scholarly journals Blocking bacterial naphthohydroquinone oxidation and ADP-ribosylation improves activity of rifamycins against Mycobacterium abscessus

2021 ◽  
Author(s):  
Uday S. Ganapathy ◽  
Tian Lan ◽  
Philipp Krastel ◽  
Marissa Lindman ◽  
Matthew D. Zimmerman ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Mycobacterium Abscessus ◽  
Bacterial Metabolism ◽  
Proof Of Concept ◽  
Intrinsic Resistance ◽  
Bacterial Oxidation ◽  
Adp Ribosylation ◽  
Dual Mechanism ◽  
Adp Ribosyltransferase

Rifampicin is an effective drug for treating tuberculosis (TB) but is not used to treat M. abscessus infections due to poor in vitro activity. While rifabutin, another rifamycin, has better anti- M. abscessus activity, its activity is far from the nanomolar potencies of rifamycins against M. tuberculosis . Here, we asked i) why is rifabutin more active against M. abscessus than rifampicin, and ii) why is rifabutin’s anti- M. abscessus activity poorer than its anti-TB activity. Comparative analysis of naphthoquinone versus naphthohydroquinone-containing rifamycins suggested that the improved activity of rifabutin over rifampicin is linked to its less readily oxidizable naphthoquinone core. Although rifabutin is resistant to bacterial oxidation, metabolite and genetic analyses showed that this rifamycin is metabolized by the ADP-ribosyltransferase Arr Mab like rifampicin, preventing it from achieving the nanomolar activity it displays against M. tuberculosis . Based on the identified dual mechanism of intrinsic rifamycin resistance, we hypothesized that rifamycins more potent than rifabutin should contain the molecule’s naphthoquinone core plus a modification that blocks ADP-ribosylation at its C23. To test these predictions, we performed a blinded screen of a diverse collection of 189 rifamycins and identified two molecules more potent than rifabutin. As predicted, these compounds contained both a more oxidatively-resistant naphthoquinone core and C25 modifications that blocked ADP-ribosylation. Together, this work revealed dual bacterial metabolism as the mechanism of intrinsic resistance of M. abscessus to rifamycins and provides proof of concept for the repositioning of rifamycins for M. abscessus disease by developing derivatives that resist both bacterial oxidation and ADP-ribosylation.

scholarly journals ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

2004 ◽  
Vol 385 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Zhefeng ZHAO ◽  
Joanna GRUSZCZYNSKA-BIEGALA ◽  
Anna ZOLKIEWSKA
Keyword(s):  
C2c12 Myotubes ◽  
Post Translational Modification ◽  
Integrin Β1 ◽  
Adp Ribosylation ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
C2c12 Skeletal Muscle Cells ◽  
Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

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 Genome-Wide Essentiality Analysis of Mycobacterium abscessus by Saturated Transposon Mutagenesis and Deep Sequencing

mBio ◽  
2021 ◽  
Author(s):  
Dalin Rifat ◽  
Liang Chen ◽  
Barry N. Kreiswirth ◽  
Eric L. Nuermberger
Keyword(s):  
Deep Sequencing ◽  
Mycobacterium Abscessus ◽  
Transposon Mutagenesis ◽  
Comprehensive Analysis ◽  
Data Sets ◽  
Similar Data ◽  
Intrinsic Resistance ◽  
Effective Strategies ◽  
Genome Wide

Limited knowledge regarding Mycobacterium abscessus pathogenesis and intrinsic resistance to most classes of antibiotics is a major obstacle to developing more effective strategies to prevent and mitigate disease. Using optimized procedures for Himar1 transposon mutagenesis and deep sequencing, we performed a comprehensive analysis to identify M. abscessus genetic elements essential for in vitro growth and compare them to similar data sets for M. tuberculosis and M. avium subsp. hominissuis .

scholarly journals In vitro activity of the new β-lactamase inhibitors relebactam and vaborbactam in combination with β-lactams against Mycobacterium abscessus complex clinical isolates

2018 ◽  
Author(s):  
Amit Kaushik ◽  
Nicole C. Ammerman ◽  
Jin Lee ◽  
Olumide Martins ◽  
Barry N Kreiswirth ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Mycobacterium Abscessus ◽  
Vitro Activity ◽  
Clinical Isolates ◽  
Phase Iii ◽  
In Vitro Activity ◽  
Intrinsic Resistance ◽  
Phase Iii Trials ◽  
Lactamase Inhibitor

Pulmonary disease due to infection with Mycobacterium abscessus complex (MABC) is notoriously difficult to treat, in large part due to MABC’s intrinsic resistance to most antibiotics, including β-lactams. MABC organisms express a broad-spectrum β-lactamase that is resistant to traditional β-lactam-based β-lactamase inhibitors but inhibited by a newer non-β-lactam-based β-lactamase inhibitor, avibactam. Consequently, the susceptibility of MABC to some β-lactams is increased in the presence of avibactam. Therefore, we hypothesized that two new non-β-lactam-based β-lactamase inhibitors, relebactam and vaborbactam, would also increase susceptibility of MABC to β-lactams. The objective of the present study was to evaluate the in vitro activity of various marketed β-lactams alone and in combination with either relebactam or vaborbactam against multidrug-resistant MABC clinical isolates. Our data demonstrate that both β-lactamase inhibitors significantly improved the anti-MABC activity of many carbapenems (including imipenem and meropenem) and cephalosporins (including cefepime, ceftaroline, and cefuroxime). As a meropenem/vaborbactam combination is now marketed and an imipenem/relebactam combination is currently in phase III trials, these fixed combinations may become the β-lactams of choice for the treatment of MABC infections. Furthermore, given the evolving interest in dual β-lactam regimens, our results identify select cephalosporins, such as cefuroxime, with superior activity in the presence of a β-lactamase inhibitor, deserving of further evaluation in combination with these carbapenem/β-lactamase inhibitor products.

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

scholarly journals Independent and Coordinate Effects of ADP-Ribosyltransferase and GTPase-Activating Activities of Exoenzyme S on HT-29 Epithelial Cell Function

2001 ◽  
Vol 69 (9) ◽  
pp. 5318-5328 ◽  
Author(s):  
Jennifer E. Fraylick ◽  
Jeannine R. La Rocque ◽  
Timothy S. Vincent ◽  
Joan C. Olson
Keyword(s):  
Dna Synthesis ◽  
Cell Function ◽  
Double Mutation ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Cell Rounding ◽  
Inhibition Of Dna Synthesis ◽  
Adp Ribosyltransferase ◽  
Ht 29

ABSTRACT Type III-mediated translocation of exoenzyme S (ExoS) into HT-29 epithelial cells by Pseudomonas aeruginosa causes complex alterations in cell function, including inhibition of DNA synthesis, altered cytoskeletal structure, loss of readherence, microvillus effacement, and interruption of signal transduction. ExoS is a bifunctional protein having both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) functional domains. Comparisons of alterations in HT-29 cell function caused by P. aeruginosastrains that translocate ExoS having GAP or ADPRT mutations allowed the independent and coordinate functions of the two activities to be assessed. An E381A ADPRT mutation revealed that ExoS ADPRT activity was required for effects of ExoS on DNA synthesis and long-term cell rounding. Conversely, the R146A GAP mutation appeared to have little impact on the cellular effects of ExoS. While transient cell rounding was detected following exposure to the E381A mutant, this rounding was eliminated by an E379A-E381A ADPRT double mutation, implying that residual ADPRT activity, rather than GAP activity, was effecting transient cell rounding by the E381A mutant. To explore this possibility, E381A and R146A-E381A mutants were examined for their ability to ADP-ribosylate Ras in vitro or in vivo. While no ADP-ribosylation of Ras was detected by either mutant in vitro, both mutants were able to modify Ras when translocated by the bacteria, with the R146A-E381A mutant causing more efficient modification than the E381A mutant, in association with increased inhibition of DNA synthesis. Comparisons of Ras ADP-ribosylation by wild-type and E381A mutant ExoS by two-dimensional electrophoresis found the former to ADP-ribosylate Ras at two sites, while the latter modified Ras only once. These studies draw attention to the key role of ExoS ADPRT activity in causing the effects of bacterially translocated ExoS on DNA synthesis and cell rounding. In addition, the studies provide insight into the enhancement of ExoS ADPRT activity within the eukaryotic cell microenvironment and into possible modulatory roles that the GAP and ADPRT domains might have on the function of each other.

scholarly journals Efficacy of epetraborole against Mycobacterium abscessus is increased with norvaline

2021 ◽  
Vol 17 (10) ◽  
pp. e1009965
Author(s):  
Jaryd R. Sullivan ◽  
Andréanne Lupien ◽  
Elias Kalthoff ◽  
Claire Hamela ◽  
Lorne Taylor ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Dissociation Constants ◽  
Cell Activity ◽  
Trna Synthetase ◽  
Mycobacterium Abscessus ◽  
Intrinsic Resistance ◽  
Clp Proteases ◽  
The Unfolded Protein Response

Mycobacterium abscessus is the most common rapidly growing non-tuberculous mycobacteria to cause pulmonary disease in patients with impaired lung function such as cystic fibrosis. M. abscessus displays high intrinsic resistance to common antibiotics and inducible resistance to macrolides like clarithromycin. As such, M. abscessus is clinically resistant to the entire regimen of front-line M. tuberculosis drugs, and treatment with antibiotics that do inhibit M. abscessus in the lab results in cure rates of 50% or less. Here, we identified epetraborole (EPT) from the MMV pandemic response box as an inhibitor against the essential protein leucyl-tRNA synthetase (LeuRS) in M. abscessus. EPT protected zebrafish from lethal M. abscessus infection and did not induce self-resistance nor against clarithromycin. Contrary to most antimycobacterials, the whole-cell activity of EPT was greater against M. abscessus than M. tuberculosis, but crystallographic and equilibrium binding data showed that EPT binds LeuRSMabs and LeuRSMtb with similar residues and dissociation constants. Since EPT-resistant M. abscessus mutants lost LeuRS editing activity, these mutants became susceptible to misaminoacylation with leucine mimics like the non-proteinogenic amino acid norvaline. Proteomic analysis revealed that when M. abscessus LeuRS mutants were fed norvaline, leucine residues in proteins were replaced by norvaline, inducing the unfolded protein response with temporal changes in expression of GroEL chaperonins and Clp proteases. This supports our in vitro data that supplementation of media with norvaline reduced the emergence of EPT mutants in both M. abscessus and M. tuberculosis. Furthermore, the combination of EPT and norvaline had improved in vivo efficacy compared to EPT in a murine model of M. abscessus infection. Our results emphasize the effectiveness of EPT against the clinically relevant cystic fibrosis pathogen M. abscessus, and these findings also suggest norvaline adjunct therapy with EPT could be beneficial for M. abscessus and other mycobacterial infections like tuberculosis.

scholarly journals The TetR Family Transcription Factor MAB_2299c Regulates the Expression of Two Distinct MmpS-MmpL Efflux Pumps Involved in Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Ana Victoria Gutiérrez ◽  
Matthias Richard ◽  
Françoise Roquet-Banères ◽  
Albertus Viljoen ◽  
Laurent Kremer
Keyword(s):  
Efflux Pump ◽  
Respiratory Infections ◽  
Efflux Pumps ◽  
Mycobacterium Abscessus ◽  
Cross Resistance ◽  
Intrinsic Resistance ◽  
Mobility Shift ◽  
Pump System ◽  
Content Type

ABSTRACT Mycobacterium abscessus is a human pathogen responsible for severe respiratory infections, particularly in patients with underlying lung disorders. Notorious for being highly resistant to most antimicrobials, new therapeutic approaches are needed to successfully treat M. abscessus-infected patients. Clofazimine (CFZ) and bedaquiline (BDQ) are two antibiotics used for the treatment of multidrug-resistant tuberculosis and are considered alternatives for the treatment of M. abscessus pulmonary disease. To get insights into their mechanisms of resistance in M. abscessus, we previously characterized the TetR transcriptional regulator MAB_2299c, which controls expression of the MAB_2300-MAB_2301 genes, encoding an MmpS-MmpL efflux pump. Here, in silico studies identified a second mmpS-mmpL (MAB_1135c-MAB_1134c) target of MAB_2299c. A palindromic DNA sequence upstream of MAB_1135c, sharing strong homology with the one located upstream of MAB_2300, was found to form a complex with the MAB_2299c regulator in electrophoretic mobility shift assays. Deletion of MAB_1135c-1134c in a wild-type strain led to increased susceptibility to both CFZ and BDQ. In addition, deletion of these genes in a CFZ/BDQ-susceptible mutant lacking MAB_2299c as well as MAB_2300-MAB_2301 further exacerbated the sensitivity of this strain to both drugs in vitro and inside macrophages. Overall, these results indicate that MAB_1135c-1134c encodes a new MmpS-MmpL efflux pump system involved in the intrinsic resistance to CFZ and BDQ. They also support the view that MAB_2299c controls the expression of two separate MmpS-MmpL efflux pumps, substantiating the importance of MAB_2299c as a marker of resistance to be considered when assessing drug susceptibility in clinical isolates.

scholarly journals Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity

2018 ◽  
Vol 475 (23) ◽  
pp. 3827-3846 ◽  
Author(s):  
Alvin Gomez ◽  
Christian Bindesbøll ◽  
Somisetty V. Satheesh ◽  
Giulia Grimaldi ◽  
David Hutin ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Zinc Finger ◽  
Electron Transfer Dissociation ◽  
Biochemical Characterization ◽  
Zinc Finger Domain ◽  
Adp Ribosylation ◽  
Aryl Hydrocarbon ◽  
Adp Ribosyltransferase

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400–657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.

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