Dataset Information

Fish Liver Immune Response LC-MSMS

ABSTRACT: High-throughput proteomics was used to determine the role of the fish liver in defense responses to bacterial infection, done using a rainbow trout (Oncorhynchus mykiss) model following infection with Aeromonas salmonicida, the causative agent of furunculosis. The vertebrate liver has multifaceted roles in innate immunity, metabolism, and growth; we hypothesize this tissue serves a dual function in supporting host defense in parallel to metabolic adjustments that promote effective immune function. While past studies have reported mRNA responses to A. salmonicida in salmonids, the impact of bacterial infectionon the liver proteome remains uncharacterized in fish.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Oncorhynchus Mykiss (rainbow Trout) (salmo Gairdneri)

TISSUE(S): Liver

SUBMITTER: Dwight Causey

LAB HEAD: Daniel Macqueen

PROVIDER: PXD010186 | Pride | 2018-10-08

REPOSITORIES: Pride

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High-throughput proteomic profiling of the fish liver following bacterial infection.

Causey Dwight R DR Pohl Moritz A N MAN Stead David A DA Martin Samuel A M SAM Secombes Christopher J CJ Macqueen Daniel J DJ

BMC genomics 20181001 1

<h4>Background</h4>High-throughput proteomics was used to determine the role of the fish liver in defense responses to bacterial infection. This was done using a rainbow trout (Oncorhynchus mykiss) model following infection with Aeromonas salmonicida, the causative agent of furunculosis. The vertebrate liver has multifaceted functions in innate immunity, metabolism, and growth; we hypothesize this tissue serves a dual role in supporting host defense in parallel to metabolic adjustments that prom ...[more]

PMID: 30285610

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Project description:We evaluated the expression profiles of turbot in spleen, liver and head kidney across five temporal points of the Philasterides dicentrarchi infection process using an 8x15K Agilent oligo-microarray. The microarray included 2,176 different 5-fold replicated gene probes designed from a turbot 3’ sequenced EST database. We were able to identify 221 differentially expressed (DE) genes (8.1% of the whole microarray), 113 in spleen, 83 in liver and 90 in head kidney, in at least one of the 5 temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using GO terms (69.1%) after the additional sequencing of DE genes from the 5’ end. Many DE genes were related to innate and acquired immune functions. A high proportion of DE genes were organ-specific (70.6%), although their associated GO functions showed notable similarities in the three organs. The most striking difference in functional distribution was observed between the up- and down-regulated gene groups. Up-regulated genes were mostly associated to immune functions, while down-regulated ones mainly involved metabolism-related genes. Genetic response appeared clustered in a few groups of genes with similar expression profiles along the temporal series. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to P. dicentrarchi to achieve more resistant broodstocks for turbot industry. We were interested in analyzing the response of turbot as species to P. dicentrarchi in three of the main immune organs (spleen, liver and head kidney) along a temporal series representing the main episodes of infection (1d, 3d, 7d, 15d, 25d). Accordingly, individual samples were pooled at each sampling point to average interindividual variation. A single control point (time 0; non-injected) was used since a very slight effect of injection on gene expression was previously reported in these three organs in turbot. Five fish were sacrificed on day 0 to be used as the unique control in the experiment and one hundred fifty were challenged with a highly virulent strain of P. dicentrarchi as previously described (Paramá et al., 2003). Groups of five fish were sacrificed at 1d, 3d, 7d, 15d and 25d post-challenging. Equal amounts of spleen, liver and head kidney were sampled from each fish, pooled per organ at each sampling point and immediately stored in liquid nitrogen.

Project description:Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the macrophage responses to A. salmonicida grown in vivo and in vitro were compared. Enriched macrophages isolated from head kidney of Atlantic salmon (Salmo salar) were infected in vitro in 96-well microtitre dishes and changes in gene expression during the infection process were monitored using a custom Atlantic salmon cDNA microarray. A. salmonicida cultures grown in tryptic soy broth and in peritoneal implants were used to infect the macrophages. The macrophages were harvested at 0.5, 1.0 and 2.0 h after addition of the bacteria to the medium. Significant changes in gene expression were evident by microarray analysis at 2.0 h post-infection in macrophages infected with broth-grown and implant-grown bacteria; however, qPCR analysis revealed earlier up-regulation of JunB and TNF-alpha in macrophages exposed to the implant-grown bacteria. Up-regulation of those genes and others is consistent with the effects of extracellular products of aeromonad bacteria on macrophages and also suggests initiation of the innate immune response. Keywords: time course Enriched macrophages from 24 responder fish that showed positive respiratory burst in response to phorbol myristate acetate were plated in individual wells of 96-well flat-bottom polystyrene tissue culture plates. A. salmonicida were added to the macrophages, and incubated for 0.5, 1.0 or 2.0 h. Control wells received 10 ul of HBSS. Three replicate infections were performed for each type of bacteria. Hybridizations were carried out in duplicate, reversing the fluors for each sample on the second chip.

Project description:With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. A total number of 204 juvenile turbot were divided into 3 groups, two of them containing 72 fish and the last one 60 fish. Turbot were anaesthetized by immersion in 50 mg/ml buffered tricaine methanesulfonate (MS-222; Sigma) and then, fish from the first two groups were intramuscularly (i.m.) injected with 50 µl of PBS containing 2 µg of pMCV1.4 or pMCV1.4-G860. Turbot from the last batch were i.m. inoculated with 50 µl of PBS. At 8, 24 and 72 h after injection, 12 fish were removed from the first two tanks and, at 8 h after PBS inoculation, other 12 fish were taken from the last tank. These turbot were sacrificed by anaesthetic overdose and the head kidney was removed. Equal amounts of tissue from three fish belonging to the same tank and sampling point were pooled, obtaining 4 biological replicates for each treatment and time point (3 turbot/replicate). The remaining fish (36 in the plasmid-injected groups and 48 in the PBS-inoculated tank) were maintained during one month and then, 12 fish from the PBS injected group were separated to another tank. This new group of fish was intraperitoneally (i.p.) injected with 50 µl of MEM + penicillin and streptomycin + 2% FBS (PBS - MEM group), whereas the other turbot were i.p. infected with a dose of VHSV860 of 5 x 105 TCID50/fish (pMCV1.4 - VHSV and pMCV1.4-G860 - VHSV groups). At 8, 24 and 72 hours after infection, 12 fish were removed from the VHSV-infected tanks, and at 8 h after MEM injection the 12 fish were taken from the non-infected tank. The fish were sacrificed by anaesthetic overdose and the head kidney was removed. Equal amounts of tissue from three fish belonging to the same tank and sampling point were pooled, obtaining 4 biological replicates for treatment and time point (3 turbot/replicate)

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