scholarly journals Maturing Mycobacterial Peptidoglycan Requires Non-canonical Crosslinks to Maintain Shape

2018 ◽  
Author(s):  
Catherine Baranowski ◽  
Michael A. Welsh ◽  
Lok-To Sham ◽  
Haig A. Eskandarian ◽  
Hoong C. Lim ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Single Cell ◽  
Uniform Distribution ◽  
Drug Targets ◽  
Polar Growth ◽  
Cell Distribution ◽  
Penicillin Binding Proteins ◽  
Aging Cell

AbstractIn most well studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin binding proteins (PBPs). However, in mycobacteria, L,D-transpeptidase (LDT)-mediated crosslinks are highly abundant. To elucidate the role of these unusual crosslinks, we characterized mycobacterial cells lacking all LDTs. We find that LDT-mediated crosslinks are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this,Mycobacterium tuberculosis(Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT-inhibition. Thus, knowledge about the single-cell distribution of drug targets can be exploited to more effectively treat this pathogen.

scholarly journals Maturing Mycobacterium smegmatis peptidoglycan requires non-canonical crosslinks to maintain shape

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Catherine Baranowski ◽  
Michael A Welsh ◽  
Lok-To Sham ◽  
Haig A Eskandarian ◽  
Hoong Chuin Lim ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Uniform Distribution ◽  
Genetic Relationship ◽  
Mycobacterium Smegmatis ◽  
Drug Targets ◽  
Polar Growth ◽  
Penicillin Binding Proteins ◽  
Aging Cell

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

2011 ◽  
Vol 436 (3) ◽  
pp. 729-739 ◽  
Author(s):  
Marcio V. B. Dias ◽  
William C. Snee ◽  
Karen M. Bromfield ◽  
Richard J. Payne ◽  
Satheesh K. Palaninathan ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Structural Investigation ◽  
Catalytic Mechanism ◽  
Shikimate Pathway ◽  
Structural Rearrangement ◽  
Competitive Inhibitors ◽  
Structural Insights ◽  
Potential Drug Targets

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19–24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

scholarly journals Molecular Investigation of Resistance to the Antituberculous Drug Ethionamide in Multidrug-Resistant Clinical Isolates ofMycobacterium tuberculosis

2010 ◽  
Vol 55 (1) ◽  
pp. 355-360 ◽  
Author(s):  
F. Brossier ◽  
N. Veziris ◽  
C. Truffot-Pernot ◽  
V. Jarlier ◽  
W. Sougakoff
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Nadh Dehydrogenase ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Cell Wall Biosynthesis ◽  
Molecular Investigation ◽  
Antituberculous Drug ◽  
Resistant Cells

ABSTRACTEthionamide (ETH) needs to be activated by the mono-oxygenase EthA, which is regulated by EthR, in order to be active againstMycobacterium tuberculosis. The activated drug targets the enzyme InhA, which is involved in cell wall biosynthesis. Resistance to ETH has been reported to result from various mechanisms, including mutations altering EthA/EthR, InhA and its promoter, the NADH dehydrogenase encoded byndh, and the MshA enzyme, involved in mycothiol biosynthesis. We searched for such mutations in 87 clinical isolates: 47 ETH-resistant (ETHr) isolates, 24 ETH-susceptible (ETHs) isolates, and 16 isolates susceptible to ETH but displaying an intermediate proportion of resistant cells (ETHSip; defined as ≥1% but <10% resistant cells). In 81% (38/47) of the ETHrisolates, we found mutations inethA,ethR, orinhAor its promoter, which mostly corresponded to new alterations inethAandethR. The 9 ETHrisolates without a mutation in these three genes (9/47, 19%) had no mutation inndh, and a single isolate had a mutation inmshA. Of the 16 ETHSipisolates, 7 had a mutation inethA, 8 had no detectable mutation, and 1 had a mutation inmshA. Finally, of the 24 ETHsisolates, 23 had no mutation in the studied genes and 1 displayed a yet unknown mutation in theinhApromoter. Globally, the mechanism of resistance to ETH remained unknown for 19% of the ETHrisolates, highlighting the complexity of the mechanisms of ETH resistance inM. tuberculosis.

scholarly journals Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

scholarly journals A Microbiological, Toxicological, and Biochemical Study of the Effects of Fucoxanthin, a Marine Carotenoid, on Mycobacterium tuberculosis and the Enzymes Implicated in Its Cell Wall: A Link Between Mycobacterial Infection and Autoimmune Diseases

Marine Drugs ◽  
2019 ◽  
Vol 17 (11) ◽  
pp. 641 ◽  
Author(s):  
Miroslava Šudomová ◽  
Mohammad Shariati ◽  
Javier Echeverría ◽  
Ioana Berindan-Neagoe ◽  
Seyed Nabavi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Autoimmune Diseases ◽  
Drug Targets ◽  
Bacterial Infections ◽  
Animal Studies ◽  
Biochemical Study ◽  
Mycobacterial Infection ◽  
Susceptible Host ◽  
Standard Drug

This study explored the antitubercular properties of fucoxanthin, a marine carotenoid, against clinical isolates of Mycobacterium tuberculosis (Mtb). Two vital enzymes involved in Mtb cell wall biosynthesis, UDP-galactopyranose mutase (UGM) and arylamine-N-acetyltransferase (TBNAT), were selected as drug targets to reveal the mechanism underlying the antitubercular effect of fucoxanthin. The obtained results showed that fucoxanthin showed a clear bacteriostatic action against the all Mtb strains tested, with minimum inhibitory concentrations (MIC) ranging from 2.8 to 4.1 µM, along with a good degree of selectivity index (ranging from 6.1 to 8.9) based on cellular toxicity evaluation compared with standard drug isoniazid (INH). The potent inhibitory actions of fucoxanthin and standard uridine-5’-diphosphate against UGM were recorded to be 98.2% and 99.2%, respectively. TBNAT was potently inactivated by fucoxanthin (half maximal inhibitory concentration (IC50) = 4.8 µM; 99.1% inhibition) as compared to INH (IC50 = 5.9 µM; 97.4% inhibition). Further, molecular docking approaches were achieved to endorse and rationalize the biological findings along with envisaging structure-activity relationships. Since the clinical evidence of the last decade has confirmed the correlation between bacterial infections and autoimmune diseases, in this study we have discussed the linkage between infection with Mtb and autoimmune diseases based on previous clinical observations and animal studies. In conclusion, we propose that fucoxanthin could demonstrate great therapeutic value for the treatment of tuberculosis by acting on multiple targets through a bacteriostatic effect as well as by inhibiting UGM and TBNAT. Such outcomes may lead to avoiding or decreasing the susceptibility to autoimmune diseases associated with Mtb infection in a genetically susceptible host.

scholarly journals Novel drug targets for Mycobacterium tuberculosis: 2-heterostyrylbenzimidazoles as inhibitors of cell wall protein synthesis

2017 ◽  
Vol 11 (1) ◽  
Author(s):  
Mohana Rao Anguru ◽  
Ashok Kumar Taduri ◽  
Rama Devi Bhoomireddy ◽  
Malathi Jojula ◽  
Shravan Kumar Gunda
Keyword(s):  
Cell Wall ◽  
Protein Synthesis ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Cell Wall Protein ◽  
Novel Drug ◽  
Novel Drug Targets

scholarly journals Structural and molecular dynamics of Mycobacterium tuberculosis malic enzyme, a potential anti-TB drug target

2020 ◽  
Author(s):  
Kalistyn H. Burley ◽  
Bonnie J. Cuthbert ◽  
Piyali Basu ◽  
Jane Newcombe ◽  
Ervin M. Irimpan ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Malic Enzyme ◽  
Drug Targets ◽  
Reducing Power ◽  
Md Simulations ◽  
Oligomeric State ◽  
Effects Of Ph ◽  
Multiple Conformations

AbstractTuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, Mycobacterium tuberculosis (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ provides the required reducing power for lipid biosynthesis. Here, we present the X-ray crystal structure of MEZ to 3.6 Å resolution and compare it with known structures of prokaryotic and eukaryotic malic enzymes. We use biochemical assays to determine its oligomeric state and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn2+ and NAD(P)+, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually disordered, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that MEZ subunits alternate between open and closed states and that MEZ can stably bind its NAD(P)+ cofactor in multiple conformations, including an inactive, compact NAD+ form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb.

scholarly journals Mycobacterium tuberculosis Thymidylyltransferase RmlA Is Negatively Regulated by Ser/Thr Protein Kinase PknB

2021 ◽  
Vol 12 ◽  
Author(s):  
Dehui Qu ◽  
Xiaohui Zhao ◽  
Yao Sun ◽  
Fan-Lin Wu ◽  
Sheng-Ce Tao
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Catalytic Triad ◽  
Mass Spectrometry Analysis ◽  
Wall Structure ◽  
Cell Wall Formation ◽  
Phenotypic Analysis ◽  
Spectrometry Analysis ◽  
Wall Formation

Ser/Thr phosphorylation by serine/threonine protein kinases (STPKs) plays significant roles in molecular regulation, which allows Mycobacteria to adapt their cell wall structure in response to the environment changes. Identifying direct targets of STPKs and determining their activities are therefore critical to revealing their function in Mycobacteria, for example, in cell wall formation and virulence. Herein, we reported that RmlA, a crucial L-rhamnose biosynthesis enzyme, is a substrate of STPK PknB in Mycobacterium tuberculosis (M. tuberculosis). Mass spectrometry analysis revealed that RmlA is phosphorylated at Thr-12, Thr-54, Thr-197, and Thr-12 is located close to the catalytic triad of RmlA. Biochemical and phenotypic analysis of two RmlA mutants, T12A/T12D, showed that their activities were reduced, and cell wall formation was negatively affected. Moreover, virulence of RmlA T12D mutant was attenuated in a macrophage model. Overall, these results provide the first evidence for the role of PknB-dependent RmlA phosphorylation in regulating cell wall formation in Mycobacteria, with significant implications for pathogenicity.

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