Abstract
Tuberculosis (TB) continues to be an important public health threat worldwide, due in part to drug resistant Mycobacterium tuberculosis (Mtb) strains. The United States recently reported a shortage of isoniazid (INH), which could drive higher INH resistance rates. Changes in the Mtb proteome before and after acquisition of INH resistance in a clean genetic background remain understudied and may elucidate alternate drug targets. Here, we focused on Mtb clonal strains to characterize the consequences of INH resistance on mycobacterial metabolism. Proteomic analysis was conducted by liquid-chromatography tandem mass spectrometry (LC-MS/MS) of cellular and secreted fractions, followed by a normalized spectral counting (NSAF) analysis (data are available via ProteomeXchange with identifier PXD009549). Two different Mtb clonal pairs representing a specific genetic lineage (one clinical and one generated in the laboratory) but sharing a katG mutation associated with INH resistance, were used in our analysis. Overall, we found 26 Mtb proteins with altered abundances after acquisition of INH resistance across both Mtb genetic lineages studied. These proteins were involved in ATP synthesis, lipid metabolism, regulatory events, and virulence, detoxification, and adaptation processes. Proteomic findings were validated by Western blotting analyses whenever possible. Mycolic acid (MA) analysis through LC/MS in the clonal Mtb pairs did not reveal a common trend in the alteration of these fatty acids across both INHr strains but revealed a significant reduction in levels of the two more abundant α-MA features in the clinical INHr strain. Interestingly, the clinical clonal pair demonstrated more variation in the abundance of the proteins involved in the FAS II pathway. Together, the proteomic and lipidomic data highlight the identification of potential drug targets such as alternative lipid biosynthetic pathways that may be exploited to combat clinically relevant Mtb INHr strains.
Highlights
Despite global efforts to control tuberculosis (TB),1 the emergence of drug-resistant cases has been detrimental for the successful control of this disease
This study presents a snapshot of genetically related Mycobacterium tuberculosis (Mtb) strains under the same in vitro conditions to detect specific changes related to the INHr phenotype
It is important to emphasize two points that should be considered in the analysis of the findings presented here; first, with the exception of KatG levels, none of the other protein changes described in this study have associated mutations in their respective genes, as previously revealed by whole genomics sequencing of these strains [12]. This shotgun proteomics approach presents several limitations in that changes in protein abundance may be attributed to decreased expression, differences in protein half-lives, and losses in protein abundance due to aggregation and post-translational modifications with concomitant changes in protein function
Summary
Despite global efforts to control tuberculosis (TB),1 the emergence of drug-resistant cases has been detrimental for the successful control of this disease. AtpH was increased in both INHr strains, this difference was statistically significant only in the membrane and cell wall fractions of the laboratory pair (Fig. 3A).
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