LFQ proteome comparsion of Burkholderia cenocepacia glycosylation mutants
ABSTRACT: Characterisation of the effect of glycosylation disruption within Burkholderia cenocepacia k56-2. Five strain comparsion (WT vs delta pglL vs delta OGC vs Delta pglL OGC vs Delta pglL complemented S7-pglL-his)
Project description:Characterisation of the effect of multiple pglL muants and complement within Burkholderia cenocepacia k56-2 to confirm glycosylation phenotype. Six strain comparsion (WT vs delta pglL 1 vs delta pglL 2 vs delta pglL 1 complement native pglL vs delta pglL 1 complement S7-pglL-his vs delta pglL 2 complement native pglL
Project description:Comparsion of multiple BC k56-2 muants to assess protoeme chnages. seven strain comparsion between WT vs delta pglL vs delta 1086 vs delta 2974 vs delta CepI vs delta CepR vs delta pglL complemented AmrAB::S7-pglL-his. LFQ based quantification
Project description:Comparsion of DNA interacting proteome of BC muants to assess chnages in transcriptional proteins. Four strain comparsion between WT vs delta pglL vs delta ogc vs delta pglL complemented AmrAB::S7-pglL-his. LFQ based quantification
Project description:Comparsion of the endogenous peptide pool within Burkholderia Cenocepacia strains to identify evidence for glycoprotein degradation in the absent of glycosylation. WT vs delta pglL vs pglL complement were compared (4 biological of each)
Project description:Comparsion of proteomes of Campylobacter fetus subsp. fetus to compare protein level via iBAQ analysis, expression (by LFQ) and coverage between Campylobacter fetus subsp. fetus strain82-40 vs Campylobacter fetus subsp. fetus strain ATCC 27374
Project description:Burkholderia cenocepacia is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. B. cenocepacia survives in macrophages in membrane-bound vacuoles; however, the mechanism by which B. cenocepacia gains entry into macrophages remains unknown. After macrophage internalization, survival of B. cenocepacia within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that B. cenocepacia induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of B. cenocepacia contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of Burkholderia to enter macrophages through the induction of macropinocytosis.
Project description:Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1?, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1? processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1? release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1? release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1? response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1? processing and release.
Project description: Transcription profiling of one Burkholderia cenocepacia clinical isolate, J2315, versus a soil isolate, HI2424, in conditions mimicking CF sputum  Transcription profiling of Burkholderia cenocepacia isolates J2315 and HI2424 in media mimicking CF sputum or the soil environment Overall design:  J2315 vs. HI2424 cells in the same condition.  Two-condition experiment. Biological replicates: 4 replicates.
Project description:Strains of the Burkholderia cepacia complex can survive within macrophages by arresting the maturation of phagocytic vacuoles. The bacteria preclude fusion of the phagosome with lysosomes by a process that is poorly understood. Using murine macrophages, we investigated the stage at which maturation is arrested and analyzed the underlying mechanism. Vacuoles containing B. cenocepacia strain J2315, an isolate of the transmissible ET12 clone, recruited Rab5 and synthesized phosphatidylinositol-3-phosphate, indicating progression to the early phagosomal stage. Despite the fact that the B. cenocepacia-containing vacuoles rarely fused with lysosomes, they could nevertheless acquire the late phagosomal markers CD63 and Rab7. Fluorescence recovery after photobleaching and use of a probe that detects Rab7-guanosine triphosphate indicated that activation of Rab7 was impaired by B. cenocepacia, accounting at least in part for the inability of the vacuole to merge with lysosomes. The Rab7 defect was not due to excessive cholesterol accumulation and was confined to the infected vacuoles. Jointly, these experiments indicate that B. cenocepacia express virulence factors capable of interfering with Rab7 function and thereby with membrane traffic.
Project description:B. cenocepacia is an opportunistic human pathogen that is particularly problematic for patients suffering from cystic fibrosis (CF). In the CF lung bacteria grow to high densities within the viscous mucus that is limited in oxygen. Pseudomonas aeruginosa, the dominant pathogen in CF patients, is known to grow and survive under oxygen-limited to anaerobic conditions by using micro-oxic respiration, denitrification and fermentative pathways. In contrast, inspection of the genome sequences of available B. cenocepacia strains suggested that B. cenocepacia is an obligate aerobic and non-fermenting bacterium. In accordance with the bioinformatics analysis we observed that B. cenocepacia H111 is able to grow with as little as 0.1% O2 but not under strictly anoxic conditions. Phenotypic analyses revealed that H111 produced larger amounts of biofilm, pellicle and proteases under micro-oxic conditions (0.5%-5% O2, i.e. conditions that mimic those encountered in CF lung infection), and was more resistant to several antibiotics. RNA-Seq and shotgun proteomics analyses of cultures of B. cenocepacia H111 grown under micro-oxic and aerobic conditions showed up-regulation of genes involved in the synthesis of the exopolysaccharide (EPS) cepacian as well as several proteases, two isocitrate lyases and other genes potentially important for life in micro-oxia.RNA-Seq raw data files are accessible through the GEO Series accession number GSE48585. MS data have been deposited in the ProteomeXchange database (PXD000270).