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

Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, TX, USA.
PLoS Pathogens (Impact Factor: 7.56). 08/2009; 5(7):e1000534. DOI: 10.1371/journal.ppat.1000534
Source: PubMed


Author Summary
The human pathogen Mycobacterium tuberculosis (Mtb) survives in the hostile intracellular environment, in part, by withstanding the actions of host-induced cationic antimicrobial peptides (CAMPs). Membrane phospholipid composition and the resultant charge could play an important role in Mtb survival within the host. Acidic phospholipids such as cardiolipin, phosphatidylinositol and its mannoside derivatives, phosphatidylglycerol, and a single basic species, phosphatidylethanolamine, are constituents of the Mtb membrane bilayer. We demonstrate that lysinylated phosphatidylglycerol (L-PG) represents another basic phospholipid and that the lysX gene, which encodes a two-domain protein with lysyl transferase and lysyl-tRNA synthase activities, is necessary for L-PG production. We show that L-PG is required for maintenance of an optimal membrane potential and resistance towards CAMPs. Phagosomes containing the lysX mutant showed an increased association with lysosomes, and the lysX mutant showed growth defects in mouse and guinea pig lungs, indicating that LysX activity is required for full virulence. Collectively, our results suggest that LysX activity, which is responsible for the production of L-PG, is necessary for maintenance of an optimal membrane potential such that the pathogen can grow optimally upon infection, presumably by withstanding the actions of CAMPs.

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Available from: Murty V Madiraju,
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    • "The negatively charged phospholipid, 1,2-dimyristoyl-sn-glycero-3-phospho-rac- (1-glycerol) (DMPG) was selected because the bacterial cell membranes are predominantly negatively charged, being the phosphatidylglycerols one of the main components of the prokaryotic membranes (Pinheiro et al., 2013a). In addition, a mixture of DPPE:DPPG 8:2 (molar ratio) was chosen as a more complex bacterial membrane model, being the phosphatidylethanolamines in addition to the phosphatidylglycerols one of the main constituents of the bacterial membranes (Maloney et al., 2009). Moreover, this lipid mixture permits to study if the antimycobacterial compounds induce non-lamellar phases, one of the mechanisms of action of some antibiotics (Oszlanczi et al., 2010a). "
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    ABSTRACT: This work focuses on the interaction of N'-acetyl-rifabutin (RFB2) and N'-butanoyl-rifabutin (RFB3) with human and bacterial cell membrane models under physiological conditions. The effect of RFB2 and RFB3 on human cell membrane models was assessed using multilamellar vesicles (MLVs) composed of 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC). In order to mimic the bacterial cell membrane, 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG) and a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG) (8:2 molar ratio) were chosen. Small and Wide-Angle X-Ray Scattering (SAXS and WAXS) were used to study the effect of these antimycobacterial compounds on the structure formed in aqueous lipid dispersions. This study contributes to understanding the molecular mechanisms of the drugs delivery through the human and bacterial cells and the effect of these antimycobacterial compounds on the membrane lipids organization, which is related with their antibiotic efficacy and toxic effects.
    International Journal of Pharmaceutics 06/2013; 453(2). DOI:10.1016/j.ijpharm.2013.06.018 · 3.65 Impact Factor
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    • "LysX from M. tuberculosis is required for synthesis of lysinylated phosphatidylglycerol. A LysX mutant was shown to be sensitive to cationic antibiotics and peptides, to be more lysosome-associated and to display defective growth in mouse and guinea pig lungs [76]. "
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    ABSTRACT: Background The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. Results We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). Conclusions We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.
    BMC Microbiology 09/2012; 12(1):204. DOI:10.1186/1471-2180-12-204 · 2.73 Impact Factor
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    • "A M. tuberculosis mutant strain with a mutation that knocks out lysX is sensitive to cationic antibiotics and CAMPs. is mutant also shows an increased association with lysosome-associated membrane protein–positive vesicles and exhibits altered membrane potential compared to the wild-type strain. e complementation of the lysX gene but not mprF restored the production of Lys-PG (Maloney et al., 2009). mprF was discovered in S. aureus by screening transposon insertion libraries in mutants sensitive to gallidermin. "
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    ABSTRACT: Naturally occurring cationic antimicrobial peptides (CAMPs) have been considered as promising candidates to treat infections caused by pathogenic bacteria to animals and humans. This assumption is based on their mechanism of action, which is mainly performed through electrostatic membrane interactions. Unfortunately, the rise in the reports that describe bacterial resistance to CAMPs has redefined their role as therapeutic agents. In this review, we describe the state of the art of the most common resistance mechanisms developed by bacteria to CAMPs, making special emphasis on resistance selection. Considering most of the resistance mechanisms here reviewed, the emergence of resistance is unlikely in the short term, however we also described evidences that show the evolution of resistance to CAMPs, reevaluating their use as good antibacterial agents. Finally, the knowledge related to the description of CAMP resistance mechanisms may provide useful information for improving strategies to control infections.
    Critical Reviews in Microbiology 07/2012; 39(2). DOI:10.3109/1040841X.2012.699025 · 6.02 Impact Factor
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