Project description:In Mycobacterium tuberculosis, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Several mutations, present in phoR of Mycobacterium canettii relative to M. tuberculosis, impact the expression of the PhoP regulon and the pathogenicity of the strains. Here, we analyse by RNA-seq the expression profile of PhoP-regulated genes between the two M. tuberculosis strains H37Rv and HN878 and the two M. canettii isolates STB-Ks and STB-Kr.
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: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: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:Purpose: to determine the differentially expressed genes in the phase-variable rough and smooth colony isolates of C. difficile Methods: C. difficile R20291 was grown on BHIS agar to obtain distinct colonies. Individual rough and smooth colonies were chosen for propagation on BHIS-agar for 24 hours as described in Garrett, et al., PLoS Biology, 2019. Growth was collected from n = 2 biological replicates. RNA was purified using TriSure and chloroform, beadbeating, and isopropanol/ethanol precipitation. Quality was verified with Bioanalyzer Assay. Samples were submitted to Genewiz for depletion of rRNA using the TruSeq RiboZero Gold Kit, library preparation, and single-end sequencing on the Illumina HiSeq 2500 platform. RNA sequencing analysis was done using CLC Genomic Workbench v20. Reads were mapped to C. difficile R20291 genome using the software's default menalties for mismatch, deletion, and insertion differences from the reference genome. Transcript reads were normalized as RPKM. Results: 88 genes were differentially expressed between bacteria from rough versus smooth colonies, with equal to or greater than a 2-fold change and p < 0.05 with FDR correction.
Project description:Mycobacterium abscessus (Mabs) is a fast-growing, non-tuberculous mycobacterium responsible for serious lung infections in patients with cystic fibrosis. This bacterium evolves during the pulmonary infection between a smooth (S) and a rough morphotype (R). We have previously isolated the lsr2 gene as being differentially expressed during the transition between Mabs-S and Mabs-R. lsr2 encodes a pleiotropic transcription factor belonging to the superfamily of nucleoid-associated proteins (NAPs), which play an essential role in the hierarchical organization of bacterial chromosomes. The present study aims to unravel the molecular role of Lsr2 in regulating expression of gene involved in the adaptation and the pathobiology of Mabs using both RNA-Seq and ChIP-seq.