Bjarnheidur K. Gudmundsdottir

Characterisation of cathelicidin gene family members in divergent fish species

Cathelicidins are antimicrobial peptides, well studied in mammals and found to be multifunctional proteins, important in the fight against bacterial invasion. Cathelicidins in fish have only recently been identified and little is known about their function and importance in the immune system of fish. In this study we have identified several novel cathelicidin proteins in far related fish species such as Atlantic cod (Gadus morhua) and Arctic charr (Salvelinus alpinus). Atlantic cod was found to have at least three cathelicidin genes of which two are nearly identical except for a nine-amino acid duplication in the antimicrobial peptide region. The predicted mature peptides of cod were found to be unusual peptides, made mainly of arginine, glycine and serine (RGS) residues and form a novel class of antimicrobial peptides. Cathelicidin in Arctic charr and brook trout (Salvelinus fontinalis) were found to have an exon deletion in the cathelin region of the protein, which would lead to the deletion of the predicted loop 2 of cathelin and its adjacent beta-strands. This is the first report of a deletion of a whole exon in the family of the cathelicidins. Infection of fish with pathogenic bacteria caused an upregulation of the expression of the cathelicidins in Arctic charr and Atlantic cod and indicates a role of these proteins in fish innate immunity.

A comparison of total and specific immunoglobulin levels in healthy Atlantic salmon (Salmo salar L.) and in salmon naturally infected with Aeromonas salmonicida subsp. achromogenes

Healthy Atlantic salmon and salmon with a history of chronic natural Aeromonas salmonicida subsp. achromogenes infection were compared with respect to total serum protein and the concentration and specificity of serum immunoglobulin. The immunoglobulin level was measured using competitive ELISA and the specific antibody activity against Aeromonas salmonicida subsp. achromogenes was measured using double sandwich ELISA. Significant elevation of serum protein and immunoglobulin concentration was observed in the infected salmon compared with the healthy fish. This was accompanied by weak anti-A. salmonicida activity in the infected fish which seemed to contribute to the raised immunoglobulin level to only a limited degree.

Pathogenicity of atypical Aeromonas salmonicida in Atlantic salmon compared with protease production

The pathogenicity of extracellular products (ECPs) from 24 atypical Aeromonas salmonicida strains was studied with respect to  : lethality in Atlantic salmon, pathogenic effect in muscle, haemolytic activity, cytotoxicity in two fish cell lines and proteolytic activities. Furthermore, the relationship between lethality of ECPs and mortality caused by bacterial challenge was examined. Correlation was demonstrated between the pathogenic properties and proteolytic activities of the ECPs. Cytolytic (GCAT) activity comparable with that of the typical reference strain used (NCMB 1102) was not detected in ECPs of any of the atypical strain tested. An extracellular metallo‐caseinase, AsaP1, was linked with lethal toxicity and a strong pathogenic effect. Furuncular‐like lesions were produced by ECPs containing AsaP1 activity. One strain produced a lethal toxin which was neither caseinolytic nor with GCAT comparable activity. The examined atypical strains form at least three distinct groups based on different virulence mechanisms and extracellular proteases.

Study of the humoral response of Atlantic salmon (Salmo salar L.), naturally infected with Aeromonas salmonicida ssp. achromogenes

The humoral antibody response of healthy Atlantic salmon and of two groups of salmon, naturally infected with Aeromonas salmonicida ssp. achromogenes, was examined in some detail. One diseased group was chronically infected and the other recently infected. It was found that the humoral response of these two infected groups was quite different. The chronically infected fish showed poor specific response to the causative agent whereas the recently infected salmon produced strong specific antibody response. The chronically infected fish showed evidence of increased unspecific response including an elevated level of natural antibodies. The specific humoral response of the recently infected fish was primarily directed against two cell-associated antigens of the A. salmonicida ssp. achromogenes bacterium, the A-layer protein and the o-polysaccharide component of LPS. In the chronically infected fish the humoral response was primarily directed against the A-layer protein.

Natural antibodies of cod (Gadus morhua L.): specificity, activity and affinity.

Natural antibodies are present in the serum of vertebrates regardless of antigenic stimulation. Characteristic activity is commonly detected against haptenated proteins, single stranded DNA and thyroglobulin. Natural antibodies are believed to provide an instant protection against pathogens of a broad specificity and to participate in homeostasis. Cod is a poor antibody responder but shows a relatively high level of natural antibodies against haptenated proteins. In this project the specificity, activity and affinity of natural antibodies was studied in different groups of cod and the effects of age/size, environmental temperature, immunisation and infection examined. Antigen driven selection of natural antibodies was also studied in one group of cod. The results demonstrated a broad and yet characteristic specificity, primarily directed against haptenated proteins and possible food antigens. The antibody activity increased with increasing age and at higher temperature whereas immunostimulation by immunisation or infection resulted in variable response. The affinity index of natural antibodies of cod generally did not correlate with changes in the antibody activity but it was in the same range as the affinity index of acquired cod antibodies and that of some mammalian monoclonal acquired antibodies. Analysis of antigen driven antibody selection showed that the natural antibody repertoire of individual cod was heterogeneous with respect to its affinity for haptenated protein.

Isolation of putative probionts from cod rearing environment.

Survival problems are encountered at early stages of intensive fish rearing and antibiotics are widely used to remedy the situation. Probiotics may provide a potential alternative method to protect larvae from opportunistic and pathogenic bacteria and promote a balanced environment. This study was designed to search for new probiotics to target this critical period in cod rearing. Potential probionts were selected from the natural microbiota of cod aquacultural environment. The selection was based on several criteria: pathogen inhibition potential, growth characteristics, strain identification, metabolite production and adhesion to fish cell lines. Our study demonstrated that 14% of screened bacteria (n=188) had antagonistic properties towards fish pathogens. The majority of these isolates were Gram-positive (81%), belonging to Firmicutes (69.2%) and Actinobacteria (11.5%) phyla based on 16S rRNA gene sequencing. Only 6 (3.2%) of 188 isolates could inhibit all three pathogens tested: Vibrio anguillarum, Aeromonas salmonicida subsp. achromogenes and Vibrio salmonicida. Differences observed in activity intensity and spectrum among inhibitory isolates emphasise the need to develop probiotic mixtures for efficient prophylactic methods. Comparison of growth behaviour of inhibitory isolates and pathogens at cod rearing temperatures, metabolite production and adhesion capacity were considered for final probiont selection. Four promising isolates that could be used as a mixed supplement to rearing water were identified as putative probiotic bacteria. This study emphasises the importance and potential of lactic acid bacteria in aquaculture.

Effects of bacterial treatment at early stages of Atlantic cod (Gadus morhua L.) on larval survival and development

Aims:  To assess the effects of bacterial treatment at the earliest stages of cod rearing on the microbial load, larval development and performance, testing three bacterial strains (Carnobacterium divergens V41, Arthrobacter sp. and Enterococcus sp.) in vivo that were previously shown to have inhibitory potential towards fish pathogens in vitro.

Functional characterization of codCath, the mature cathelicidin antimicrobial peptide from Atlantic cod (Gadus morhua).

Cathelicidins are among the best characterized antimicrobial peptides and have been shown to have an important role in mammalian innate immunity. We recently isolated a novel mature cathelicidin peptide (codCath) from Atlantic cod and in the present study we functionally characterized codCath. The peptide demonstrated salt sensitivity with abrogation of activity at physiological salt concentrations. In low ionic strength medium we found activity against marine and non-marine Gram-negative bacteria with an average MIC of 10 μM, weak activity against a Gram-positive bacterium (MIC 80 μM), and pronounced antifungal activity (MIC 2.5 μM). The results suggest the kinetics and mode of action of codCath to be fast killing accompanied by pronounced cell lysis. Extracellular products (ECPs) of three marine bacteria caused breakdown of the peptide into smaller fragments and the cleaved peptide lost its antibacterial activity. Proteolysis of the peptide on the other hand was abolished by prior heat-treatment of the ECPs, suggesting a protease involvement. We observed no cytotoxicity of the peptide in fish cells up to a concentration of 40 μM and the selectivity of activity was confirmed with bacterial and mammalian membrane mimetics. We conclude that the potent broad-spectrum activity of codCath hints at a role of the peptide in cod immune defense.

Quorum sensing in Aeromonas salmonicida subsp. achromogenes and the effect of the autoinducer synthase AsaI on bacterial virulence

The Gram-negative fish pathogenic bacterium Aeromonas salmonicida possesses the LuxIR-type quorum sensing (QS) system, termed AsaIR. In this study the role of QS in A. salmonicida subsp. achromogenes virulence and pigment production was investigated. Five wild-type Asa strains induced the N-acyl-homoserinelactone (AHL) monitor bacteria. HPLC-HR-MS analysis identified only one type of AHL, N-butanoyl-L-homoserine lactone (C4-HSL). A knock out mutant of AsaI, constructed by allelic exchange, did not produce a detectable QS signal and its virulence in fish was significantly impaired, as LD(50) of the AsaI-deficient mutant was 20-fold higher than that of the isogenic wt strain and the mean day to death of the mutant was significantly prolonged. Furthermore, the expression of two virulence factors (a toxic protease, AsaP1, and a cytotoxic factor) and a brown pigment were reduced in the mutant. AsaP1 production was inhibited by synthetic QS inhibitors (N-(propylsulfanylacetyl)-L-homoserine lactone; N-(pentylsulfanylacetyl)-L-homoserine lactone; and N-(heptylsulfanylacetyl)-L-homoserine lactone) at concentrations that did not affect bacterial growth. It is a new finding that the AHL synthase of Aeromonas affects virulence in fish and QS has not previously been associated with A. salmonicida infections in fish. Furthermore, AsaP1 production has not previously been shown to be QS regulated. The simplicity of the A. salmonicida subsp. achromogenes LuxIR-type QS system and the observation that synthetic QSI can inhibit an important virulence factor, AsaP1, without affecting bacterial growth, makes A. salmonicida subsp. achromogenes an interesting target organism to study the effects of QS in disease development and QSI in disease control.

The AsaP1 Peptidase of Aeromonas salmonicida subsp. achromogenes Is a Highly Conserved Deuterolysin Metalloprotease (Family M35) and a Major Virulence Factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic Delta asaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The Delta asaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.