scholarly journals Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis

2016 ◽  
Vol 60 (10) ◽  
pp. 6091-6099 ◽  
Author(s):  
Gareth A. Prosser ◽  
Anne Rodenburg ◽  
Hania Khoury ◽  
Cesira de Chiara ◽  
Steve Howell ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Early Stage ◽  
Human Pathogens ◽  
Intact Cells ◽  
Content Type ◽  
Peptidoglycan Biosynthesis ◽  
Glutamate Racemase ◽  
Starting Point ◽  
Effective Drugs

ABSTRACTThe increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except inMycobacterium tuberculosis, where it has so far been underexploited.d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within thed-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies ofM. tuberculosis. During our studies on alternative inhibitors of thed-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro–d-alanine (BCDA) is a very poor inhibitor of recombinantM. tuberculosisAlr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit thed-alanine pathway in intact cells, consistent with its poorin vitroactivity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI inM. tuberculosisand thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.

scholarly journals Evidence for Inhibition of Topoisomerase 1A by Gold(III) Macrocycles and Chelates TargetingMycobacterium tuberculosisandMycobacterium abscessus

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Rashmi Gupta ◽  
Carolina Rodrigues Felix ◽  
Matthew P. Akerman ◽  
Kate J. Akerman ◽  
Cathryn A. Slabber ◽  
...  
Keyword(s):  
Mycobacterium Abscessus ◽  
Cross Resistance ◽  
Human Pathogens ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Starting Point ◽  
Scalable Synthesis ◽  
High Level ◽  
Novel Drug

ABSTRACTMycobacterium tuberculosisand the fast-growing speciesMycobacterium abscessusare two important human pathogens causing persistent pulmonary infections that are difficult to cure and require long treatment times. The emergence of drug-resistantM. tuberculosisstrains and the high level of intrinsic resistance ofM. abscessuscall for novel drug scaffolds that effectively target both pathogens. In this study, we evaluated the activity of bis(pyrrolide-imine) gold(III) macrocycles and chelates, originally designed as DNA intercalators capable of targeting human topoisomerase types I and II (Topo1 and Topo2), againstM. abscessusandM. tuberculosis. We identified a total of 5 noncytotoxic compounds active against both mycobacterial pathogens under replicatingin vitroconditions. We chose one of these hits, compound 14, for detailed analysis due to its potent bactericidal mode of inhibition and scalable synthesis. The clinical relevance of this compound was demonstrated by its ability to inhibit a panel of diverseM. tuberculosisandM. abscessusclinical isolates. Prompted by previous data suggesting that compound 14 may target topoisomerase/gyrase enzymes, we demonstrated that it lacked cross-resistance with fluoroquinolones, which target theM. tuberculosisgyrase.In vitroenzyme assays confirmed the potent activity of compound 14 against bacterial topoisomerase 1A (Topo1) enzymes but not gyrase. Novel scaffolds like compound 14 with potent, selective bactericidal activity againstM. tuberculosisandM. abscessusthat act on validated but underexploited targets like Topo1 represent a promising starting point for the development of novel therapeutics for infections by pathogenic mycobacteria.

scholarly journals A SpoIID Homolog Cleaves Glycan Strands at the Chlamydial Division Septum

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Nicolas Jacquier ◽  
Akhilesh K. Yadav ◽  
Trestan Pillonel ◽  
Patrick H. Viollier ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Intracellular Bacteria ◽  
Human Pathogens ◽  
Content Type ◽  
Obligate Intracellular ◽  
Peptidoglycan Biosynthesis ◽  
Peptidoglycan Synthesis ◽  
Lytic Transglycosylase ◽  
Obligate Intracellular Bacteria

ABSTRACT Chlamydiales species are obligate intracellular bacteria lacking a classical peptidoglycan sacculus but relying on peptidoglycan synthesis for cytokinesis. While septal peptidoglycan biosynthesis seems to be regulated by MreB actin and its membrane anchor RodZ rather than FtsZ tubulin in Chlamydiales, the mechanism of peptidoglycan remodeling is poorly understood. An amidase conserved in Chlamydiales is able to cleave peptide stems in peptidoglycan, but it is not clear how peptidoglycan glycan strands are cleaved since no classical lytic transglycosylase is encoded in chlamydial genomes. However, a protein containing a SpoIID domain, known to possess transglycosylase activity in Bacillus subtilis, is conserved in Chlamydiales. We show here that the SpoIID homologue of the Chlamydia-related pathogen Waddlia chondrophila is a septal peptidoglycan-binding protein. Moreover, we demonstrate that SpoIID acts as a lytic transglycosylase on peptidoglycan and as a muramidase on denuded glycan strands in vitro. As SpoIID-like proteins are widespread in nonsporulating bacteria, SpoIID might commonly be a septal peptidoglycan remodeling protein in bacteria, including obligate intracellular pathogens, and thus might represent a promising drug target. IMPORTANCE Chlamydiales species are obligate intracellular bacteria and important human pathogens that have a minimal division machinery lacking the proteins that are essential for bacterial division in other species, such as FtsZ. Chlamydial division requires synthesis of peptidoglycan, which forms a ring at the division septum and is rapidly turned over. However, little is known of peptidoglycan degradation, because many peptidoglycan-degrading enzymes are not encoded by chlamydial genomes. Here we show that an homologue of SpoIID, a peptidoglycan-degrading enzyme involved in sporulation of bacteria such as Bacillus subtilis, is expressed in Chlamydiales, localizes at the division septum, and degrades peptidoglycan in vitro, indicating that SpoIID is not only involved in sporulation but also likely implicated in division of some bacteria.

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 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.

Ethambutol targets the glutamate racemase of Mycobacterium tuberculosis—an enzyme involved in peptidoglycan biosynthesis

2018 ◽  
Vol 103 (2) ◽  
pp. 843-851 ◽  
Author(s):  
Alka Pawar ◽  
Prakash Jha ◽  
Chandrika Konwar ◽  
Uma Chaudhry ◽  
Madhu Chopra ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Peptidoglycan Biosynthesis ◽  
Glutamate Racemase

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 Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis

2020 ◽  
Vol 89 (1) ◽  
pp. e00401-20
Author(s):  
Raghuveer Singh ◽  
Jessica A. Slade ◽  
Mary Brockett ◽  
Daniel Mendez ◽  
George W. Liechti ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Diaminopimelic Acid ◽  
Competition Strategy ◽  
Content Type ◽  
E Coli ◽  
Glutamate Racemase ◽  
Heterologous System ◽  
Substrate Competition

ABSTRACTThe Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapFCt function in vivo in C. trachomatis. We reasoned that, because DapFCt utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapFCt DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapFCt is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis. Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

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.

scholarly journals De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Jees Sebastian ◽  
Sharmada Swaminath ◽  
Rashmi Ravindran Nair ◽  
Kishor Jakkala ◽  
Atul Pradhan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Hydroxyl Radical ◽  
Prolonged Exposure ◽  
De Novo ◽  
Random Mutagenesis ◽  
Content Type ◽  
Genome Wide ◽  
Resistant Mutants ◽  
Phase Population

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

Sign in / Sign up

Export Citation Format

Share Document