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Macrophage-Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti-Bacterial Function in Zebrafish

ABSTRACT: Macrophage expressed gene 1 (MPEG1) encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates, MPEG1 is an integral membrane protein of macrophages, but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1, two of which (mpeg1 and mpeg1.2) are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens, Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down-regulated during infection with both pathogens, mpeg1.2 is infection inducible. Up-regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1, and requires the Toll-like receptor adaptor molecule MyD88 and transcription factor NFκB. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection, both mpeg1 and mpeg1.2 knockdown increase bacterial burdens, but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti-bacterial function of the Mpeg1 family and indicate that the intricate cross-regulation of the two mpeg1 copies aids the zebrafish host in combatting infection

ORGANISM(S): Danio rerio

PROVIDER: GSE54885 | GEO | 2014/09/01

SECONDARY ACCESSION(S): PRJNA237966

REPOSITORIES: GEO

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Project description:Macrophage expressed gene 1 (MPEG1) encodes an evolutionary conserved protein with a predicted Membrane Attack Complex/Perforin domain associated with host defence against invading pathogens. In vertebrates, MPEG1 is an integral membrane protein of macrophages, but how it contributes to the macrophage defence mechanisms remains unknown. Zebrafish have three copies of MPEG1, two of which (mpeg1 and mpeg1.2) are expressed in macrophages whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens, Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is down-regulated during infection with both pathogens, mpeg1.2 is infection inducible. Up-regulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1, and requires the Toll-like receptor adaptor molecule MyD88 and transcription factor NFM-NM-:B. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in increased bacterial burden. In S. typhimurium infection, both mpeg1 and mpeg1.2 knockdown increase bacterial burdens, but mpeg1 morphants show an increased survival rate. The combined results of these two in vivo infection models support the anti-bacterial function of the Mpeg1 family and indicate that the intricate cross-regulation of the two mpeg1 copies aids the zebrafish host in combatting infection Embryos were injected at the one cell stage with a morpholino targeting mpeg1, or with the standard control morpholino from GeneTools, or with a morpholino targeting ptpn6 (Kanwal et al., 2013, J. Immunol 190:1631-45) for comparison. Subsequently, at 24 hours post fertilisation (hpf) the morphants and their controls were manually dechorionated at 24 hpf and at 28 hpf they were infected by injecting 200 colony forming units of M. marinum Mma20 into the caudal vein, or mock-injected with PBS/2%PVP. After injections embryos were transferred into fresh egg water containing 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) to prevent melanisation and incubated for 4 days at 28M-BM-0C. After the incubation period, infected and uninfected morphants, mutants and their controls were imaged and groups of 30 embryos were snap-frozen in liquid nitrogen and RNA was isolated for Illumina RNAseq analysis.

Project description:We use the zebrafish embryo model to study the similarities and differences in the innate immune response against three different bacterial pathogens. Therefore, we injected E. tarda, Salmonella, or M. marinum into the caudal vein of 28 hours post fertilization (hpf) zebrafish embryos and analysed their gene expression profile at 8 hours or 4 days after infection by microarrays. The results show that infections with the gram-negative bacteria E. tarda and S. typhimurium, which are lethal within 1-2 days, induce a strong early immune response at 8 hours after infection. In contrast, infection with M. marinum leads to a chronic infection that only induces a strong response at 4 days post infection. This microarray study was designed to determine the gene expression profile during infection with Salmonella typhimurium, Mycobacterium marinum, and Edwardsiella tarda. RNA was isolated from single embryos and each treatment group consisted of three embryos: (1) Wildtypes injected with PBS 8 hours post infection (hpi), (2) S. typhimurium-infected wildtypes 8hpi, (3) wildtypes injected with PBS/2%PVP 8hpi , (4) M. marinum-infected wildtypes 8hpi, (5) wildtypes injected with PBS/2%PVP 4 days post infection (dpi), (6) M. marinum-infected wildtypes 4dpi, (7) wildtypes injected with PBS 8hpi (E. tarda control), (8) E. tarda-infected wildtypes 8hpi. Embryos were grown at 28.5M-bM-^@M-^S30M-BM-0C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200 colony forming units (CFU) of S. typhimurium SL1027, E. tarda or Mycobacteria marinum M20 bacteria into the caudal vein, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h or 4 days at 28M-BM-0C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.

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Macrophage-Expressed Perforins Mpeg1 and Mpeg1.2 Have an Anti-Bacterial Function in Zebrafish

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