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Deficiency of GntR Family Regulator MSMEG_5174 Promotes Mycobacterium smegmatis Resistance to Aminoglycosides via Manipulating Purine Metabolism.

ABSTRACT: The increasing incidence of drug-resistant tuberculosis is still an emergency for global public health and a major obstacle to tuberculosis treatment. Therefore, deciphering the novel mechanisms of mycobacterial antibiotic resistance is crucial for combatting the rapid emergence of drug-resistant strains. In this study, we identified an unexpected role of Mycobacterium smegmatis GntR family transcriptional regulator MSMEG_5174 and its homologous gene Mycobacterium tuberculosis Rv1152 in aminoglycoside antibiotic resistance. Deficiency of MSMEG_5174 rendered Mycobacterium smegmatis highly resistant to aminoglycoside antibiotic treatment, and ectopic expression of Rv1152 in MSMEG_5174 mutants restored antibiotic-induced bacterial killing. We further demonstrated that MSMEG_5174 negatively regulates the expression of purine metabolism-related genes and the accumulation of purine metabolites. Moreover, overexpression of xanthine dehydrogenase MSMEG_0871 or xanthine treatment elicited a significant decrease in aminoglycoside antibiotic lethality for Mycobacterium smegmatis. Together, our findings revealed MSMEG_5174 as a metabolic regulator and hint toward unexplored crosstalk between purine metabolism and antibiotic resistance.

SUBMITTER: Deng W

PROVIDER: S-EPMC9309504 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMycobacterium smegmatis is fast growing non-pathogenic mycobacteria. This organism has been widely used as a model organism to study the biology of other virulent and extremely slow growing species like Mycobacterium tuberculosis. Based on the homology of the N-terminal DNA binding domain, the recently sequenced genome of M. smegmatis has been shown to possess several putative GntR regulators. A striking characteristic feature of this family of regulators is that they possess a conserved N-terminal DNA binding domain and a diverse C-terminal domain involved in the effector binding and/or oligomerization. Since the physiological role of these regulators is critically dependent upon effector binding and operator sites, we have analysed and classified these regulators into their specific subfamilies and identified their potential binding sites.ResultsThe sequence analysis of M. smegmatis putative GntRs has revealed that FadR, HutC, MocR and the YtrA-like regulators are encoded by 45, 8, 8 and 1 genes respectively. Further out of 45 FadR-like regulators, 19 were classified into the FadR group and 26 into the VanR group. All these proteins showed similar secondary structural elements specific to their respective subfamilies except MSMEG_3959, which showed additional secondary structural elements. Using the reciprocal BLAST searches, we further identified the orthologs of these regulators in Bacillus subtilis and other mycobacteria. Since the expression of many regulators is auto-regulatory, we have identified potential operator sites for a number of these GntR regulators by analyzing the upstream sequences.ConclusionThis study helps in extending the annotation of M. smegmatis GntR proteins. It identifies the GntR regulators of M. smegmatis that could serve as a model for studying orthologous regulators from virulent as well as other saprophytic mycobacteria. This study also sheds some light on the nucleotide preferences in the target-motifs of GntRs thus providing important leads for initiating the experimental characterization of these proteins, construction of the gene regulatory network for these regulators and an understanding of the influence of these proteins on the physiology of the mycobacteria.

Project description:UnlabelledIn the presence of alanine, AldR, which belongs to the Lrp/AsnC family of transcriptional regulators and regulates ald encoding alanine dehydrogenase in Mycobacterium smegmatis, changes its quaternary structure from a homodimer to an octamer with an open-ring conformation. Four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N2-NWW/WWN-N2-A/TC were identified upstream of the M. smegmatis ald gene by means of DNase I footprinting analysis. O2, O1, and O4 are required for the induction of ald expression by alanine, while O3 is directly involved in the repression of ald expression. In addition to O3, both O1 and O4 are also necessary for full repression of ald expression in the absence of alanine, due to cooperative binding of AldR dimers to O1, O4, and O3. Binding of a molecule of the AldR octamer to the ald control region was demonstrated to require two AldR-binding sites separated by three helical turns between their centers and one additional binding site that is in phase with the two AldR-binding sites. The cooperative binding of AldR dimers to DNA requires three AldR-binding sites that are aligned with a periodicity of three helical turns. The aldR gene is negatively autoregulated independently of alanine. Comparative analysis of ald expression of M. smegmatis and Mycobacterium tuberculosis in conjunction with sequence analysis of both ald control regions led us to suggest that the expression of the ald genes in both mycobacterial species is regulated by the same mechanism.ImportanceIn mycobacteria, alanine dehydrogenase (Ald) is the enzyme required both to utilize alanine as a nitrogen source and to grow under hypoxic conditions by maintaining the redox state of the NADH/NAD(+) pool. Expression of the ald gene was reported to be regulated by the AldR regulator that belongs to the Lrp/AsnC (feast/famine) family, but the underlying mechanism was unknown. This study revealed the regulation mechanism of ald in Mycobacterium smegmatis and Mycobacterium tuberculosis. Furthermore, a generalized arrangement pattern of cis-acting regulatory sites for Lrp/AsnC (feast/famine) family regulators is suggested in this study.

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Deficiency of GntR Family Regulator MSMEG_5174 Promotes Mycobacterium smegmatis Resistance to Aminoglycosides via Manipulating Purine Metabolism.

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