The W-Beijing lineage of Mycobacterium tuberculosis overproduces triglycerides and has the DosR dormancy regulon constitutively upregulated

Abstract

The Beijing family of Mycobacterium tuberculosis strains has been associated with epidemic spread and an increased likelihood of developing drug resistance. The characteristics that predispose this family to such clinical outcomes have not been identified, although one potential candidate, the phenolic glycolipid PGL-tb, has been shown to mediate a fulminant lethal disease in mice and rabbits due to lipid-mediated immunosuppression. However, PGL-tb is not uniformly expressed throughout the Beijing lineage and may not be the only unique virulence trait associated with this family. In an attempt to define phenotypes common to all Beijing strains, we interrogated a carefully selected set of isolates representing the five extant lineages of the Beijing family. Comparison of lipid production in this set revealed that all Beijing strains accumulated large quantities of triacylglycerides in in vitro aerobic culture. This accumulation was found to be coincident with upregulation of Rv3130c, whose product was previously characterized as a triacylglyceride synthase. Rv3130c is a member of the DosR-controlled regulon of M. tuberculosis, and further examination revealed that several members of this regulon were upregulated throughout this strain family. The upregulation of the DosR regulon may confer an adaptive advantage for growth in microaerophilic or anaerobic environments encountered by the bacillus during infection and thus may be related to the epidemiological phenomena associated with this important strain lineage.

FIG. 1.

Global population structure of M. tuberculosis. Numbers in rectangles refer to lineage-defining LSPs. The geographic regions associated with specific lineages are indicated, as is the Beijing family subgroup structure (adapted from reference with the permission of the publisher).

FIG. 2.

Triglyceride synthesis in Beijing strains. (A) Apolar lipids extracted from [1-¹⁴C]propionic acid-, [1-¹⁴C]acetic acid-, or [U-¹⁴C]glycerol-labeled cultures of the M. tuberculosis strains indicated were analyzed on silica gel TLC plates developed in petroleum ether-acetone (96:4). Radiolabeled lipids were detected by phosphorimaging. PDIM, phthiocerol dimycocerosate. (B) Apolar lipid extracts of HN878 and H37Rv cultures were fractionated as for panel A and detected by staining with 5% phosphomolybdic acid (PMA).

FIG. 3.

All Beijing sublineages accumulate triglycerides. Apolar lipids extracted from [1-¹⁴C]propionic acid-labeled in vitro cultures of the Beijing and non-Beijing strains indicated were analyzed on silica gel TLC plates developed in petroleum ether-acetone (96:4). Radiolabeled lipids were subsequently visualized by phosphorimaging. The position of the triglyceride fraction specific to all Beijing strains is shown. PDIM, phthiocerol dimycocerosate.

FIG. 4.

Constitutive dormancy regulon expression in the M. tuberculosis Beijing lineage. Gene expression levels within RNA samples isolated from early-log-phase in vitro cultures were analyzed by qRT-PCR. Mean transcript abundance (ng) was determined for Beijing (n = 11) and non-Beijing (n = 8) isolates (assayed in triplicate) relative to sigA transcript levels. Two independent RNA samples were analyzed for each M. tuberculosis strain, and data from a single representative experiment are presented. Error bars represent standard deviations.