Project description:Adaptation of C. elegans to hypertonic environments involves the accumulation of the organic osmolyte glycerol via transcriptional upregulation of the glycerol biosynthestic enzyme gpdh-1. A number of mutants, termed osmotic stress resistant (osr) mutants, have been identified. osr mutants cause constitutive upregulation of gpdh-1 and confer extreme resistance to hypertonicity. We tested the hypothesis that osr mutants broadly activate a gene expression program normally activated by osmotic stress in wild type animals using Affymterix microarray analysis of the hypertonic stress response in wild type animals and of constituitive gene expression changes in five osr mutants.
Project description:Adaptation of C. elegans to hypertonic environments involves the accumulation of the organic osmolyte glycerol via transcriptional upregulation of the glycerol biosynthestic enzyme gpdh-1. A number of mutants, termed osmotic stress resistant (osr) mutants, have been identified. osr mutants cause constitutive upregulation of gpdh-1 and confer extreme resistance to hypertonicity. We tested the hypothesis that osr mutants broadly activate a gene expression program normally activated by osmotic stress in wild type animals using Affymterix microarray analysis of the hypertonic stress response in wild type animals and of constituitive gene expression changes in five osr mutants. Experiment Overall Design: Young adult C. elegans were exposed to hypertonic growth plates for varying times prior to RNA extraction and hybridization on Affymetrix microarrays. Since we wished to separate direct response from secondary responses to osmotic stress, we collected worms following short term exposures to hypertonic conditions (15 minutes and 1 hour) and after long term exposure to hypertonic conditions (6 hours or a full generation of growth under hypertonic conditions). We also collected young adults from the osr mutants osm-7, osm-8, osm-11, dpy-9, and dpy-10 for microarray analysis. These mutants were grown under isotonic conditions to determine whether that constitutively activate genes normally regulated by hypertonic stress in wild type animals.
Project description:Young adult N2 Caenorhabditis elegans were infected with Enterococcus faecalis or Enterococcus faecium for 8 h to determine the transcriptional host response to each enterococcal species. Analysis of differential gene expression in C. elegans young adults exposed to four different bacteria: heat-killed Escherichia coli strain OP50 (control), wild-type E. faecalis MMH594, wild-type E. faecium E007, or Bacillus subtilis PY79 (sigF::kan). Samples were analyzed at 8 hours after exposure to the different bacteria. These studies identified C. elegans genes induced by pathogen infection. Brain-heart infusion agar plates (10 ug/ml kanamycin) were used.
Project description:Young adult fer-15;fem-1 Caenorhabditis elegans were infected with Staphylococcus aureus for 8 h to determine the transcriptional host response to Staphylococcus aureus. Analysis of differential gene expression in C. elegans young adults exposed to two different bacteria: E. coli strain OP50 (control), wild-type Staphylococcus aureus RN6390. Samples were analyzed at 8 hours after exposure to the different bacteria. These studies identified C. elegans genes induced by pathogen infection. Keywords: response to pathogen infection, innate immunity, host-pathogen interactions
Project description:The Caenorhabditis elegans bus (bacterial unswollen) mutants were isolated by their altered response to the nematode pathogen Microbacterium nematophilum. The bus-2, bus-4 and bus-17 mutants are resistant to infection by this bacterium and to infection by human pathogens Yersinia pestis and Yersinia pseudotuberculosis. Here we extend that list to Staphylococcus aureus. The bus-2, bus-4 and bus-17 mutants each harbors a defect in a different glycosyltransferase involved in O-glycosylation. Glycomics analysis of these strains reveals significant O-glycosylation defects. We further investigated the nature of bus mutant phenotypes in bus-2, bus-4 and bus-17 by gene expression analysis. Three distinct areas of altered expression were identified: 1) N- and O-glycosylation; 2) innate immune response; 3) protein folding and editing control. As expected N- and O-glycosylation gene expression was altered at key enzymatic steps. Innate immune system expression patterns were altered in a way that significantly overlapped with expression patterns seen in wild-type upon exposure to Staphylococuss aureus. Upon infection with S. aureus markers of innate immune activity increased significantly compared to wild-type. The abu/pqn genes, active in the non-canonical unfolded protein response (UPR) pathway were dramatically upregulated in bus when these mutants were exposed to the pathogen. This work demonstrates a genetic link between O-glycosylation and expression of key components of the innate immune response.