Lipid composition and transcriptional response of Mycobacterium tuberculosis grown under iron-limitation in continuous culture: identification of a novel wax ester

Abstract

The low level of available iron in vivo is a major obstacle for microbial pathogens and is a stimulus for the expression of virulence genes. In this study, Mycobacterium tuberculosis H37Rv was grown aerobically in the presence of limited iron availability in chemostat culture to determine the physiological response of the organism to iron-limitation. A previously unidentified wax ester accumulated under iron-limited growth, and changes in the abundance of triacylglycerol and menaquinone were also observed between iron-replete and iron-limited chemostat cultures. DNA microarray analysis revealed differential expression of genes involved in glycerolipid metabolism and isoprenoid quinone biosynthesis, providing some insight into the underlying genetic changes that correlate with cell-wall lipid profiles of M. tuberculosis growing in an iron-limited environment.

Fig. 1

2D non-phospholipid patterns of representative chemostat-grown M. tuberculosis H37Rv. (A), (C), (E) and (G) are TLC images for iron-replete culture IR3. (B), (D), (F) and (H) are TLC images for iron-limited culture IL1. (A–F) are apolar lipids. (G) and (H) are polar lipids. Profiles in (A) and (B) were resolved using solvent system A. Profiles (C) and (D) were resolved using solvent system B. Profiles (E–H) were resolved using solvent system D. FA, fatty acids; A, B and C, unknowns; SL and SL′, sulfolipids.

Fig. 2

¹H-NMR spectra of the two major components of the putative WE (Fig. 1B, WE) that accumulated under iron-limitation. (A) Least mobile component, (B) more mobile component, (C) stearyl oleate, and (D) stearyl stearate. x axes of plots show p.p.m.

Fig. 3

RT-qPCR analysis of (A) tcrX, (B) pks2 and (C) Rv0560c from M. tuberculosis grown under iron-replete or iron-limited conditions. Levels of sigA were not significantly different under iron-limitation. The other genes were significantly induced under iron-limitation (unpaired Student’s t test); *P<0.01, **P<0.001.