scholarly journals Mycobacterium abscessus Genetic Determinants Associated with the Intrinsic Resistance to Antibiotics

2021 ◽  
Vol 9 (12) ◽  
pp. 2527
Author(s):  
Mylene Gorzynski ◽  
Tiana Week ◽  
Tiana Jaramillo ◽  
Elizaveta Dzalamidze ◽  
Lia Danelishvili
Keyword(s):  
Gene Knockout ◽  
Efflux Pumps ◽  
Mycobacterium Abscessus ◽  
Transport Systems ◽  
Nontuberculous Mycobacterium ◽  
Genetic Determinants ◽  
Intrinsic Resistance ◽  
Surface Transport ◽  
Knockout Mutants ◽  
Molecule Transport

Mycobacterium abscessus subsp. abscessus (MAB) is a fast-growing nontuberculous mycobacterium causing pulmonary infections in immunocompromised and immunocompetent individuals. The treatment of MAB infections in clinics is extremely challenging, as this organism is naturally resistant to most available antibiotics. There is limited knowledge on the mechanisms of MAB intrinsic resistance and on the genes that are involved in the tolerance to antimicrobials. To identify the MAB genetic factors, including the components of the cell surface transport systems related to the efflux pumps, major known elements contributing to antibiotic resistance, we screened the MAB transposon library of 2000 gene knockout mutants. The library was exposed at either minimal inhibitory (MIC) or bactericidal concentrations (BC) of amikacin, clarithromycin, or cefoxitin, and MAB susceptibility was determined through the optical density. The 98 susceptible and 36 resistant mutants that exhibited sensitivity below the MIC and resistance to BC, respectively, to all three drugs were sequenced, and 16 mutants were found to belong to surface transport systems, such as the efflux pumps, porins, and carrier membrane enzymes associated with different types of molecule transport. To establish the relevance of the identified transport systems to antibiotic tolerance, the gene expression levels of the export related genes were evaluated in nine MAB clinical isolates in the presence or absence of antibiotics. The selected mutants were also evaluated for their ability to form biofilms and for their intracellular survival in human macrophages. In this study, we identified numerous MAB genes that play an important role in the intrinsic mechanisms to antimicrobials and further demonstrated that, by targeting components of the drug efflux system, we can significantly increase the efficacy of the current antibiotics.

scholarly journals Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1187-1195 ◽  
Author(s):  
Rachid Nessar ◽  
Jean-Marc Reyrat ◽  
Lisa B. Davidson ◽  
Thomas F. Byrd
Keyword(s):  
Pathogenic Bacteria ◽  
Biosynthetic Pathway ◽  
Gene Knockout ◽  
Cellular Level ◽  
Mycobacterium Abscessus ◽  
Bacterial Gene ◽  
Human Macrophages ◽  
Knockout Mutants ◽  
A Cell ◽  
Toll Like Receptor 2

Mycobacterium abscessus is considered to be the most virulent of the rapidly growing mycobacteria. Generation of bacterial gene knockout mutants has been a useful tool for studying factors that contribute to virulence of pathogenic bacteria. Until recently, the optimal genetic approach to generation of M. abscessus gene knockout mutants was not clear. Based on the recent identification of genetic recombineering as the preferred approach, a M. abscessus mutant was generated in which the gene mmpL4b, critical to glycopeptidolipid synthesis, was deleted. Compared to the previously well-characterized parental strain 390S, the mmpL4B deletion mutant had lost sliding motility and the ability to form biofilm, but acquired the ability to replicate in human macrophages and stimulate macrophage Toll-like receptor 2. This study demonstrates that deletion of a gene associated with expression of a cell-wall lipid can result in acquisition of an immunostimulatory, invasive bacterial phenotype and has important implications for the study of M. abscessus pathogenesis at the cellular level.

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 MAB_2355c Confers Macrolide Resistance in Mycobacterium abscessus by Ribosome Protection

2021 ◽  
Author(s):  
Qi Guo ◽  
Yongjie Zhang ◽  
Junsheng Fan ◽  
Haonan Zhang ◽  
Zhemin Zhang ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Gene Knockout ◽  
Antibiotic Sensitivity ◽  
Resistance Mechanisms ◽  
Mycobacterium Abscessus ◽  
Macrolide Resistance ◽  
Intrinsic Resistance ◽  
Family Protein ◽  
Increased Sensitivity

Macrolide resistance is always a concern when treating Mycobacterium abscessus infections. MAB_2355c was identified previously as a possible new factor that confers the intrinsic resistance of 194 clinical M. abscessus isolates to clarithromycin. Herein, the potential mechanism by which MAB_2355c exerts macrolide resistance was explored by bioinformatics analysis, MAB_2355 cloning and protein purification, ATP hydrolysis assay, gene knockout and complementation, antibiotic sensitivity, and transcription-translation assays. MAB_2355c is a putative ATP-binding cassette F (ABC-F) family protein. Purified MAB_2355c protein exhibits ATP hydrolysis activity, which can be inhibited by ribosome-targeting antibiotics. MAB_2355c mRNA expression is upregulated more significantly after exposure to macrolides than exposure to other ribosome-targeting antibiotics. MAB_2355c deleted strains showed increased sensitivity to macrolides, which was reduced by MAB_2355c complementation. Finally, MAB_2355c rescued the transcription and translation activities affected by macrolides in vitro . These findings suggest that MAB_2355c confers the resistance of M. abscessus to macrolides by ribosome protection, thus complementing other known resistance mechanisms.

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 Genetic Determinants Involved in the Susceptibility of Pseudomonas aeruginosa to β-Lactam Antibiotics

2010 ◽  
Vol 54 (10) ◽  
pp. 4159-4167 ◽  
Author(s):  
Carolina Alvarez-Ortega ◽  
Irith Wiegand ◽  
Jorge Olivares ◽  
Robert E. W. Hancock ◽  
José Luis Martínez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Detailed Analysis ◽  
Barrier Function ◽  
Mechanisms Of Action ◽  
Mutant Library ◽  
Genetic Determinants ◽  
Intrinsic Resistance ◽  
Resistance Phenotype ◽  
Mixed Phenotype ◽  
Alginate Biosynthesis

ABSTRACT The resistome of P. aeruginosa for three β-lactam antibiotics, namely, ceftazidime, imipenem, and meropenem, was deciphered by screening a comprehensive PA14 mutant library for mutants with increased or reduced susceptibility to these antimicrobials. Confirmation of the phenotypes of all selected mutants was performed by Etest. Of the total of 78 confirmed mutants, 41 demonstrated a reduced susceptibility phenotype and 37 a supersusceptibility (i.e., altered intrinsic resistance) phenotype, with 6 mutants demonstrating a mixed phenotype, depending on the antibiotic. Only three mutants demonstrated reduced (PA0908) or increased (glnK and ftsK) susceptibility to all three antibiotics. Overall, the mutant profiles of susceptibility suggested distinct mechanisms of action and resistance for the three antibiotics despite their similar structures. More detailed analysis indicated important roles for novel and known β-lactamase regulatory genes, for genes with likely involvement in barrier function, and for a range of regulators of alginate biosynthesis.

scholarly journals Specific interaction of an RNA-binding protein with the 3′-UTR of its target mRNA is critical to oomycete sexual reproduction

2021 ◽  
Vol 17 (10) ◽  
pp. e1010001
Author(s):  
Hui Feng ◽  
Chuanxu Wan ◽  
Zhichao Zhang ◽  
Han Chen ◽  
Zhipeng Li ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Rna Binding ◽  
Gene Knockout ◽  
Binding Protein ◽  
Mrna Level ◽  
Specific Interaction ◽  
Rna Binding Protein ◽  
Pythium Ultimum ◽  
Knockout Mutants ◽  
Oospore Formation

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3′ untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3′-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3′-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum.

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 Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis

2020 ◽  
Vol 71 (19) ◽  
pp. 5963-5975 ◽  
Author(s):  
Panupon Khumsupan ◽  
Marta A Kozlowska ◽  
Douglas J Orr ◽  
Andreas I Andreou ◽  
Naomi Nakayama ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Catalytic Efficiency ◽  
Small Subunit ◽  
Multiple Gene ◽  
Functional Roles ◽  
Knockout Mutants ◽  
Dna Insertion ◽  
Low Levels ◽  
Rbcs Genes ◽  
Viable Approach

Abstract The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

scholarly journals Select β-Lactam Combinations Exhibit Synergy againstMycobacterium abscessus In Vitro

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Elizabeth Story-Roller ◽  
Emily C. Maggioncalda ◽  
Gyanu Lamichhane
Keyword(s):  
Treatment Options ◽  
Unmet Need ◽  
Mycobacterium Abscessus ◽  
Nontuberculous Mycobacterium ◽  
Pulmonary Infections ◽  
Content Type ◽  
Resistant Mutants ◽  
Lactamase Inhibitor ◽  
Selection Of

ABSTRACTMycobacterium abscessusis a nontuberculous mycobacterium that causes invasive pulmonary infections in patients with structural lung disease.M. abscessusis intrinsically resistant to several classes of antibiotics, and an increasing number of strains isolated from patients exhibit resistance to most antibiotics considered for treatment of infections by this mycobacterium. Therefore, there is an unmet need for new regimens with improved efficacy to treat this disease. Synthesis of the essential cell wall peptidoglycan inM. abscessusis achieved via two enzyme classes,l,d- andd,d-transpeptidases, with each class preferentially inhibited by different subclasses of β-lactam antibiotics. We hypothesized that a combination of two β-lactams that comprehensively inhibit the two enzyme classes will exhibit synergy in killingM. abscessus. Paired combinations of antibiotics tested forin vitrosynergy againstM. abscessusincluded dual β-lactams, a β-lactam and a β-lactamase inhibitor, and a β-lactam and a rifamycin. Of the initial 206 combinations screened, 24 pairs exhibited synergy. A total of 13/24 pairs were combinations of two β-lactams, and 12/24 pairs brought the MICs of both drugs to within the therapeutic range. Additionally, synergistic drug pairs significantly reduced the frequency of selection of spontaneous resistant mutants. These novel combinations of currently available antibiotics may offer viable immediate treatment options against highly-resistantM. abscessusinfections.

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