Upregulation of the Phthiocerol Dimycocerosate Biosynthetic Pathway by Rifampin-Resistant,rpoBMutant Mycobacterium tuberculosis

Abstract

h Multidrug-resistant tuberculosis has emerged as a major threat to tuberculosis control. Phylogenetically related rifampin-resistant actinomycetes with mutations mapping to clinically dominant Mycobacterium tuberculosis mutations in the rpoB gene show upregulation of gene networks encoding secondary metabolites. We compared the expressed proteomes and metabolomes of two fully drug-susceptible clinical strains of M. tuberculosis (wild type) to those of their respective rifampin-resistant, rpoB mutant progeny strains with confirmed rifampin monoresistance following antitubercular therapy. Each of these strains was also used to infect gamma interferon-and lipopolysaccharide-activated murine J774A.1 macrophages to analyze transcriptional responses in a physiologically relevant model. Both rpoB mutants showed significant upregulation of the polyketide synthase genes ppsA-ppsE and drrA, which constitute an operon encoding multifunctional enzymes involved in the biosynthesis of phthiocerol dimycocerosate and other lipids in M. tuberculosis, but also of various secondary metabolites in related organisms, including antibiotics, such as erythromycin and rifamycins. ppsA (Rv2931), ppsB (Rv2932), and ppsC (Rv2933) were also found to be upregulated more than 10-fold in the Beijing rpoB mutant strain relative to its wild-type parent strain during infection of activated murine macrophages. In addition, metabolomics identified precursors of phthiocerol dimycocerosate, but not the intact molecule itself, in greater abundance in both rpoB mutant isolates. These data suggest that rpoB mutation in M. tuberculosis may trigger compensatory transcriptional changes in secondary metabolism genes analogous to those observed in related actinobacteria. These findings may assist in developing novel methods to diagnose and treat drug-resistant M. tuberculosis infections.A pproximately 9 million people develop active tuberculosis (TB) each year, resulting in nearly 2 million deaths annually (75). Recent progress controlling drug-susceptible TB has been made in many regions (50); however, drug resistance in Mycobacterium tuberculosis, including strains resistant to both first-line drugs, isoniazid and rifampin (multidrug-resistant [MDR] TB), has emerged as a threat to TB control worldwide (26, 65). Although international surveillance systems are inadequate, data from over 80 countries indicate that approximately 1 in 10 new cases of active TB have primary drug resistance (77), and MDR TB has been reported in essentially every country in which drug resistance has been studied (76). Treatment of MDR TB requires the use of costly and toxic second-line drugs for nearly 2 years and is associated with high rates of morbidity and mortality (65), which makes the spread of drug-resistant TB a major public health concern.Drug resistance in M. tuberculosis is due primarily to singlenucleotide polymorphisms in genes encoding key mycobacterial enzymes (6). The rpoB gene encodes the ␤-subunit of bacterial RNA polymerase, which is the target of rifampin (12...

Metabolomics: Applications and Promise in Mycobacterial Disease

“…LC-MS-based metabolomics showed differences in the metabolic profile of rifampin-resistant M. tuberculosis: 99 molecular features are different in the rifampin-resistant strains. In addition, the studies showed the major role of rpoB mutations in the M. tuberculosis metabolism (46).…”

Rifampin Resistance Mutations Are Associated with Broad Chemical Remodeling of Mycobacterium tuberculosis

“…Transcriptomic analysis of M. tuberculosis after rpoB mutation likewise demonstrates durable transcriptional change in this species (12,13). However, the specific downstream conse-* This work was supported by National Institutes of Health Grants U19 AI076217, U19 AI111224, R01 AI049313, and P30 AI060354 and the Burroughs Wellcome Foundation.…”

Biochemical Characterization of Isoniazid-resistant Mycobacterium tuberculosis: Can the Analysis of Clonal Strains Reveal Novel Targetable Pathways?

“…Cells and culture filtrate were harvested after three weeks of culture as described elsewhere [14]. Culture…”

“…and used to qualify and provide similar biological reagents to the mycobacteria research community through BEI resources (https://www.beiresources.org/About/QualityControl.aspx). Before the digestion of proteins, the CW fraction was delipidated as described elsewhere [14]. All the resulting proteins were resuspended in 10mM ammonium bicarbonate (Sigma-Aldrich, A6141, US).…”

“…Total protein concentration of all subcellular fractions and CFPs was measured using the bicinchoninic acid method (BCA) (Thermo Scientific™Pierce™BCA Protein Assay, US). In-solution trypsin digestion of 30 μg of each protein sample was performed as described previously, using ProteaseMax surfactant (Fisher Scientific, PRV2071, US) followed by a final desalting step using Pierce® C-18 spin columns (Thermo Fisher Scientific, 89870, US) [14]. Peptides were finally resuspended in solvent A (0.1% formic acid (Thermo Scientific™ Pierce™, PI28905, US), 3%…”