Genetic and functional characterisation of irg1 and irg1-like, two genes which are highly expressed during bacterial infection in zebrafish (Danio rerio)

Abstract

We aimed to identify two novel immune responsive genes in zebrafish (Danio rerio) and subsequently characterise their expression patterns during the innate immune response to infection. Reverse transcriptase-PCR was used to clone the cDNA of two novel zebrafish genes, identified by differential microarray expression analysis, irg1 (ENSDART00000101982) and irg1-like (NM_001077607). The open reading frame of irg1 consists of 1161 basepairs, while the irg1-like open reading frame is 1961 basepairs long. The deduced amino acid sequence of each gene contained a single PrpD methylcitrate dehydratase domain which is highly conserved, implying an important function. We then used quantitative-PCR to verify the upregulation of these genes following bacterial infection with Salmonella enterica serovar typhimurium, confirming that these immune responsive genes are highly inducible by bacterial infection as detailed in the literature. Whole mount in situ hybridisation was used to characterise the expression of irg1 following hindbrain or yolk bacterial infection. Rapid (1 hour post infection) induction of irg1 expression was observed both in the aorta-gonad-mesonephros, a known haematopoietic region, and at the infection site. Over time the number of irg1-expresing cells in the aorta-gonadmesonephros decreased, and infection site expression became restricted to discrete regions of intense expression that persisted until at least 3 days post infection. The immune responsive migratory character of these irg1-expressing cells suggests they are macrophages. No irg1 expression was observed in control phosphate buffered saline injected larvae or in a tail wounding assay, demonstrating that irg1 expression is induced by bacterial infection, but not by sterile inflammation. We also investigated the requirement for irg1 and irg1-like during infection. Splice blocking morpholino oligonucleotide injection induced production of a truncated altered splicing product, and was sufficient to decrease survival rates in infected morphant larvae, implying that both irg1 and irg1-like are required to mount an appropriate innate immune response to bacterial infection. This study reports the first spatio-temporal characterisation of these two novel immune responsive genes in a whole animal vertebrate model of infection, and provides evidence which suggests irg1 is expressed by macrophages following infection, and that both irg1 and irg1-like are required during the innate immune response to bacterial infection.