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Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish.

ABSTRACT: Mycobacterium marinum-infected zebrafish are used to study tuberculosis pathogenesis, as well as for antitubercular drug discovery. The small size of zebrafish larvae coupled with their optical transparency allows for rapid analysis of bacterial burdens and host survival in response to genetic and pharmacological manipulations of both mycobacteria and host. Automated fluorescence microscopy and automated plate fluorimetry (APF) are coupled with facile husbandry to facilitate large-scale, repeated analysis of individual infected fish. Both methods allow for in vivo screening of chemical libraries, requiring only 0.1 ?mol of drug per fish to assess efficacy; they also permit a more detailed evaluation of the individual stages of tuberculosis pathogenesis. Here we describe a 16-h protocol spanning 22 d, in which zebrafish larvae are infected via the two primary injection sites, the hindbrain ventricle and caudal vein; this is followed by the high-throughput evaluation of pathogenesis and antimicrobial efficacy.

SUBMITTER: Takaki K

PROVIDER: S-EPMC3919459 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish.

Takaki Kevin K Davis J Muse JM Winglee Kathryn K Ramakrishnan Lalita L

Nature protocols 20130516 6

Mycobacterium marinum-infected zebrafish are used to study tuberculosis pathogenesis, as well as for antitubercular drug discovery. The small size of zebrafish larvae coupled with their optical transparency allows for rapid analysis of bacterial burdens and host survival in response to genetic and pharmacological manipulations of both mycobacteria and host. Automated fluorescence microscopy and automated plate fluorimetry (APF) are coupled with facile husbandry to facilitate large-scale, repeate ...[more]

PMID: 23680983

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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.

Dataset Information

Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish.

Publications

Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish.

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