Innate and adaptive immune responses of Arctic charr (Salvelinus alpinus, L.) during infection with Aeromonas salmonicida subsp. achromogenes and the effect of the AsaP1 toxin.

Aeromonas salmonicida subsp. achromogenes, the causative agent of atypical furunculosis in many fish species, secretes the toxic metalloendopeptidase AsaP1. This study aimed to analyze innate and adaptive immune parameters induced in Arctic charr (Salvelinus alpinus, L.) infected with wild type (wt) A. salmonicida subsp. achromogenes and its isogenic asaP1 deletion mutant (AsaP1-deficient). Head-kidney, liver and spleen were obtained from i.p. infected charr (wt, AsaP1-deficient), during a time schedule of 7 d post infection. Reverse transcription quantitative real-time PCR (RT-qPCR) was applied to study the expression of immune parameters: pro-inflammatory cytokines IL-1ß and TNF-α; anti-inflammatory cytokine IL-10; chemokines CXCL-8 (IL-8) and CC-chemokine; the cytokines IFN-γ and IL-4/13A as tracers for Th1 and Th2 immune responses, respectively; and the cell markers CD8α and CD83. In addition, lymphoid organs were histopathologically examined at days 3 and 7 post infection, including B (IgM) and T (CD3ε) cell staining. The detected immune responses were initially driven by innate mechanisms represented by the up-regulation of pro-inflammatory cytokines and chemokines and later on by adaptive Th2 related responses cumulating in B-cell recruitment as shown by regulation of immune parameters in spleen and head-kidney, with significant differences between mutant and wt infected fish. Histological sections revealed IgM-positive cells around ellipsoid arterioles in spleen, while CD3ε positive cells were found in clusters scattered all over the section. However, histopathological differences were only detected between infected and non-infected fish, but not between AsaP1-deficient mutant and wt infected fish. This work represents the first study on innate and adaptive immune responses of Arctic charr induced by a bacterial infection.

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.

Effects of Moritella viscosa antigens on pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) cell line (SHK-1).

Moritella viscosa is the causative agent of winter ulcer disease in salmonids reared in North-Atlantic countries. In this study the effects of selected M. viscosa antigens on cytotoxicity and pro-inflammatory gene expression in an Atlantic salmon (Salmo salar Linnaeus) macrophage-like cell line (SHK-1) were examined. SHK-1 cells were stimulated with live and heat-killed bacterial cells, extracellular products (ECP) and an extracellular vibriolysin, termed MvP1. Following incubation, cytotoxicity and expression levels of interleukin-1 beta (IL-1 beta) and interleukin-8 (IL-8) were examined at different time points. Both live M. viscosa cells and ECP were cytotoxic, but neither heat-killed cells, nor the MvP1 peptidase caused cell death. Expression levels of both IL-1 beta and IL-8 increased significantly after stimulation with live cells, but heat-killed cells only caused increased IL-8 expression. ECP did not affect IL-1 beta expression, but did stimulate IL-8 expression. The isolated MvP1 peptidase stimulated both IL-1 beta and IL-8 expression at the highest concentration tested. This study reveals a difference in the induction of pro-inflammatory gene expression in salmon SHK-1 cells between live and heat-killed M. viscosa cells, and also that an unknown secreted factor is the main stimulant of IL-beta and IL-8 expression.

Crystallization and preliminary X-ray diffraction studies of AsaP1_E294A and AsaP1_E294Q, two inactive mutants of the toxic zinc metallopeptidase AsaP1 from Aeromonas salmonicida subsp. achromogenes.

Two mutants of the toxic extracellular zinc endopeptidase AsaP1 (AsaP1_E294Q and AsaP1_E294A) of Aeromonas salmonicida subsp. achromogenes were expressed in Escherichia coli and crystallized by the vapour-diffusion method. Crystals were obtained using several precipitants and different protein concentrations. Protein crystals were found in a monoclinic (C2) as well as an orthorhombic (P2(1)2(1)2(1)) space group. The crystals belonging to the monoclinic space group C2 had unit-cell parameters a = 103.4, b = 70.9, c = 54.9 A, beta = 109.3 degrees for AsaP1_E294A, and a = 98.5, b = 74.5, c = 54.7 A, beta = 112.4 degrees for AsaP1_E294Q. The unit-cell parameters of the orthorhombic crystal obtained for AsaP1_E294A were a = 57.9, b = 60.2, c = 183.6 A. The crystals of the two different mutants diffracted X-rays beyond 2.0 A resolution.

Characterisation of an extracellular vibriolysin of the fish pathogen Moritella viscosa.

Moritella viscosa causes winter ulcer disease in salmonids. The aim of the present work was to isolate and partially characterise an extracellular peptidase from M. viscosa, and to study its role in virulence. The peptidase, termed MvP1, was a 38-kDa metallopeptidase produced in late exponential growth. The optimum temperature for MvP1 was 40 degrees C, but the enzyme was active over a broad range of temperatures. MvP1 was non-lethal to salmon at concentrations up to 0.22microg/g fish, but extracellular products were lethal to salmon. MvP1 degraded casein, gelatin and collagen from lumpfish skin. It caused considerable tissue necrosis and hemorrhages at the site of injection, and affected cell-cell adhesions in EPC and BF-2 cell lines, but was not highly cytotoxic. The peptidase partially degraded fish IgM heavy chain but was non-hemolytic. The mvp1 gene was sequenced and encoded a 734-aa polypeptide containing a signal sequence, an N-terminal propeptide, a mature peptidase domain and a C-terminal propeptide. The MvP1 propeptide undergoes both N-terminal and C-terminal processing and different C-terminal processing results in the formation of several active isoforms of the mature peptidase. The catalytic domain showed highest sequence similarity with several vibriolysins (EC 3.4.24.25) originating from Pseudoalteromonas strains, showing up to 80% aa identity. The results indicate that MvP1 is a previously unknown vibriolysin that might affect M. viscosa virulence by aiding in the invasion and dissemination of the bacterium in its host, by causing tissue destruction.

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.

Structural evidence of intramolecular propeptide inhibition of the aspzincin metalloendopeptidase AsaP1.

The Gram-negative bacterium Aeromonas salmonicida is a fish pathogen for various fish species worldwide. Aeromonas salmonicida subsp. achromogenes produces the extracellular, toxic zinc endopeptidase AsaP1. Crystal structure analyses at 2.0 Å resolution of two proteolytically inactive AsaP1 variants show the polypeptide folding of the protease domain and the propeptide domain. These first crystal structure analyses of a precursor of a deuterolysin-like aspzincin protease provide insights into propeptide function, and specific substrate binding. A lysine side chain of the propeptide binds in the hydrophobic S1'-pocket interacting with three carboxylate side chains. An AsaP1 variant with a lysine to alanine exchange identifies the chaperone function of the propeptide.

Toxoid construction of AsaP1, a lethal toxic aspzincin metalloendopeptidase of Aeromonas salmonicida subsp. achromogenes, and studies of its activity and processing.

AsaP1 is a toxic aspzincin metalloendopeptidase secreted by the fish pathogen Aeromonas salmonicida subsp. achromogenes. The protease is highly immunogenic and antibodies against AsaP1 evoke a passive protection against infection with A. salmonicida subsp. achromogenes. The protease is expressed as 37 kDa pre-pro-protein and processed to an active enzyme of 19kDa in A. salmonicida subsp. achromogenes. Recombinant expression of AsaP1(rec) in E. coli results in a protease of 22 kDa that is not secreted. AsaP1(rec) induces comparable pathological changes in Atlantic salmon (Salmo salar L.) to native AsaP1(wt). The aim of the study was to construct AsaP1 toxoids by exchanging catalytically important amino acids in the active site region of the protease. Four different AsaP1 mutants (AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A), and AsaP1(Y309F)) were successfully constructed by one step site directed mutagenesis, expressed in E. coli BL21 C43 as pre-pro-proteins and purified by His-tag affinity chromatography and gel filtration. Three of the resulting mutants (AsaP1(E294A), AsaP1(E294Q), and AsaP1(Y309A)) were not caseinolytic active and are detected as unprocessed pre-pro-proteins of 37 kDa. Caseinolytic active AsaP1(rec) and a mutant with reduced activity, AsaP1(Y309F), were processed to a size of 22 kDa. Furthermore, AsaP1(rec) is able to process the inactive mutants to the mature size of 22 kDa, allowing the conclusion that AsaP1 is autocatalytically processed. All four mutants AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A) and AsaP1(Y309F) are non-toxic in fish but induce a specific anti-AsaP1 antibody response in Arctic charr (Salvelinus alpinus L.) and are therefore true toxoids and possible vaccine additives.

Identification of type VI secretion systems in Moritella viscosa.

The study describes the identification of type VI secretion systems (T6SSs) in Moritella viscosa, the aetiological agent of winter ulcer disease. Despite the availability of commercial vaccines, M. viscosa causes significant financial losses in salmonid farming. The T6SS transports bacterial proteins from the cell into the environment or directly into host cells, and has been implicated with bacterial virulence. The aim of the study was to identify potential T6SSs in M. viscosa and to determine whether it possesses active T6S, providing further insight into the biology of the bacterium. The genome of M. viscosa 06/09/139 was screened for homology with known T6SS encoding genes. Two genetically distinct loci, termed Moritella Type Six Secretion 1 and 2 (mts1 and mts2), were identified as encoding putative T6SSs. Each locus contained known T6S core genes. The mts2 locus contained species specific genes, some of which have not previously been connected with T6S. The mts1 locus showed sequence homology and synteny to T6SSs of the fish pathogen Aliivibrio salmonicida and a non-pathogenic Moritella sp. PE36. The mts2 locus was more similar to a Vibrio parahaemolyticus T6SS. A functional T6SS was confirmed through identification of secreted Mts1-M, a hemolysin coregulated protein (Hcp) which is a part of the secretion system. Both virulent and avirulent M. viscosa isolates expressed two genes encoding Hcp, mts1-M and mts2-M. The results show that M. viscosa has a functional T6S, but the role of the secretion system and possible connections with virulence need further examination.

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.

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.

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.

Vaccination against atypical furunculosis and winter ulcer disease of fish.

Atypical furunculosis is a problem in farming of salmonids and various other fish species caused by a heterogeneous group of atypical Aeromonas salmonicida strains. Winter ulcer is a disease of salmonids and cod caused by Moritella viscosa, but a number of fish species are susceptible to the infection. Vaccines are available against atypical furunculosis of salmonids, but their efficacy is dependent on the characteristics of the infective strain. Vaccines for non-salmonid fish are currently not commercially available. Furunculosis vaccines for salmon can induce cross protection against some atypical A. salmonicida infections and only in some fish species. Polyvalent injection vaccines based on inactivated bacterial cells are available against winter ulcer disease of salmonids. Outbreaks of winter ulcer disease in vaccinated salmon are, however, continuously reported.