Alterations in Phospholipid Catabolism in Mycobacterium Tuberculosis LysX Mutant

Dorota Stankowska,Erin Maloney,Haidan Guo,Malini Rajagoapalan,Murty V Madiraju,Shichun Lun,William R Bishai

doi:10.3389/fmicb.2011.00019

Abstract

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.

Highlights

Mycobacterium tuberculosis is a pathogenic bacterium that causes the infectious disease tuberculosis
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 cationic antimicrobial peptides (CAMPs) and for maintaining optimal membrane potential (Maloney et al, 2009)
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

Summary

Introduction

Mycobacterium tuberculosis is a pathogenic bacterium that causes the infectious disease tuberculosis. The effect of PMNB on survival of Ptet-lysX complement strain We have previously shown that the lysX mutant is sensitive to PMNB, a CAMP whereas Pami-lysX complement is as proficient for survival as the wild type at 100 units/mL of PMNB (Maloney et al, 2009). To test if Ptet-lysX complement reverses lysX sensitivity to PMNB at higher doses, we grew M. tuberculosis strains in the presence of 500 units/mL PMNB and recorded change in optical density as a measurement of growth (Figure 2).

Results

Conclusion