximately 750 bp fragment that hybridizes with the 5′-end of lysX and 160 bp of the lysX coding region. The ClaI fragment bearing the mutant lysX gene disrupted with the gentamycin cassette (0.9 kb) is also shown. The dark band designated as “probe 2” is the 900 bp gentamycin gene that hybridizes with the mutant lysX gene. B-ii: Southern blot analysis of ClaI-digested Mtb genomic DNA hybridized with probe 1. The 7 kb and 4 kb band positions represent Rv-03 and Rv-80lys, respectively. Note that the complemented copy contains a band corresponding to the integrated copy of lysX gene plus the flanking plasmid sequence. B-iii: Southern blot analysis of ClaI-digested Mtb genomic DNA (see Fig. 1B-iii) hybridized with probe 2. pMMR85 is a positive control plasmid containing the mutant lysX gene plus flanking regions.
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ppat-1000534-g001: Polar lipid and Southern blot analysis of the lysX mutant strain.A: Mtb strains were grown in the presence and absence of 14C-lysine. Total lipids were extracted in chloroform∶methanol (2∶1 v/v) and resolved by TLC on Silcia Gel 60 (EMD Chemicals, New Jersey) in a solvent system of chloroform∶methanol∶water (65∶25∶4 v/v/v). TLC plates were either visualized by autoradiography (lanes i, iv, vii and x), exposed to iodine vapors (lanes ii, v, viii and xi), or stained with ninhydrin (lanes iii, vi, ix and xii). B: Southern blot analysis of Mtb lysX mutant strains. B-i: The ClaI fragment bearing the wild type lysX gene (3.5 kb) with the locations of the mprF and lysU regions marked. The dark box designated as “probe 1” is an approximately 750 bp fragment that hybridizes with the 5′-end of lysX and 160 bp of the lysX coding region. The ClaI fragment bearing the mutant lysX gene disrupted with the gentamycin cassette (0.9 kb) is also shown. The dark band designated as “probe 2” is the 900 bp gentamycin gene that hybridizes with the mutant lysX gene. B-ii: Southern blot analysis of ClaI-digested Mtb genomic DNA hybridized with probe 1. The 7 kb and 4 kb band positions represent Rv-03 and Rv-80lys, respectively. Note that the complemented copy contains a band corresponding to the integrated copy of lysX gene plus the flanking plasmid sequence. B-iii: Southern blot analysis of ClaI-digested Mtb genomic DNA (see Fig. 1B-iii) hybridized with probe 2. pMMR85 is a positive control plasmid containing the mutant lysX gene plus flanking regions.

Mentions: In order to detect lysinylated PLs in Mtb, actively growing cultures were incubated with 14C-lysine for 3 days; total lipids were extracted, and PoLs were resolved by thin layer chromatography (TLC). A distinct radiolabeled lipid was evident, indicating that lysinylated PLs are members of the Mtb PoL pool (Fig. 1A, lane i). In S. aureus, the mprF gene is responsible for L-PG production [8]. Homology searches of the Mtb genome identified Rv1640c as lysX, which encodes an mprF-like gene as a fusion to a lysyl-tRNA synthetase (lysU). The latter gene is distinct from the essential housekeeping tRNA synthetase (Rv3598c). The mprF gene in S. aureus encodes a protein with potential lysyl transferase activity [10]. In order to evaluate the function of lysX, we created a lysX mutant strain, Rv-80lys, by replacing the majority of the coding region comprising the mprF and lysU domains with a gentamycin resistance cassette using homologous recombination (see Methods section). A complementing derivative of this strain, Rv-81ami, was created by integrating a plasmid expressing the intact lysX gene under the control of the amidase promoter [11]. The lysX mutant strain was found to be defective in the production of L-PoLs (Fig. 1A, lane iv compared with lane i). L-PoL production was restored, however, in the lysX complemented strain Rv-81ami (Fig. 1A, see lane vii), confirming that the lysX gene product is responsible for the production of L-PoLs. Staining TLC plates with iodine (lanes ii, v, viii and xi) or ninhydrin (lanes iii, vi, ix and xii), on the other hand, did not detect L-PoLs, indicating that they may not be an abundant lipid species. We cultured Mtb in the presence of 14C-acetic acid and extracted total lipids, followed by TLC separation and subsequent quantification of L-PoL relative to total input radioactivity, and found that L-PoL accounts for approximately 0.3% of the total lipids (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)

Polar lipid and Southern blot analysis of the lysX mutant strain.A: Mtb strains were grown in the presence and absence of 14C-lysine. Total lipids were extracted in chloroform∶methanol (2∶1 v/v) and resolved by TLC on Silcia Gel 60 (EMD Chemicals, New Jersey) in a solvent system of chloroform∶methanol∶water (65∶25∶4 v/v/v). TLC plates were either visualized by autoradiography (lanes i, iv, vii and x), exposed to iodine vapors (lanes ii, v, viii and xi), or stained with ninhydrin (lanes iii, vi, ix and xii). B: Southern blot analysis of Mtb lysX mutant strains. B-i: The ClaI fragment bearing the wild type lysX gene (3.5 kb) with the locations of the mprF and lysU regions marked. The dark box designated as “probe 1” is an approximately 750 bp fragment that hybridizes with the 5′-end of lysX and 160 bp of the lysX coding region. The ClaI fragment bearing the mutant lysX gene disrupted with the gentamycin cassette (0.9 kb) is also shown. The dark band designated as “probe 2” is the 900 bp gentamycin gene that hybridizes with the mutant lysX gene. B-ii: Southern blot analysis of ClaI-digested Mtb genomic DNA hybridized with probe 1. The 7 kb and 4 kb band positions represent Rv-03 and Rv-80lys, respectively. Note that the complemented copy contains a band corresponding to the integrated copy of lysX gene plus the flanking plasmid sequence. B-iii: Southern blot analysis of ClaI-digested Mtb genomic DNA (see Fig. 1B-iii) hybridized with probe 2. pMMR85 is a positive control plasmid containing the mutant lysX gene plus flanking regions.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000534-g001: Polar lipid and Southern blot analysis of the lysX mutant strain.A: Mtb strains were grown in the presence and absence of 14C-lysine. Total lipids were extracted in chloroform∶methanol (2∶1 v/v) and resolved by TLC on Silcia Gel 60 (EMD Chemicals, New Jersey) in a solvent system of chloroform∶methanol∶water (65∶25∶4 v/v/v). TLC plates were either visualized by autoradiography (lanes i, iv, vii and x), exposed to iodine vapors (lanes ii, v, viii and xi), or stained with ninhydrin (lanes iii, vi, ix and xii). B: Southern blot analysis of Mtb lysX mutant strains. B-i: The ClaI fragment bearing the wild type lysX gene (3.5 kb) with the locations of the mprF and lysU regions marked. The dark box designated as “probe 1” is an approximately 750 bp fragment that hybridizes with the 5′-end of lysX and 160 bp of the lysX coding region. The ClaI fragment bearing the mutant lysX gene disrupted with the gentamycin cassette (0.9 kb) is also shown. The dark band designated as “probe 2” is the 900 bp gentamycin gene that hybridizes with the mutant lysX gene. B-ii: Southern blot analysis of ClaI-digested Mtb genomic DNA hybridized with probe 1. The 7 kb and 4 kb band positions represent Rv-03 and Rv-80lys, respectively. Note that the complemented copy contains a band corresponding to the integrated copy of lysX gene plus the flanking plasmid sequence. B-iii: Southern blot analysis of ClaI-digested Mtb genomic DNA (see Fig. 1B-iii) hybridized with probe 2. pMMR85 is a positive control plasmid containing the mutant lysX gene plus flanking regions.
Mentions: In order to detect lysinylated PLs in Mtb, actively growing cultures were incubated with 14C-lysine for 3 days; total lipids were extracted, and PoLs were resolved by thin layer chromatography (TLC). A distinct radiolabeled lipid was evident, indicating that lysinylated PLs are members of the Mtb PoL pool (Fig. 1A, lane i). In S. aureus, the mprF gene is responsible for L-PG production [8]. Homology searches of the Mtb genome identified Rv1640c as lysX, which encodes an mprF-like gene as a fusion to a lysyl-tRNA synthetase (lysU). The latter gene is distinct from the essential housekeeping tRNA synthetase (Rv3598c). The mprF gene in S. aureus encodes a protein with potential lysyl transferase activity [10]. In order to evaluate the function of lysX, we created a lysX mutant strain, Rv-80lys, by replacing the majority of the coding region comprising the mprF and lysU domains with a gentamycin resistance cassette using homologous recombination (see Methods section). A complementing derivative of this strain, Rv-81ami, was created by integrating a plasmid expressing the intact lysX gene under the control of the amidase promoter [11]. The lysX mutant strain was found to be defective in the production of L-PoLs (Fig. 1A, lane iv compared with lane i). L-PoL production was restored, however, in the lysX complemented strain Rv-81ami (Fig. 1A, see lane vii), confirming that the lysX gene product is responsible for the production of L-PoLs. Staining TLC plates with iodine (lanes ii, v, viii and xi) or ninhydrin (lanes iii, vi, ix and xii), on the other hand, did not detect L-PoLs, indicating that they may not be an abundant lipid species. We cultured Mtb in the presence of 14C-acetic acid and extracted total lipids, followed by TLC separation and subsequent quantification of L-PoL relative to total input radioactivity, and found that L-PoL accounts for approximately 0.3% of the total lipids (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