The Orphan Response Regulator Rv3143 Modulates the Activity of the NADH Dehydrogenase Complex (Nuo) in Mycobacterium tuberculosis via Protein–Protein Interactions

Plocinska, Renata; Wasik, Karolina; Płociński, Przemysław; Lechowicz, Ewelina; Antczak, Magdalena; Błaszczyk, Ewelina; Dziadek, Bożena; Słomka, Marcin; Rumijowska-Galewicz, Anna; Dziadek, Jarosław

doi:10.3389/fcimb.2022.909507

Cited by 6 publications

(3 citation statements)

References 61 publications

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“…MSMEG_2064 is found in the same operon as other Complex I genes 5′ of nuoA and was proposed to be a constituent of mycobacterial Complex I (12). Furthermore, recent crosslinking experiments suggested that MSMEG_2064 associates with NuoD and deletion of the MSMEG_2064 gene affects expression of some electron transport chain components (51). The structure of MSMEG_2064 predicted by AlphaFold (52) fits into the unidentified density with high fidelity (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…MSMEG_2064 is a two-component response regulator protein that is considered to be an “orphan” because its cognate kinase has not been identified (51, 53). Sequence comparison and structural alignment with the PDBeFold server (54, 55) showed similar folds but low sequence identity (<30%) between MSMEG_2064 and other two-component response proteins with known structures.…”

Section: Resultsmentioning

confidence: 99%

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Structure of mycobacterial respiratory Complex I

Liang

Plourde

Bueler

et al. 2022

Preprint

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Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found at low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the "orphan" two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel and suggests that menaquinone interacts more extensively than ubiquinone with a key catalytic histidine residue in the enzyme.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structure of mycobacterial respiratory Complex I

Liang

Plourde

Bueler

et al. 2022

Preprint

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“…mtrA is essential in Mtb due its role in replication, whereas in Mab mtrA was reported to be non-essential, pointing to potential differences in the role of this TCS between the two species ( Fol et al., 2006 ; Akusobi et al., 2022 ). The Mtb ortholog (Rv3143) of orphan RR Mab _ 2133 is implicated in nitrate metabolism in the absence of oxygen, binds to nuo subunits required for electron transport and is within nuo operon ( Plocinska et al., 2022 ). Mab’s nuo operon displays synteny with the Mtb nuo operon including the orphan RR Mab _ 2133 .…”

Section: Resultsmentioning

confidence: 99%

Mycobacterium abscessus DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia

Simcox

Tomlinson²,

Shaw³

et al. 2023

Front. Cell. Infect. Microbiol.

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Mycobacterium abscessus (Mab), an emerging opportunistic pathogen, predominantly infects individuals with underlying pulmonary diseases such as cystic fibrosis (CF). Current treatment outcomes for Mab infections are poor due to Mab’s inherent antibiotic resistance and unique host interactions that promote phenotypic tolerance and hinder drug access. The hypoxic, mucus-laden airways in the CF lung and antimicrobial phagosome within macrophages represent hostile niches Mab must overcome via alterations in gene expression for survival. Regulatory mechanisms important for the adaptation and long-term persistence of Mab within the host are poorly understood, warranting further genetic and transcriptomics study of this emerging pathogen. DosRSMab, a two-component signaling system (TCS), is one proposed mechanism utilized to subvert host defenses and counteract environmental stress such as hypoxia. The homologous TCS of Mycobacterium tuberculosis (Mtb), DosRSMtb, is known to induce a ~50 gene regulon in response to hypoxia, carbon monoxide (CO) and nitric oxide (NO) in vitro and in vivo. Previously, a small DosRMab regulon was predicted using bioinformatics based on DosRMtb motifs however, the role and regulon of DosRSMab in Mab pathogenesis have yet to be characterized in depth. To address this knowledge gap, our lab generated a Mab dosRS knockout strain (MabΔdosRS) to investigate differential gene expression, and phenotype in an in vitro hypoxia model of dormancy. qRT-PCR and lux reporter assays demonstrate Mab_dosR and 6 predicted downstream genes are induced in hypoxia. In addition, RNAseq revealed induction of a much larger hypoxia response comprised of >1000 genes, including 127 differentially expressed genes in a dosRS mutant strain. Deletion of DosRSMab led to attenuated growth under low oxygen conditions, a shift in morphotype from smooth to rough, and down-regulation of 216 genes. This study provides the first look at the global transcriptomic response of Mab to low oxygen conditions encountered in the airways of CF patients and within macrophage phagosomes. Our data also demonstrate the importance of DosRSMab for adaptation of Mab to hypoxia, highlighting a distinct regulon (compared to Mtb) that is significantly larger than previously described, including both genes conserved across mycobacteria as well as Mab-specific genes.

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