RESUMEN
Mycobacterium abscessus subsp. massiliense (Mycma) is a rapidly growing Mycobacterium belonging to the M. abscessus complex that is often associated with lung and soft tissue infection outbreaks. Mycma is resistant to many antimicrobials, including those used for treating tuberculosis. Therefore, Mycma infections are difficult to treat and may lead to high infectious complication rates. Iron is essential for bacterial growth and establishment of infection. During infection, the host reduces iron concentrations as a defense mechanism. To counteract the host-induced iron deficiency, Mycma produces siderophores to capture iron. Mycma has two ferritins (encoded by mycma_0076 and mycma_0077) modulated by different iron concentrations, which allow the survival of this pathogen during iron scarcity. In this study, we constructed knockout (Mycma 0076KO) and complemented (Mycma 0076KOc) gene strains for mycma_0076 to understand the function of 0076 ferritin. Deletion of mycma_0076 in Mycma led to the transition in colony morphology from smooth to rough, alteration of the glycopeptidolipids spectra, increased permeability of the envelope, reduction in biofilm formation, increased susceptibility to antimicrobials and hydrogen peroxide-induced oxidative stress, and decreased internalization by macrophages. This study shows that Mycma_0076 ferritin in Mycma is involved in resistance to oxidative stress and antimicrobials, and alteration of cell envelope architecture. KEY POINTS: ⢠Deletion of the mycma_0076 gene altered colony morphology to rough; ⢠Mycma 0076KO changed GPL profile; ⢠Absence of Mycma_0076 ferritin results in increased susceptibility to antimicrobials and oxidative stress in Mycma. Legend: a In wild-type M. abscessus subsp. massiliense strain, iron is captured from the environment by carboxymycobactins and mycobactins (1). Iron-dependent regulator (IdeR) proteins bind to ferrous iron (Fe+2) in the bacterial cytoplasm leading to the activation of the IdeR-Fe+2 complex (2). The activated complex binds to the promoter regions of iron-dependent genes, called iron box, which in turn help in the recruitment of RNA polymerase to promote transcription of genes such as mycma_0076 and mycma_0077 ferritin genes (3). Mycma_0076 and Mycma_0077 ferritins bind to excess iron in the medium and promote Fe2+ oxidation into ferric iron (Fe3+) and store iron molecules to be released under iron scarcity conditions. (4) Genes related to biosynthesis and transport of glycopeptidolipids (GPL) are expressed normally and the cell envelope is composed of different GPL species (colored squares represented on the cell surface (GPLs). Consequently, WT Mycma present smooth colony phenotype (5). b In Mycma 0076KO strain, the lack of ferritin 0076 causes overexpression of mycma_0077 (6), but does not restore wild-type iron homeostasis and thus may result in free intracellular iron, even in the presence of miniferritins (MaDps). The excess iron potentiates oxidative stress (7) by generating hydroxyl radicals through Fenton Reaction. During this process, through an unknown mechanism, that could involve Lsr2 (8), the expression of GPL synthesis locus is regulated positively and/or negatively, resulting in alteration of GPL composition in the membrane (as represented by different colors of squares on the cell surface), resulting in a rough colony phenotype (9). The changes of GPL can increase cell wall permeability, contributing to antimicrobial susceptibility (10).
Asunto(s)
Ferritinas , Mycobacterium abscessus , Ferritinas/genética , Ferritinas/metabolismo , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Antibacterianos/farmacología , Hierro/metabolismo , Estrés OxidativoRESUMEN
Mycolic acids, a hallmark of the genus Mycobacterium, are unique branched long-chain fatty acids produced by a complex biosynthetic pathway. Due to their essentiality and involvement in various aspects of mycobacterial pathogenesis, the synthesis of mycolic acids-and the identification of the enzymes involved-is a valuable target for drug development. Although most of the core pathway is comparable between species, subtle structure differences lead to different structures delineating the mycolic acid repertoire of tuberculous and some nontuberculous mycobacteria. We here report the characterization of an α'-mycolic acid-deficient Mycobacterium smegmatis mutant obtained by chemical mutagenesis. Whole-genome sequencing and bioinformatic analysis identified a premature stop codon in MSMEG_4301, encoding an acyl-CoA synthetase. Orthologs of MSMEG_4301 are present in all mycobacterial species containing α'-mycolic acids. Deletion of the Mycobacterium abscessus ortholog MAB_1915 abrogated synthesis of α'-mycolic acids; likewise, deletion of MSMEG_4301 in an otherwise wild-type M. smegmatis background also caused loss of these short mycolates. IMPORTANCE Mycobacterium abscessus is a nontuberculous mycobacterium responsible for an increasing number of hard-to-treat infections due to the impervious nature of its cell envelope, a natural barrier to several antibiotics. Mycolic acids are key components of that envelope; thus, their synthesis is a valuable target for drug development. Our results identify the first enzyme involved in α'-mycolic acids, a short-chain member of mycolic acids, loss of which greatly affects growth of this opportunistic pathogen.
Asunto(s)
Mycobacterium abscessus , Mycobacterium , Vías Biosintéticas/genética , Ácidos Grasos/metabolismo , Mycobacterium/metabolismo , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos/metabolismo , Micobacterias no TuberculosasRESUMEN
Mycobacterium abscessus subsp. massiliense (Mycma) belongs to the Mycobacterium abscessus complex and is a rapidly growing non-tuberculous mycobacterium. The chronic pulmonary, skin, and soft tissue infections that it causes may be difficult to treat due to its intrinsic resistance to the commonly used antimicrobial drugs, making it a serious world public health problem. Iron is an essential nutrient for the growth of microorganisms; nonetheless, it can be toxic when in excess. Thus, bacteria require an iron homeostasis mechanism to succeed in different environments. DNA-binding proteins from starved cells (Dps) are miniferritins with the property to act as additional iron storage proteins but also can bind to DNA, protecting it against hydroxyl radical. Annotation of the Mycma genome revealed the gene mycma_03135 with 79% sequential identity when compared to MSMEG_3242 gene from M. smegmatis mc2 155, which codifies for a known Dps. Recombinant Dps from M. abscessus (rMaDps) was produced in Escherichia coli, purified in soluble form and shown to form high mass oligomers in solution with ferroxidase activity, DNA binding, and protection against damage. The expression of the mycma_03135 gene was induced during Mycma growth in the presence of hydrogen peroxide (H2O2). Additionally, the expression of rMaDps by E. coli conferred greater resistance to H2O2. Thus, this study is the first to identify and characterize a Dps from M. abscessus. KEY POINTS: Mycobacterium abscessus subsp. massiliense express a miniferritin protein (Dps). Mycma Dps binds to DNA and protects against oxidative stress.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Estrés Fisiológico , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Peróxido de Hidrógeno/farmacología , Mycobacterium abscessus/efectos de los fármacos , Análisis de Secuencia de ADNRESUMEN
Herein, we evaluated tetrahydropyridine (THP) compounds (NUNM) as antimicrobials and inhibitors of the efflux mechanism in M. abscessus. subsp. abscessus. The modulation factor (MF) of efflux inhibitors was calculated from the minimum inhibitory concentrations (MICs) of amikacin (AMI), ciprofloxacin (CIP) and clarithromycin (CLA) in the absence and presence of subinhibitory concentrations of the NUNM compounds and canonical inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and verapamil (VP). The kinetics of the intracellular accumulation of the fluorimetric substrate ethidium bromide (EtBr) was evaluated and calculated by the relative final fluorescence (RFF). In addition, molecular modeling simulations for the MmpL5 and Tap efflux transporters with ligands (CLA, NUNM, CCCP, VP and EtBr) were performed to better understand the efflux mechanism. We highlight the NUNM01 compound because it reduced the MICs of AMI, CIP and CLA by 4-, 4- and 16-fold, respectively, had the highest effect on EtBr accumulation (RFFâ¯=â¯3.1) and showed a significant in silico affinity for the evaluated proteins in docking simulations. Based on the analyses performed in vitro and in silico, we propose that NUNM01 is a potential pharmacophore candidate for the development of a therapeutic adjuvant for M. abscessus infections.
Asunto(s)
Antibacterianos/farmacología , Mycobacterium abscessus/efectos de los fármacos , Pirrolidinas/farmacología , Transporte Biológico/efectos de los fármacos , Simulación por Computador , Etidio/farmacocinética , Fluorometría/métodos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Simulación del Acoplamiento Molecular/métodos , Mycobacterium abscessus/metabolismoRESUMEN
New drugs are needed to treat infections with antimicrobial-resistant Mycobacterium abscessus; therefore, we evaluated usnic acid as an antimicrobial agent and efflux inhibitor (EI) against M. abscessus. Usnic acid showed antimicrobial activity, and synergistically, the EI verapamil increased this activity. In addition, when we evaluated the interaction of antimicrobials with usnic acid, the increase of their activity was observed. Finally, usnic acid showed an efflux inhibitory effect between the classical EIs verapamil and carbonyl cyanide m-chlorophenylhydrazine. In conclusion, usnic acid showed both antimicrobial and EI activity, indicating that this natural compound may be a promising scaffold for new drugs against this difficult-to-treat microorganism.