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

Abstract

Global control of tuberculosis has become increasingly complicated with the emergence of multidrug-resistant strains of Mycobacterium tuberculosis First-line treatments are anchored by two antibiotics, rifampin and isoniazid. Most rifampin resistance occurs through the acquisition of missense mutations in the rifampin resistance-determining region, an 81-base pair region encoding the rifampin binding site on the β subunit of RNA polymerase (rpoB). Although these mutations confer a survival advantage in the presence of rifampin, they may alter the normal process of transcription, thereby imposing significant fitness costs. Because the downstream biochemical consequences of the rpoB mutations are unknown, we used an organism-wide screen to identify the number and types of lipids changed after rpoB mutation. A new mass spectrometry-based profiling platform systematically compared ∼10,000 cell wall lipids in a panel of rifampin-resistant mutants within two genetically distinct strains, CDC1551and W-Beijing. This unbiased lipidomic survey detected quantitative alterations (>2-fold, p < 0.05) in more than 100 lipids in each mutant. By focusing on molecular events that change among most mutants and in both genetic backgrounds, we found that rifampin resistance mutations lead to altered concentrations of mycobactin siderophores and acylated sulfoglycolipids. These findings validate a new organism-wide lipidomic analysis platform for drug-resistant mycobacteria and provide direct evidence for characteristic remodeling of cell wall lipids in rifampin-resistant strains of M. tuberculosis The specific links between rifampin resistance and named lipid factors provide diagnostic and therapeutic targets that may be exploited to address the problem of drug resistance.

Keywords: MS; Mycobacterium tuberculosis; antibiotic resistance; lipidomics; multidrug resistance; rifampin; siderophore; tuberculosis.

FIGURE 1.

Lipidomics data pipeline. A, drug-sensitive (sensitive) parental and RpoB mutant strains identified according to the position of mutation (H526Y, S531L, and Q513E) were grown in antibiotic-free liquid medium. O.D., optical density. B, triplicate lipidomic analysis of the drug-sensitive S531L mutant in the CDC1551 (left panel) and W-Beijing (right panel) backgrounds shows the total number of detected events. After excluding events that are also detectable in solvent blanks or not seen in all three HPLC runs, the list of “filtered events” more closely approximates the mycobacterial lipid profile. C, after aligning filtered events with equivalent mass and retention time values, paired values were assigned as intensity ratios (dots). Paired events with -fold change values of >2 with corrected p < 0.05 are considered to be biologically significant (red dots). The number of changed events divided by the number of filtered events gives a global value of lipid change (red).

FIGURE 2.

Identification of globally changed events. A, lists of all changed events for each of three mutants were compared to identify the subset of events changed in every mutant within each strain background. B, using mass values and the MycoMass database to preliminarily identify events corresponding to sulfoglycolipid, mycobactin, and carboxymycobactin, named (black) and unnamed unchanged (gray) events are shown.

FIGURE 3.

Validation of automated detection of mycobactins using analytical chemistry methods. A, focusing on ions whose detected m/z values match the theoretical values for ferrimycobactin and ferricarboxymycobactin [M+Fe-2H] within experimental error, positive mode CID-MS generated the indicated fragment ions. B and C, software-derived intensity values using XCMS curve fitting software shown in Fig. 2 were validated using raw data generated as ion chromatograms corresponding to the known masses of mycobactin (909.4) and carboxymycobactin (785.3). B, W-Beijing. C, CDC1551.

FIGURE 4.

Association of the rpoB mutation with sulfoglycolipid expression (Ac2SGL). A, focusing on ions whose detected m/z values match the theoretical values for Ac2SGL [M+NH4⁺]⁺, CID-MS, detecting the indicated fragment ions corresponding to the loss of hexose sulfate, fatty acyl units, and the indicated phthioceranic acid. B and C, to validate intensity values generated using curve fitting software, ion chromatograms corresponding to the known masses of AC2SGL (m/z 1277.9) were generated manually.