Alterations in phospholipid catabolism in Mycobacterium tuberculosis lysX mutant

Original Research Article published: 11 February 2011 doi: 10.3389/fmicb.2011.00019 Alterations in phospholipid catabolism in Mycobacterium tuberculosis lysX mutant Erin Maloney1, Shichun Lun2, Dorota Stankowska1, Haidan Guo2, Malini Rajagoapalan1, William R. Bishai2 and Murty V. Madiraju1* 1 2 Biomedical Research, The University of Texas Health Science Center, Tyler, TX, USA Center for Tuberculosis Research, Johns Hopkins School of Medicine, Baltimore, MD, USA Edited by: Rey Carabeo, Imperial College London, UK Reviewed by: Tanya Parish, Queen Mary University of London, UK Jose A. Bengoechea, Fundacion Caubet-CIMERA Illes Balears, Spain *Correspondence: Murty V. Madiraju, Biomedical Research, The University of Texas Health Science Center, 11937 US Highway 271, Tyler, TX 75708-3154, USA. e-mail: Mycobacterium tuberculosis lysX mutant, defective for production of lysinylated phosphatidylglycerol, is sensitive to cationic antimicrobial peptides, is not proficient for proliferation in mice lungs, and exhibits altered membrane potential (Maloney et al., 2009). In the present study we show that a lysX complement strain expressing lysX from inducible tet promoter is proficient in restoring lysX phenotypes, confirming that the observed phenotypes are specific to lysX. To evaluate the correlation between changes in membrane potential and lysX activity, we visualized regions of cardiolipin (CL), one of the abundant phospholipids of mycobacteria, by staining with fluorescent dye 10-N-nonyl acridine orange and found that CL is localized as bright spots at septal regions and poles of actively dividing cells, but not in stationary phase cells. lysX mutants were elongated and showed more numerous and brighter CL staining at both mid cell and quarter cell septa, compared with wild type, indicating a defect in the cell division process. Evaluation of 14C-acetic acid incorporation into major phospholipids such as CL, phosphatidylethanolamine (PE), phosphatidylinositol (PI), and their degradation between lysX mutant and its parent revealed differences in the turnover of PE and PI. Our results favor a hypothesis that alterations in phospholipid metabolism could be contributing to changes in membrane potential, hence the observed phenotype of lysX mutant. Keywords: tuberculosis, mycobacteria, lysX, phospholipids, cardiolipin, cell division Introduction Mycobacterium tuberculosis is a pathogenic bacterium that causes the infectious disease tuberculosis. Estimates indicate that approximately one-third of the world’s population is infected with M. tuberculosis and approximately 1.8 million deaths were attributable to tuberculosis in 2008 (WHO, 2010)1. Survival of M. tuberculosis following its uptake by alveolar macrophages and subsequent replication and multiplication in the hostile environment is a challenging task to the pathogen (Smith, 2003; Tischler and McKinney, 2010). Within the hostile macrophage environment M. tuberculosis is believed to face reactive oxygen and nitrogen species (ROS, RNS), acidic pH of phagolysosome compartments and possibly antimicrobial peptides. ROS and RNS damage DNA, lipids, and proteins and thereby promote pathogen killing. Mycobacterium tuberculosis multiplies in this hostile environment by efficiently operating multiple stress resistance pathways (reviewed in (Smith, 2003; Tischler and McKinney, 2010). One of these elegant strategies includes the operation of proteasome machinery for processing RNS damaged proteins (Darwin et al., 2003). This pathway includes the activities of proteasomal core subunit PrcBA, accessory factors Mpa and PafA that recognize specific proteins for targeted degradation by the activity of prokaryotic ubiquitin-like protein, Pup. It is expected that the net charge or membrane potential of the bacterial cell wall is determined by its composition, including the ratio of acidic to basic phospholipids. Thus, another survival strategy 1 http://www.who.int/mediacentre/news/releases/2010/drug_resistant_tb 20100318/ en/index.html www.frontiersin.org involves the modulation of membrane surface charge so that the pathogen can weather the action of host cationic antimicrobial peptides (CAMPs; Peschel, 2002; Kraus and Peschel, 2006). Gram positive pathogens, such as Staphylococcus aureus, decrease net membrane charge by adding lysine groups on acidic phospholipid phosphatidylglycerol (PG) and thereby resist the action of CAMPs (Peschel et al., 2001). It is also known that other acidic phospholipids, such as cardiolipin (CL), are subject to such modification (Thedieck et al., 2006). We recently showed that a two-domain lysyltransferase and lysyl-tRNA-synthetase protein encoded by lysX gene of M. tuberculosis is necessary for PG lysinylation, optimal survival in lungs of mice and guinea pigs, resistance to the action of CAMPs and for maintaining optimal membrane potential (Maloney et al., 2009). While the above study shows that lysX plays an important role in M. tuberculosis survival upon infection, several questions remain with respect to lysX in vivo phenotype. For example, these studies revealed that lysinylated phosphatidylglycerol (L-PG) is a constituent of M. tuberculosis, although it is unknown if other mycobacterial species also produce lysinylated phospholipids (L-PL). Also, the lysX complement strain, wherein lysX expression was achieved from M. smegmatis amidase promoter (Triccas et al., 1998), was partially proficient in restoring lysX defect (Maloney et al., 2009). While studies based on fluorescent probes allowed the visualization of acidic phospholipids such as PG and CL in Escherichia coli and other bacteria as defined domains (Kawai et al., 2004; Romantsov et al., 2007; Mileykovskaya and Dowhan, 2009), it is unknown whether the absence of L-PG production is associated with changes in membrane lipid domain February 2011 | Volume 2 | Article 19 | 1 Maloney et al. organization and possibly their turnover thereby contributing to the observed changes in membrane potential (Maloney et al., 2009). The present study is undertaken to address some of these issues. We found that the domain organization of the major acidic phospholipid CL, as revealed by 10-nonyl acridine orange (NAO) staining, is not significantly affected in lysX mutants. However, differences in the turnover of select membrane phospholipids were found between lysX and its parent. Furthermore, our results indicate that expression of lysX from a strong inducible tet promoter allowed full complementation in vivo and that radiolabeled lysine is incorporated in other mycobacterial phospholipids. Our studies favor a hypothesis that alterations in phospholipid turnover are in part responsible for the observed lysX phenotype. Materials and Methods Cloning Unless otherwise noted all genes used in this study were generated by polymerase chain reaction (PCR) using genomic DNA as a template and Phusion DNA polymerase (New England Biolabs). As needed, PCR products were (...truncated)