Dataset Information

Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance.

ABSTRACT: In recent years, the treatment of tuberculosis is once again facing a severe situation because the existing antituberculosis drugs have become weaker and weaker with the emergence of drug-resistant Mycobacterium tuberculosis (Mtb). The studies of cell division and cell cycle-related factors in Mtb are particularly important for the development of new drugs with broad-spectrum effects. Mycobacterium smegmatis (Msm) has been used as a model organism to study the molecular, physiological, and drug-resistant mechanisms of Mtb. Bioinformatics analysis has predicted that MSMEG_6171 is a MinD-like protein of the septum site-determining protein family associated with cell division in Mycobacterium smegmatis. In our study, we use ultrastructural analysis, proteomics, metabolomics, and molecular biology techniques to comprehensively investigate the function of MSMEG_6171. Overexpression of MSMEG_6171 in Msm resulted in elongated cells, suggesting an important role of MSMEG_6171 in regulating cell wall morphology. The MSMEG_6171 overexpression could enhance the bacterial resistance to vancomycin, ethionamide, meropenem, and cefamandole. The MSMEG_6171 overexpression could alter the lipid metabolism of Msm to cause the changes on cellular biofilm property and function, which enhances bacterial resistance to antibiotics targeting cell wall synthesis. MSMEG_6171 could also induce the glyceride and phospholipid alteration in vivo to exhibit the pleiotropic phenotypes and various cellular responses. The results showed that amino acid R249 in MSMEG_6171 was a key site that can affect the level of bacterial drug resistance, suggesting that ATPase activity is required for function.

SUBMITTER: Wu Z

PROVIDER: S-EPMC7393984 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance.

Wu Zhuhua Z Wei Wenjing W Zhou Ying Y Guo Huixin H Zhao Jiao J Liao Qinghua Q Chen Liang L Zhang Xiaoli X Zhou Lin L

Frontiers in microbiology 20200724

In recent years, the treatment of tuberculosis is once again facing a severe situation because the existing antituberculosis drugs have become weaker and weaker with the emergence of drug-resistant Mycobacterium tuberculosis (Mtb). The studies of cell division and cell cycle-related factors in Mtb are particularly important for the development of new drugs with broad-spectrum effects. Mycobacterium smegmatis (Msm) has been used as a model organism to study the molecular, physiologi ...[more]

PMID: 32793136

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Project description:Despite the availability of effective drug treatment, Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, kills ~1. 5 million people annually, and the rising prevalence of drug resistance increasingly threatens to worsen this plight. We previously showed that sublethal exposure to the frontline anti-TB drug, rifampicin, resulted in substantial adaptive remodeling of the proteome of the model organism, Mycobacterium smegmatis, in the drug-sensitive mc2155 strain [wild type (WT)]. In this study, we investigate whether these responses are conserved in an engineered, isogenic mutant harboring the clinically relevant S531L rifampicin resistance-conferring mutation (SL) and distinguish the responses that are specific to RNA polymerase β subunit- (RpoB-) binding activity of rifampicin from those that are dependent on the presence of rifampicin alone. We verified the drug resistance status of this strain and observed no phenotypic indications of rifampicin-induced stress upon treatment with the same concentration as used in WT (2.5 μg/ml). Thereafter, we used a cell wall-enrichment strategy to focus attention on the cell wall proteome and observed 253 proteins to be dysregulated in SL bacteria in comparison with 716 proteins in WT. We observed that decreased abundance of ATP-binding cassette (ABC) transporters and increased abundance of ribosomal machinery were conserved in the SL strain, whereas the upregulation of transcriptional machinery and the downregulation of numerous two-component systems were not. We conclude that the drug-resistant M. smegmatis strain displays some of the same proteomic responses observed in WT and suggest that this evidence supports the hypothesis that rifampicin exercises effects beyond RpoB-interaction alone and that mycobacteria recognise rifampicin as a signaling molecule in an RpoB-independent manner at sublethal doses. Taken together, our data indicates mixed RpoB-independent and RpoB-dependent proteomic remodeling in WT mycobacteria, with evidence for RpoB-independent ABC transporter downregulation, but drug activity-based transcriptional upregulation and two-component system downregulation.

Project description:BackgroundTranscriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, is in part mediated by several nucleic-acid associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acid-associated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation.ResultsAP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification.ConclusionsEffective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA- and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase β-subunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress.

Dataset Information

Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of Mycobacterium smegmatis Leading to Increased Vancomycin Resistance.

Publications

Integrated Quantitative Proteomics and Metabolome Profiling Reveal MSMEG_6171 Overexpression Perturbing Lipid Metabolism of <i>Mycobacterium smegmatis</i> Leading to Increased Vancomycin Resistance.

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