Project description:Novel high-throughput deep sequencing technology has dramatically changed the way that the functional complexity of transcriptomes can be studied. Here we report on the first use of this technology to gain insight into the wide range of transcriptional alterations that are associated with an infectious disease process. Using Solexa/IlluminaM-bM-^@M-^Ys digital gene expression (DGE) system, a tag-based transcriptome sequencing method, we investigated mycobacterium-induced transcriptome changes in a model vertebrate species, the zebrafish. Our DGE data substantiate recent RNA-seq results from other models indicating a much larger extent of genome transcription than previously thought, and demonstrate that the host response to bacterial infection adds a further degree of complexity to the transcriptome. We obtained a sequencing depth of over 5 million tags per sample with strong correlation between replicates. Tag mapping indicated that mycobacterium-infected adult zebrafish express over 70% of all genes represented in transcript databases. Comparison of our DGE data with a previous multiplatform microarray analysis showed that both types of technologies identified regulation of similar functional groups of genes, more specifically the up-regulation of different classes of immune response genes concomitant with a broad down-regulation of metabolic genes. However, the unbiased nature of DGE analysis provided insights that microarray analysis could not have achieved. As demonstrated here, DGE data are useful for the verification of predicted gene models and allowed us to detect mycobacterium-regulated switching between different transcript isoforms. Moreover, genomic mapping of infection-induced DGE tags revealed novel transcript forms for which any previous EST-based evidence of expression was lacking. Adult male zebrafish were infected by intraperitoneal inoculation with approximately 1x 10-3 M. marinum bacteria. All four infected fish were sacrificed when they showed overt signs of fish tuberculosis, including lethargy, skin ulcers and extensive granuloma formation in organs such as liver and kidney. Histological examination of fish from the same experiments confirmed that the pathology of infected fish corresponded to fish tuberculosis and that no characteristics of the disease were present in the control fish. For DGE analysis RNA samples from the four control adult zebrafish (c1,c2,c3,c4) were pooled, and RNA samples from the four M.marinum-infected adult zebrafish (i1,i2,i3,i4) were pooled. Before pooling the individual RNA samples had been checked by microarray analysis for correlation between biological replicates.
Project description:Mycobacteria infect macrophages that aggregate with additional macrophages and lymphocytes to form granulomas. We have used a functional genomics approach to identify immune response genes expressed during granuloma formation in Mycobacterium marinum-infected transparent zebrafish larvae where individual infection steps can be viewed in real time. We assessed RNA expression profiles from zebrafish larvae that were either infected with Mycobacterium marinum or mock-infected. Zebrafish infections were performed at 1 day post-fertilization (dpf), and samples were derived from pools of 6dpf zebrafish larvae. Keywords: host response to infection
Project description:Mycobacteria infect macrophages that aggregate with additional macrophages and lymphocytes to form granulomas. We have used a functional genomics approach to identify immune response genes expressed during granuloma formation in Mycobacterium marinum-infected transparent zebrafish larvae where individual infection steps can be viewed in real time. We assessed RNA expression profiles from zebrafish larvae that were either infected with Mycobacterium marinum, mock-infected, or uninfected. Zebrafish infections were performed at 1 day post-fertilization (dpf), and samples were derived from pools of 6dpf zebrafish larvae. Keywords: host response to infection
Project description:A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.