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Ex vivo proteomics of Mycobacterium marinum

ABSTRACT: Proteins expressed and exported out of the mycobacterial biofilm cells into granulomatous environment during infection were identified by LC-MS/MS. To this end, adult 5- to 10-month-old female AB wild-type zebrafish (Danio rerio) were infected with 4-day old M. marinum ATCC927 (carrying the pTEC27 plasmid expressing the tdTomato fluorescent protein) cells. After 8 weeks post infection, ten zebrafish were euthanized, and red fluorescent mycobacterial granulomas were carefully dissected from the zebrafish ovaries. Ten granulomas were collected per tube, frozen on dry ice and stored at -80 °C until preparation for proteomics. Altogether, ten replicate samples, each with ten mycobacterial granulomas, were subjected to protein extraction and LC-MS/MS identification.

INSTRUMENT(S): timsTOF fleX

ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)

SUBMITTER: Tuula Nyman

LAB HEAD: Tuula Nyman

PROVIDER: PXD039416 | Pride | 2023-03-06

REPOSITORIES: Pride

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Project description:Pathogenic mycobacteria have the ability to survive within macrophages and persist inside granulomas composed of host immune cells. The complex host-pathogen interactions that determine the outcome of a mycobacterial infection process result in marked alterations of the host gene expression profile. Here we used the zebrafish model to investigate the specificity of the host response to infections with two mycobacterium strains that give distinct disease outcomes: an acute disease with early lethality or a chronic disease with granuloma formation, caused by Mycobacterium marinum strains Mma 20 and E11, respectively. We performed a microarray study of different stages of disease progression in adult zebrafish and found that the acute and the chronic strains evoked partially overlapping host transcriptome signatures, despite that they induce profoundly different disease phenotypes. Both strains affected many signaling cascades, including Wnt and Tlr pathways. Interestingly, the strongest differences were observed at the initial stage of the disease. The immediate response to the acute strain was characterized by higher expression of genes encoding MHC class I proteins, matrix metalloproteinases, transcription factors, cytokines and other common immune response proteins. In contrast, small GTPase and histone gene groups showed higher expression in response to the chronic strain. We also found that nearly 1,000 mycobacterium-responsive genes overlapped between the expression signatures of infected zebrafish adults and embryos at different stages of granuloma formation. Since adult zebrafish possess an adaptive immune system similar to mammals and zebrafish embryos rely solely on innate immunity, this overlap indicates a major contribution of the innate component of the immune system in the response to mycobacterium infection. Taken together, our comparison of the transcriptome responses involved in acute versus chronic infections and in the embryonic versus adult situation provides important new leads for investigating the mechanism of mycobacterial pathogenesis.

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Ex vivo proteomics of Mycobacterium marinum

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