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 ␤ 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 ϳ10,000 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.

Altered Mycobacterium tuberculosis Cell Wall Metabolism and Physiology Associated With RpoB Mutation H526D

“…For instance, transcriptomic and proteomic analysis of Mtb clinical isolates containing rpoB mutations demonstrated upregulation of polyketide synthases, which are enzymes involved in the biosynthesis of phthiocerol dimycocerosate (PDIM) ( Bisson et al, 2012 ). More recently, lipidomic profiling of rpoB mutant Mtb showed altered concentrations of mycobactin siderophores and acylated sulfoglycolipids ( Lahiri et al, 2016 ), while metabolomics revealed decreased synthesis of various branched-chain fatty acids ( du Preez and Loots du, 2012 ). These cell wall-associated lipids, including PDIM, mycobactin, sulfoglycolipids, and fatty acids, have been implicated in cell wall permeability and virulence ( Camacho et al, 2001 ; Converse et al, 2003 ; Yeruva et al, 2006 ; Gebhardt et al, 2007 ; Gilmore et al, 2012 ; Reddy et al, 2013 ; Lahiri et al, 2016 ; Quigley et al, 2017 ).…”

The Physiology of Mycobacterium tuberculosis in the Context of Drug Resistance: A System Biology Perspective

“…In Neisseria meningitidis and Staphylococcus aureus, rpoB mutations lead to a decrease permeability of the cell wall, which can be related to a subsequent increase in tolerance to certain antibiotics such as vancomycin [96][97][98]. Interestingly, after exposure to RIF, Mtb also appears to have an increased tolerance to ofloxacin, probably because of an increase activity of efflux pumps [99], although recent findings from our laboratory as well as others also suggest a potential role for cell permeability [75,100]. In our study, isogenic Mtb pairs with two different rpoB mutations and representing two different genetic lineages (Beijing and Haarlem) showed an increased abundance of proteins involved polyketide synthesis.…”

“…The analysis revealed reduced levels of di-acylated sulfoglycolipid (Ac2SGL) and mycobactins (including carboxymycobactins), while increased levels of PDIM compared to their DS parental strain. These compounds were identified among 172 features in the W-Beijing group and 102 features in the CDC1551 group analyzed by high performance liquid chromatography (HPLC) mass quadrupole-time-of-flight (QTOF) MS and suggesting a global remodeling of the cell wall after acquisition of RIF resistance [100]. This study supports previous findings from our group, which included a significant increase of diacylglycerol phosphocholines and PDIM precursors as observed by ultra-performance liquid chromatography (UPLC)-QTOF [75].…”

Emerging Approaches to Tuberculosis Drug Development: At Home in the Metabolome

“…Using GC-MS on a small number of rpoB mutants, du Preez and Loots found decreased levels of 10-methyl branched-chain fatty acids and cell wall lipids, suggestive evidence of increased metabolism of short fatty acids [70,71]. Using LC-MS lipidomics, Lahiri et al reported altered levels of mycobactin siderophores and acylated sulfoglycolipids [72]. Mutations in catalase-peroxidase (katG) that render resistance to the frontline drug isoniazid, by preventing activation into its biochemically active form, were similarly associated with increased levels of several fatty acids, alcohols and alkanes [73].…”