scholarly journals Characterization of Guided Entry of Tail-Anchored Proteins 3 Homologues in Mycobacterium tuberculosis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Kuan Hu ◽  
Ashley T. Jordan ◽  
Susan Zhang ◽  
Avantika Dhabaria ◽  
Amanda Kovach ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Structure Prediction ◽  
Bacterial Species ◽  
Normal Growth ◽  
Anion Transport ◽  
Content Type ◽  
Reading Frame ◽  
Anion Transporters ◽  
Anion Transporter

ABSTRACT We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode “anion transporter ATPase” homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined. IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.

scholarly journals Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Jordan B. Jastrab ◽  
Marie I. Samanovic ◽  
Richard Copin ◽  
Bo Shopsin ◽  
K. Heran Darwin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Heat Shock ◽  
Protein Quality ◽  
Normal Growth ◽  
Culture Conditions ◽  
Growth Defect ◽  
Loss Of Function ◽  
Content Type ◽  
Accessory Factor

ABSTRACT Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosis proteasome accessory factor E (PafE). Recently, we found that a Mycobacterium tuberculosis pafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (heat shock protein repressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE + bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR. IMPORTANCE Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis. In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress.

scholarly journals Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

2016 ◽  
Vol 60 (9) ◽  
pp. 5232-5237 ◽  
Author(s):  
Xia Yu ◽  
Guirong Wang ◽  
Suting Chen ◽  
Guomei Wei ◽  
Yuanyuan Shang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Bacterial Species ◽  
Drug Susceptibility Testing ◽  
World Health ◽  
Wild Type ◽  
Resistance Mutations ◽  
Content Type ◽  
Lowenstein Jensen

ABSTRACTAntofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying itsin vitroactivity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinicalMycobacterium tuberculosisstrains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions ofgyrA(Rv0006) andgyrB(Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

scholarly journals Urease Activity Represents an Alternative Pathway for Mycobacterium tuberculosis Nitrogen Metabolism

2012 ◽  
Vol 80 (8) ◽  
pp. 2771-2779 ◽  
Author(s):  
Wenwei Lin ◽  
Vanessa Mathys ◽  
Emily Lei Yin Ang ◽  
Vanessa Hui Qi Koh ◽  
Julia María Martínez Gómez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitrogen Source ◽  
Nitrogen Metabolism ◽  
Urease Activity ◽  
Alternative Pathway ◽  
Bacterial Species ◽  
Deficient Mutant ◽  
Content Type ◽  
Metabolic Versatility

ABSTRACTUrease represents a critical virulence factor for some bacterial species through its alkalizing effect, which helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease, but its role during infection has yet to be characterized. In this study, we constructed a urease-deficientMycobacterium tuberculosisstrain and confirmed the alkalizing effect of the urease activity within the mycobacterium-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage onM. tuberculosisin macrophages, as evidenced by comparable growth profiles for the mutant, wild-type (WT), and complemented strains. In contrast, the urease-deficient mutant exhibited impairedin vitrogrowth compared to the WT and complemented strains when urea was the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen, and liver in mice. Together, our data demonstrate a role for the urease activity inM. tuberculosisnitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion thatM. tuberculosisvirulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

scholarly journals Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Zhaojing Zong ◽  
Wei Jing ◽  
Jin Shi ◽  
Shu'an Wen ◽  
Tingting Zhang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Novel Mutation ◽  
Multidrug Resistant ◽  
23S Rrna ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Significant Difference ◽  
Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Mycobacterium tuberculosis Alters the Metalloprotease Activity of the COP9 Signalosome

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Lia Danelishvili ◽  
Lmar Babrak ◽  
Sasha J. Rose ◽  
Jamie Everman ◽  
Luiz E. Bermudez
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cop9 Signalosome ◽  
Virulence Determinants ◽  
Bacterial Survival ◽  
Phagocytic Cells ◽  
Content Type ◽  
Metalloprotease Activity ◽  
Macrophage Protein

ABSTRACT Inhibition of apoptotic death of macrophages by Mycobacterium tuberculosis represents an important mechanism of virulence that results in pathogen survival both in vitro and in vivo. To identify M. tuberculosis virulence determinants involved in the modulation of apoptosis, we previously screened a transposon bank of mutants in human macrophages, and an M. tuberculosis clone with a nonfunctional Rv3354 gene was identified as incompetent to suppress apoptosis. Here, we show that the Rv3354 gene encodes a protein kinase that is secreted within mononuclear phagocytic cells and is required for M. tuberculosis virulence. The Rv3354 effector targets the metalloprotease (JAMM) domain within subunit 5 of the COP9 signalosome (CSN5), resulting in suppression of apoptosis and in the destabilization of CSN function and regulatory cullin-RING ubiquitin E3 enzymatic activity. Our observation suggests that alteration of the metalloprotease activity of CSN by Rv3354 possibly prevents the ubiquitin-dependent proteolysis of M. tuberculosis-secreted proteins. IMPORTANCE Macrophage protein degradation is regulated by a protein complex called a signalosome. One of the signalosomes associated with activation of ubiquitin and protein labeling for degradation was found to interact with a secreted protein from M. tuberculosis, which binds to the complex and inactivates it. The interference with the ability to inactivate bacterial proteins secreted in the phagocyte cytosol may have crucial importance for bacterial survival within the phagocyte.

scholarly journals Identification of Functional LsrB-Like Autoinducer-2 Receptors

2009 ◽  
Vol 191 (22) ◽  
pp. 6975-6987 ◽  
Author(s):  
Catarina S. Pereira ◽  
Anna K. de Regt ◽  
Patrícia H. Brito ◽  
Stephen T. Miller ◽  
Karina B. Xavier
Keyword(s):  
Structure Prediction ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Autoinducer 2 ◽  
Communication Signal ◽  
Independent Events ◽  
Key Species ◽  
Serovar Typhimurium

ABSTRACT Although a variety of bacterial species have been reported to use the interspecies communication signal autoinducer-2 (AI-2) to regulate multiple behaviors, the molecular mechanisms of AI-2 recognition and signal transduction remain poorly understood. To date, two types of AI-2 receptors have been identified: LuxP, present in Vibrio spp., and LsrB, first identified in Salmonella enterica serovar Typhimurium. In S. Typhimurium, LsrB is the ligand binding protein of a transport system that enables the internalization of AI-2. Here, using both sequence analysis and structure prediction, we establish a set of criteria for identifying functional AI-2 receptors. We test our predictions experimentally, assaying key species for their abilities to import AI-2 in vivo, and test their LsrB orthologs for AI-2 binding in vitro. Using these experimental approaches, we were able to identify AI-2 receptors in organisms belonging to phylogenetically distinct families such as the Enterobacteriaceae, Rhizobiaceae, and Bacillaceae. Phylogenetic analysis of LsrB orthologs indicates that this pattern could result from one single origin of the functional LsrB gene in a gammaproteobacterium, suggesting possible posterior independent events of lateral gene transfer to the Alphaproteobacteria and Firmicutes. Finally, we used mutagenesis to show that two AI-2-interacting residues are essential for the AI-2 binding ability. These two residues are conserved in the binding sites of all the functional AI-2 binding proteins but not in the non-AI-2-binding orthologs. Together, these results strongly support our ability to identify functional LsrB-type AI-2 receptors, an important step in investigations of this interspecies signal.

scholarly journals Ambroxol Induces Autophagy and Potentiates Rifampin Antimycobacterial Activity

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Seong Won Choi ◽  
Yuexi Gu ◽  
Ryan Scott Peters ◽  
Padmini Salgame ◽  
Jerrold J. Ellner ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Antimycobacterial Activity ◽  
Lead Compound ◽  
Content Type ◽  
Tuberculosis Model

ABSTRACT Host-directed therapy in tuberculosis is a potential adjunct to antibiotic chemotherapy directed at Mycobacterium tuberculosis. Ambroxol, a lead compound, emerged from a screen for autophagy-inducing drugs. At clinically relevant doses, ambroxol induced autophagy in vitro and in vivo and promoted mycobacterial killing in macrophages. Ambroxol also potentiated rifampin activity in a murine tuberculosis model.

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