Project description:Mycobacterium abscessus is an emerging pathogen causing pulmonary infections in those with inflammatory lung disorders, such as Cystic Fibrosis (CF), and is associated with the highest fatality rate among rapidly growing mycobacteria (RGM). Phenotypically, MAB manifests as either a Smooth (MAB-S) or a Rough (MAB-R) morphotype, which differ in their levels of cell wall glycopeptidolipids (GPLs) and in their pathogenicity in vivo. As one of the primary immune cells encountered by MAB, we sought to examine the early transcriptional events within macrophages, following infection with both MAB-S or MAB-R. We sampled the small RNA (sRNA) transcriptome of THP-1-derived macrophages infected with both MAB-R and MAB-S at 1, 4 and 24 hours post-infection (hpi) using RNA-seq. MAB-S elicited a more robust transcriptional response at the miRNA level, reflecting higher cytokine levels in culture supernatants. However, and a direct comparison identified no differentially expressed miRNAs between MAB-R- and MAB-S-infected cells. Most of the induced miRNAs have previously been associated with mycobacterial infection and overall miRNA expression patterns were similarly highly correlated between the morphotypes.
Project description:Mycobacterium abscessus is an emerging pathogen causing pulmonary infections in those with inflammatory lung disorders, such as Cystic Fibrosis (CF), and is associated with the highest fatality rate among rapidly growing mycobacteria (RGM). Phenotypically, MAB manifests as either a Smooth (MAB-S) or a Rough (MAB-R) morphotype, which differ in their levels of cell wall glycopeptidolipids (GPLs) and in their pathogenicity in vivo. As one of the primary immune cells encountered by MAB, we sought to examine the early transcriptional events within macrophages, following infection with both MAB-S or MAB-R. We sampled the small RNA (sRNA) transcriptome of THP-1-derived macrophages infected with both MAB-R and MAB-S at 1, 4 and 24 hours post-infection (hpi) using RNA-seq. MAB-S elicited a more robust transcriptional response at the miRNA level, reflecting higher cytokine levels in culture supernatants. However, and a direct comparison identified no differentially expressed miRNAs between MAB-R- and MAB-S-infected cells. Most of the induced miRNAs have previously been associated with mycobacterial infection and overall miRNA expression patterns were similarly highly correlated between the morphotypes.
Project description:Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a novel nucleoid-associated protein that specifically binds AT-rich regions of the genome, including regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM/PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain swamping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA binding domain. NMR analysis of the DNA binding domain of Lsr2 and its interaction with DNA reveals a novel structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve an equivalent function as the Enterobacteriaceae H-NS, coordinating global gene regulation and virulence in this group of medically important bacteria.
Project description:During our efforts to isolate potantial binding partners of Esat6, we isolated few peptides rich in phenylalanine residues that strongly interacted with Esat6. All peptides were less than fifty amino acids in length, One of them, Hcl1, when expressed in mycobacteria showed significant retardation in growth and survival within macrophages. Microarray analysis showed that Hcl1 affects a host of genes and cellular pathways. RNA was isolated from exponentially growing mycobacteria containing either plasmid vector pVV16 encoding peptide or vector pVV16 alone. Comparisons were made between Experimental (Mtb/Hcl1) and control (Mtb/pVV16) samples by extracting raw intensity values from multiple arrays.
Project description:Mycobacterium abscessus is an emerging pathogen causing pulmonary infections in those with inflammatory lung disorders, such as Cystic Fibrosis (CF), and is associated with the highest fatality rate among rapidly growing mycobacteria (RGM). Phenotypically, MAB manifests as either a Smooth (MAB-S) or a Rough (MAB-R) morphotype, which differ in their levels of cell wall glycopeptidolipids (GPLs) and in their pathogenicity in vivo. As one of the primary immune cells encountered by MAB, we sought to examine the early transcriptional events within macrophages, following infection with both MAB-S or MAB-R. We sampled the small RNA (sRNA) transcriptome of THP-1-derived macrophages infected with both MAB-R and MAB-S at 1, 4 and 24 hours post-infection (hpi) using RNA-seq. MAB-S elicited a more robust transcriptional response at the miRNA level, reflecting higher cytokine levels in culture supernatants. However, and a direct comparison identified no differentially expressed miRNAs between MAB-R- and MAB-S-infected cells. Most of the induced miRNAs have previously been associated with mycobacterial infection and overall miRNA expression patterns were similarly highly correlated between the morphotypes. THP-1-derived macrophages were infected in parallel with the MAB-R and MAB-S morphotypes. Poly-A selected RNAs were purified and sequenced at 1, 4 and 24 hours post-infection, and compared with uninfected controls.
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. 38 new analogs of Cyclipostins & Cyclophostin (CyC), compounds naturally produced by Streptomyces species, have been synthesized. Their antibacterial activities against clinical isolates belonging to the M. chelonae-abscessus clade, as well as Gram-negative and Gram-positive bacteria have been evaluated by the REMA method. The intracellular activities of the CyC against intramacrophagic M. abscessus have also been investigated and compared to those of imipenem. The CyCs displayed very low toxicity towards host cells and their inhibitory activity was exclusively restricted to mycobacteria. The best candidate, CyC17, showed a high selectivity for mycobacteria with MIC values (<2 up to 40 µg/mL) comparable to those of most classical antibiotics used to treat M. abscessus infections. Of importance, several CyCs were active against extracellular M. abscessus growth (i.e., CyC17 / CyC18β / CyC25 / CyC26) or against intracellular mycobacteria inside macrophages (i.e., CyC7α,β / CyC8α,β) with MIC values similar to or better than those of standard antibiotics. Based on these results, we intended to identify the potential target enzymes of CyC17/CyC26 in M. abscessus by activity-based protein profiling (ABPP) approach coupled with mass spectrometry differential analysis.
Project description:Mycobacterium abscessus is an emerging pathogen causing pulmonary infections in those with inflammatory lung disorders, such as Cystic Fibrosis (CF), and is associated with the highest fatality rate among rapidly growing mycobacteria (RGM). Phenotypically, MAB manifests as either a Smooth (MAB-S) or a Rough (MAB-R) morphotype, which differ in their levels of cell wall glycopeptidolipids (GPLs) and in their pathogenicity in vivo. As one of the primary immune cells encountered by MAB, we sought to examine the early transcriptional events within macrophages, following infection with both MAB-S or MAB-R. We sampled the small RNA (sRNA) transcriptome of THP-1-derived macrophages infected with both MAB-R and MAB-S at 1, 4 and 24 hours post-infection (hpi) using RNA-seq. MAB-S elicited a more robust transcriptional response at the miRNA level, reflecting higher cytokine levels in culture supernatants. However, and a direct comparison identified no differentially expressed miRNAs between MAB-R- and MAB-S-infected cells. Most of the induced miRNAs have previously been associated with mycobacterial infection and overall miRNA expression patterns were similarly highly correlated between the morphotypes. THP-1-derived macrophages were infected in parallel with the MAB-R and MAB-S morphotypes. Size-selected small RNAs were purified and sequenced at 1, 4 and 24 hours post-infection, and compared with uninfected controls.