Reconstruction of diaminopimelic acid biosynthesis allows characterisation of Mycobacterium tuberculosis N-succinyl-L,L-diaminopimelic acid desuccinylase

Usha, Veeraraghavan; Lloyd, Adrian J.; Roper, David I.; Dowson, Christopher G.; Kozlov, Guennadi; Gehring, Kalle; Chauhan, Smita; Imam, Hasan T.; Blindauer, Claudia A.; Besra, Gurdyal S.

doi:10.1038/srep23191

Cited by 13 publications

(10 citation statements)

References 36 publications

(74 reference statements)

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“…These mutants were significantly underrepresented by up to 8-fold upon RIF treatment specifically in cholesterol growth conditions across multiple sspB doses and drug concentrations. To further explore the effect of cell wall inhibition, we tested individual mutants with defects in peptidoglycan ( dapE ), arabinogalactan ( aftB ), and mycolic acid ( hadB ) synthesis (45, 47–49). In this format, we found that perturbation in each of these cell wall layers produced a similar effect, as they all preferentially sensitized the bacterium to RIF in cholesterol growth conditions (Fig4BCD) .…”

Section: Resultsmentioning

confidence: 99%

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy

Koh

Ruecker

et al. 2021

Preprint

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Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug-drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilize conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment, and probe the relationship between Mtb carbon metabolism and chemical-genetic interactions (CGI). We examined the anti-tubercular drugs isoniazid, rifampicin and moxifloxacin, and found that CGI are differentially responsive to the metabolic state, defining both environment independent and dependent synergies. Specifically, growth on the in vivo relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGI could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of novel chemical genetic environmental synergies that can be used to optimize drug-drug interactions as well as provide a framework for understanding in vitro correlates of in vivo efficacy.

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Section: Resultsmentioning

confidence: 99%

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy

Koh

Ruecker

et al. 2021

Preprint

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“…Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) (Perkin-Elmer Optima 5300 DV, Model S10) was used to determine the S, Zn, Cd, and Cu contents of the purified proteins as described previously [ 73 , 74 ]. In addition, the protein concentration was also routinely determined from the concentration of free thiols (cysteine) in the EDTA-demetallated proteins by Ellman’s reagent, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) at 412 nm by UV–visible spectroscopy using N -acetyl-cysteine as standard (Biomate 3 spectrophotometer, Fisher Scientific, UK).…”

Section: Methodsmentioning

confidence: 99%

Differential reactivity of closely related zinc(II)-binding metallothioneins from the plant Arabidopsis thaliana

Imam

Blindauer

2017

J Biol Inorg Chem

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The dynamics of metal binding to and transfer from metalloproteins involved in metal homeostasis are important for understanding cellular distribution of metal ions. The dicotyledonous plant Arabidopsis thaliana has two type 4 seed-specific metallothionein homologues, MT4a and MT4b, with likely roles in zinc(II) homeostasis. These two metallothioneins are 84% identical, with full conservation of all metal-binding cysteine and histidine residues. Yet, differences in their spatial and temporal expression patterns suggested divergence in their biological roles. To investigate whether biological functions are reflected in molecular properties, we compare aspects of zinc(II)-binding dynamics of full-length MT4a and MT4b, namely the pH dependence of zinc(II) binding and protein folding, and zinc(II) transfer to the chelator EDTA. UV–Vis and NMR spectroscopies as well as native electrospray ionisation mass spectrometry consistently showed that transfer from Zn6MT4a is considerably faster than from Zn6MT4b, with pseudo-first-order rate constants for the fastest observed step of k obs = 2.8 × 10−4 s−1 (MT4b) and k obs = 7.5 × 10−4 s−1 (MT4a) (5 µM protein, 500 µM EDTA, 25 mM Tris buffer, pH 7.33, 298 K). 2D heteronuclear NMR experiments allowed locating the most labile zinc(II) ions in domain II for both proteins. 3D homology models suggest that reactivity of this domain is governed by the local environment around the mononuclear Cys2His2 site that is unique to type 4 MTs. Non-conservative amino acid substitutions in this region affect local electrostatics as well as whole-domain dynamics, with both effects rendering zinc(II) ions bound to MT4a more reactive in metal transfer reactions. Therefore, domain II of MT4a is well suited to rapidly release its bound zinc(II) ions, in broad agreement with a previously suggested role of MT4a in zinc(II) transport and delivery to other proteins.Electronic supplementary materialThe online version of this article (10.1007/s00775-017-1516-6) contains supplementary material, which is available to authorized users.

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“…Mycobacterium tuberculosis DapE was found to be insensitive to the known DapE inhibitor L-captopril (Usha et al . 2016). Finally, DapF in Chlamydia trachomatis is bifunctional and involved in both mDAP synthesis and the racemisation of glutamate (Liechti et al .…”

Section: Introductionmentioning

confidence: 99%

Cell wall peptidoglycan inMycobacterium tuberculosis: An Achilles’ heel for the TB-causing pathogen

Maitra

Munshi

Healy³

et al. 2019

FEMS Microbiology Reviews

150

122

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Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.

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Reconstruction of diaminopimelic acid biosynthesis allows characterisation of Mycobacterium tuberculosis N-succinyl-L,L-diaminopimelic acid desuccinylase

Cited by 13 publications

References 36 publications

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy

Differential reactivity of closely related zinc(II)-binding metallothioneins from the plant Arabidopsis thaliana

Cell wall peptidoglycan inMycobacterium tuberculosis: An Achilles’ heel for the TB-causing pathogen

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