Project description:We used microarrays to describe the gene expression in response to two isogenic morphotypes of M. abscessus (Mab) and to Staphylococcus aureus. Human neutrophils from healthy donors were exposed to rough Mab (ATCC 19977T), smooth Mab (ATCC 19977T) and S. aureus (CF clinical strain) for two hours; control cells were exposed to saline.
Project description:The response of human neutrophils to the emerging pathogen Mycobacterium abscessus has not been described. However, M. abscessus infections are frequently associated with neutrophil-rich abscesses. To better understand the reponse of neutrophils to M. abscessus we performed gene expression analysis using Affymetrix HG-U133A Plus 2.0 microarrays. Human neutrophils from healthy donors were stimulated with isogenic rough and smooth morphotypes of M. abscessus. Staphylococcus aureus was used as a control. Gene expression was compared to neutrophils left unstimulated. Neutrophils from four individual donors were isolated on separate days and stimulated with freshly prepared bacteria. Neutrophils (stimulated and control) were left for 2 hours before total RNA was isolated, and biotinylated cRNA was prepared by standard methods. Analysis indicates that M. abscessus morphotypes induce a limited number of genes, when compared to S. aureus, which are enriched in genes for cytokines and chemokines, including neutrophil-specific chemokines. These data suggest that neutrophils have a limited response to M. abscessus, which may contribute to neutrophil-rich abscess formation.!Series_overall_design = Human neutrophils from healthy donors were exposed to rough Mab (ATCC 19977T), smooth Mab (ATCC 19977T) and S. aureus (CF clinical strain) for two hours; control cells were exposed to saline.
Project description:In this study, we were interested to get deeper insights into the molecular mechanisms that govern the formation and selection of the different colony morphologies in Mycobacterium abscessus strains, including the potential reversibility of the rough (R) phenotype into a smooth (S) phenotype. We used next generation sequencing (NGS) and micro-array / RNAseq approaches to determine the genome sequences and transcriptomic profiles of three isogenic S/R strain couples of M. abscessus. One clinical isolate strain named CF and two collection strains referred as 19977-AT and 19977-IP. To perform the transcriptomic comparison of the rough variant versus the smooth variant for each M. abscessus strain, a customized micro-array has been manufactured by Agilent (8 x 15k format). The design of oligonucleotides covering all protein coding sequences was done using OligoArray version 2.1 on the basis of the 4920 predicted coding sequences composing the entire M. abscessus genome. The experimental data for each of the 3 strains consisted of 6 hybridizations (3 biological replicates with dye-swap).
Project description:Members of the Mycobacterium (M.) abscessus complex (MABC) are rapidly growing mycobacteria showing smooth and/or rough colony morphotype. While not as virulent as M. tuberculosis, they can cause soft tissue infection and fatal pulmonary disease, especially in patients with cystic fibrosis. Diagnosing MABC pulmonary disease is challenging since the isolation of M. abscessus from respiratory samples is in itself not diagnostic and the clinical features are often non-specific. Immunologic assays, which could aid in the understanding and diagnosis of the disease, are not available. In this study eight rough and six smooth colony morphotype isolates were collected from seven clinical MABC strains and the M. abscessus reference strain ATCC19977, as six strains showed both morphotypes simultaneously and two strains only showed a rough morphotype. Clinical isolates were submitted to whole genome sequencing. Quantitative proteomic analysis was performed on bacterial lysates and the culture supernatant of all 14 isolates. Supernatant proteins present in all isolates were compared in a BLAST search against other clinically significant mycobacterial species to determine species-specific proteins of MABC. In silico B- and T-cell epitope prediction was performed for species-specific proteins. All clinical strains were found to be M. abscessus ssp. abscessus. Six of seven rough colony clinical isolates contained genetic changes in the MAB_4099c gene, which is a likely genetic basis for the rough morphotype. Proteomic analysis detected 3 137 different proteins in total of which 79 proteins were found in the culture supernatants of all isolates. BLAST analyses of these 79 proteins identified 12 of those exclusively encoded by all members of MABC plus M. immunogenum. In silico prediction of epitopes predicted B- and T-cell epitopes in all these 12 species-specific proteins, rendering them promising candidates for future studies on immune pathogenesis and immune diagnostic tools for MABC disease.
Project description:Mycobacterium abscessus is a major human pathogen, mostly infecting people with pre-existing pathological lung conditions such as cystic fibrosis. Only little is known about the ways M. abscessus regulates its virulence factors and pathogenesis phenotypes. The production of glycopeptidolipids (GPL) is a major determinant of virulence of this bacterium, with clinical isolates that lack GPL synthesis and export generally exhibiting more aggressive clinical behavior. The current paradigm is that GPL production is abolished in vivo via irreversible, spontaneous mutations taking place as part of in-host evolution. Little is known about the mechanisms or extent to which GPL production may be regulated. We have found an unusual TetR-like transcription factor of M. abscessus, mab_1638, identified via screening of a comprehensive transposon-mutant library, that appears to be a strong positive regulator of the entire GPL biosynthesis and export gene cluster through a combination of direct and indirect mechanisms. The inactivation of mab_1638 by a transposon led to stable and visually obvious rough colony morphology, as well as increased virulence in infection models, characteristic of rough, non-GPL-producers. Transcriptome analysis found the mab_1638:tn mutant to have 118 differentially expressed genes, including the GPL locus. It appears mab_1638 encodes an unusual TetR transcription factor required for the production of GPL in M. abscessus and therefore having a profound effect on virulence traits. This finding raises the important possibility that M. abscessus strains appearing smooth in laboratory growth conditions may nonetheless downregulate GPL-cluster genes in other conditions and thus acquire the phenotypic characteristics of rough strains.
Project description:In this study, we were interested to get deeper insights into the molecular mechanisms that govern the formation and selection of the different colony morphologies in Mycobacterium abscessus strains, including the potential reversibility of the rough (R) phenotype into a smooth (S) phenotype. We used next generation sequencing (NGS) and micro-array / RNAseq approaches to determine the genome sequences and transcriptomic profiles of three isogenic S/R strain couples of M. abscessus. One clinical isolate strain named CF and two collection strains referred as 19977-AT and 19977-IP.
Project description:Mycobacterium abscessus is a major human pathogen, mostly infecting people with pre-existing pathological lung conditions such as cystic fibrosis. Only little is known about the ways M. abscessus regulates its virulence factors and pathogenesis phenotypes. The production of glycopeptidolipids (GPL) is a major determinant of virulence of this bacterium, with clinical isolates that lack GPL synthesis and export generally exhibiting more aggressive clinical behavior. The current paradigm is that GPL production is abolished in vivo via irreversible, spontaneous mutations taking place as part of in-host evolution. Little is known about the mechanisms or extent to which GPL production may be regulated. We have found an unusual TetR-like transcription factor of M. abscessus, mab_1638, identified via screening of a comprehensive transposon-mutant library, that appears to be a strong positive regulator of the entire GPL biosynthesis and export gene cluster through a combination of direct and indirect mechanisms. The inactivation of mab_1638 by a transposon led to stable and visually obvious rough colony morphology, as well as increased virulence in infection models, characteristic of rough, non-GPL-producers. Transcriptome analysis found the mab_1638:tn mutant to have 118 differentially expressed genes, including the GPL locus. It appears mab_1638 encodes an unusual TetR transcription factor required for the production of GPL in M. abscessus and therefore having a profound effect on virulence traits. This finding raises the important possibility that M. abscessus strains appearing smooth in laboratory growth conditions may nonetheless downregulate GPL-cluster genes in other conditions and thus acquire the phenotypic characteristics of rough strains.
Project description:Mycobacteroides abscessus (Mabc) is a rapidly growing nontuberculous mycobacterium that poses a considerable challenge as a multidrug-resistant pathogen causing chronic human infection. Effective therapeutics that enhance protective immune responses to Mabc are urgently needed. This study introduces trans-3,5,4′-trimethoxystilbene (V46), a novel resveratrol analogue with autophagy-activating properties and antimicrobial activity against Mabc infection, including multidrug-resistant strains. Among the resveratrol analogues tested, V46 markedly inhibited the growth of both rough and smooth Mabc strains in murine bone marrow-derived macrophages and in the lungs of infected mice. Additionally, V46 significantly reduced Mabc-induced increases in chemokine and pro inflammatory cytokine levels in macrophages and in vivo during infection. Mechanistic analysis showed that V46 suppressed the activation of the AKT-mammalian target of rapamycin signaling pathway and enhanced AMP-activated protein kinase signaling in Mabc-infected cells. Notably, V46 activated autophagy and nuclear translocation of transcription factor EB, which is crucial for antimicrobial host defenses against Mabc. Furthermore, V46 upregulated genes associated with autophagy and lysosomal biogenesis in Mabc-infected bone marrow-derived macrophages. The combination of V46 and rifabutin exerted a synergistic antimicrobial effect. These findings identify V46 as a candidate host-directed therapeutic for Mabc infection that activates autophagy and lysosomal function via transcription factor EB.
Project description:Mycobacterium abscessus is nowadays under the spotlight of the scientific community. This pathogenic mycobacteria is indeed responsible for a wide spectrum of infections involving mostly pulmonary infections in patients with cystic fibrosis. M. abscessus is intrinsically resistant to a broad range of antibiotics, including most antitubercular drugs, and is considered the most pathogenic and chemotherapy-resistant rapidly growing mycobacterium. Consequently, with very limited treatment options, the development of new therapeutic approaches to fight this pathogen are urgently needed. In this context, 19 oxadiazolone (OX) derivatives have been investigated for their antibacterial activity against both the rough (R) and smooth (S) variants of M. abscessus. Several OXs were active against extracellular M. abscessus growth with moderated minimal inhibitory concentrations (MIC), or intracellularly by inhibiting M. abscessus growth inside infected macrophages with MIC values similar to those of imipenem. Such promising results prompted us to identify the potential target enzymes of the sole extra and intracellular inhibitor of M. abscessus growth, i.e., iBpPPOX via activity-based protein profiling combined with mass spectrometry. This approach led to the identification of 21 potential protein candidates being mostly involved in M. abscessus lipid metabolism and/or in cell wall biosynthesis.