Transcriptome response following administration of a live bacterial vaccine in Atlantic salmon (Salmo salar)

Abstract

Antibacterial responses have been studied in Atlantic salmon following an acute intra peritoneal injection of a genetically attenuated (aroA⁻) strain of Aeromonas salmonicida known to elicit protective immunity. Three tissues were studied for transcriptional changes, the liver, head kidney and the gill. RNA was collected from fish 6, 12, 24 and 48 h following infection or at the same time points from fish injected with PBS as non-infected control. PCR-select cDNA subtraction libraries were constructed from pooled 24 and 48 h post infection RNA to identify up-regulated mRNAs. One thousand four hundred and eighty six cDNA clones were sequenced from enriched cDNA libraries, of which 71% had significant homologies to known functional proteins. Many of these clones have previously been uncharacterised in Atlantic salmon. A salmonid cDNA microarray was used to further analyse the gene expression profile as the library construction in itself does not answer the dynamics of the response. The greatest increase in expression identified in the array analysis was a liver antibacterial peptide, hepcidin that was increased 11-fold following the challenge. A panel of clones were chosen for semiquantitative reverse transcriptase PCR from all time points sampled. These results indicated there were both temporal differences and tissue differences in the transcriptional response to bacterial exposure, potentially of relevance to the establishment of protection.

Introduction

Genomic approaches are enabling the analysis of complex genomic responses that may lead to both novel gene discovery and coordinated transcriptional responses. Nowhere is this information more relevant than in the inflammatory immune response. During the early stage of an infection the host needs to determine if the invading pathogen is bacterial, viral or parasitic and activate the relevant immune response accordingly (Janeway and Medzhitov, 2002). The immune response to pathogens by teleost fish have many common features with other higher vertebrates, but the ability of the fish to respond depends on the environmental conditions (Bols et al., 2001, Nikoskelainen et al., 2004) giving unique features to the lower vertebrate immune system. A particular difference is the dependence on the innate immune system at lower environmental temperatures experienced by fish (Le Morvan et al., 1998, Pylkko et al., 2002). Following infection a number of different defence mechanisms come into action, which include production of antibacterial peptides, the acute phase response and finally targeted killing of the pathogen which includes elements of the adaptive immune response. The acute phase response is characterised by the production of major plasma proteins to defend against the pathogen (Bayne et al., 2001). Such proteins have been studied in detail in salmonids and are known to include complement factors, serum amyloid A, transferrin, lectins, α microglobulin, haptaglobin and anti proteases (reviewed by Bayne and Gerwick, 2001). Lectins and their receptors (Soanes et al., 2004) are also produced in large quantities during the early infection to identify bacterial surfaces and aid neutralization of pathogens and foreign bodies in the host (Holmskov et al., 2003). In mammals, this response is stimulated by factors such as LPS, which triggers the production of proinflammatory cytokines including IL-1, IL-6 and TNFα that in turn are responsible for production of acute phase proteins (Mizel, 1989), similar mechanisms are thought to occur in fish. In parallel to the increase in acute phase proteins, there is a decrease in production of other “normal” plasma proteins such as apolipoproteins (Hoffman and Benditt, 1982).

Specific to the bacterial responses is the production of antibacterial peptides. These proteins are synthesised as preproproteins that are cleaved to produce a mature peptide that can bind to and kill invading bacterial pathogens (Boman, 2003). These proteins have been found in all animal and plant phyla to date and about 800 have so far been discovered (Bulet et al., 2004). There is a growing number of antibacterial peptides being characterised in fish, that include hepcidin (Douglas et al., 2003), cathelicidin (Chang et al., 2005) oncorhyncin (Fernandes et al., 2004), and liver expressed antimicrobial peptide 2 (Zhang et al., 2004) amongst others. Further antimicrobial peptides are reviewed by Patrzykat and Douglas (2003). Other enzymes that destroy invading bacteria include lysozymes (Mitra et al., 2003) and enzymes with lytic properties such as cathepsins (Tahtinen et al., 2002).

In recent years, the genomic information for Atlantic salmon has increased dramatically, with >110,000 EST sequences now publicly available (NCBI, 2005). However, there are still many differentially expressed genes not within the data bank. In this paper, we report on the construction and bioinformatics analysis of enriched cDNA libraries from liver, head kidney and gill tissues following exposure to a genetically attenuated strain of Aeromonas salmonicida (Marsden et al., 1996). In addition, the same RNA has been used to probe an Atlantic salmon cDNA micro array (Rise et al., 2004a, Rise et al., 2004b) to allow the expression in parallel of large numbers of mRNAs to be monitored. The array analysis served two purposes, firstly to confirm, using a limited number of genes cloned from the enriched libraries, that they are indeed up-regulated in response to bacterial exposure and secondly to observe expression of other genes not identified in our suppressive subtraction hybridization (SSH) study, as only a fraction of the libraries were sequenced. Lastly, a selected number of genes were chosen to perform temporal mRNA expression analysis following bacterial exposure, by reverse transcription-PCR. The strain of A. salmonicida used in this study is rapidly cleared from the host between 5 and 9 days following injection (Marsden et al., 1996) and elicits a protective immune response. Therefore, this study aims to identify molecules induced during the early stages of a bacterial infection of potential relevance to protective responses.