RESUMO
Piscirickettsiosis is a fish disease caused by the facultative intracellular bacterium, Piscirickettsia salmonis. Even though entry routes of P. salmonis in fish are not fully clear yet, the skin seems to be the main portal in some salmonid species. Despite the importance of fish mucous skin barrier in fighting waterborne pathogens, the interaction between salmonid skin mucus and the bacterium is unknown. This study seeks to determine the in vitro changes in the growth of two Chilean P. salmonis strains (LF-89-like and EM-90-like genotypes) and the type strain LF-89T under exposures to skin mucus from Salmo salar and Oncorhynchus mykiss, as well as changes in the cytotoxic effect of P. salmonis on the SHK-1 cells following exposures. The results suggest that the growth of three P. salmonis strains was not significantly negatively affected under exposures to skin mucus (adjusted at 100 µg total protein ml-1 ) of O. mykiss (69 ± 18 U lysozyme ml-1 ) and S. salar (48 ± 33 U lysozyme ml-1 ) over time. However, the cytotoxic effect of P. salmonis, pre-exposed to salmonid skin mucus, on the SHK-1 cell line was reliably identified only towards the end of the incubation period, suggesting that the mucus had a delaying effect on the cytotoxic response of the cell line to the bacterium. These results represent a baseline knowledge to open new avenues of research intended to understand how P. salmonis faces the fish mucous skin barrier.
Assuntos
Muco/imunologia , Piscirickettsia/crescimento & desenvolvimento , Infecções por Piscirickettsiaceae/veterinária , Animais , Linhagem Celular , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Genótipo , Muco/microbiologia , Oncorhynchus mykiss/imunologia , Piscirickettsia/genética , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/microbiologia , Salmo salar/imunologia , Pele/imunologia , Pele/microbiologiaRESUMO
Early detection of piscirickettsiosis is an important purpose of government- and industry-based surveillance for the disease in Atlantic salmon farms in Chile. Real-time qPCRs are currently used for surveillance because bacterial isolation is inadequately sensitive or rapid enough for routine use. Since no perfect tests exist, we used Bayesian latent class models to estimate diagnostic sensitivity (DSe) and specificity (DSp) of qPCR and culture using separate two-test, single-population models for three farms (n = 148, 151, 44). Informative priors were used for DSp (culture (beta(999,1); qPCR (beta(98,2)), and flat priors (beta 1,1) for DSe and prevalence. Models were run for liver and kidney tissues combined and separately, based on the presence of selected gross-pathological signs. Across all models, qPCR DSe was 5- to 30-fold greater than for culture. Combined-tissue qPCR median DSe was highest in Farm 3 (sampled during P. salmonis outbreak (DSe = 97.6%)) versus Farm 1 (DSe = 85.6%) or Farm 2 (DSe = 83.5%), both sampled before clinical disease. Median DSe of qPCR was similar for liver and kidney, but higher when gross-pathological signs were evident at necropsy. High DSe and DSp and rapid turnaround-time indicate that the qPCR is fit for surveillance programmes and diagnosis during an outbreak. Targeted testing of salmon with gross-pathological signs can enhance DSe.
Assuntos
Doenças dos Peixes/diagnóstico , Piscirickettsia/isolamento & purificação , Infecções por Piscirickettsiaceae/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Salmo salar/microbiologia , Animais , Aquicultura , Técnicas Bacteriológicas , Teorema de Bayes , Chile , Doenças dos Peixes/microbiologia , Análise de Classes Latentes , Piscirickettsia/crescimento & desenvolvimento , Infecções por Piscirickettsiaceae/veterinária , Sensibilidade e EspecificidadeRESUMO
Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-economic impacts for Chilean salmonid aquaculture. The identification of major environmental reservoirs for P. salmonis has long been ignored. Most microbial life occurs in biofilms, with possible implications in disease outbreaks as pathogen seed banks. Herein, we report on an in vitro analysis of biofilm formation by P. salmonis Psal-103 (LF-89-like genotype) and Psal-104 (EM-90-like genotype), the aim of which was to gain new insights into the ecological role of biofilms using multiple approaches. The cytotoxic response of the salmon head kidney cell line to P. salmonis showed interisolate differences, depending on the source of the bacterial inoculum (biofilm or planktonic). Biofilm formation showed a variable-length lag-phase, which was associated with wider fluctuations in biofilm viability. Interisolate differences in the lag phase emerged regardless of the nutritional content of the medium, but both isolates formed mature biofilms from 288 h onwards. Psal-103 biofilms were sensitive to Atlantic salmon skin mucus during early formation, whereas Psal-104 biofilms were more tolerant. The ability of P. salmonis to form viable and mucus-tolerant biofilms on plastic surfaces in seawater represents a potentially important environmental risk for the persistence and dissemination of piscirickettsiosis.
Assuntos
Biofilmes/crescimento & desenvolvimento , Piscirickettsia/crescimento & desenvolvimento , Infecções por Piscirickettsiaceae/microbiologia , Animais , Aquicultura/métodos , Linhagem Celular , Chile , Doenças dos Peixes/microbiologia , Genótipo , Rim Cefálico/microbiologia , Piscirickettsia/genética , Salmo salar/microbiologiaRESUMO
Piscirickettsia salmonis is a facultative Gram-negative intracellular bacterium that produces piscirickettsiosis, disease that causes a high negative impact in salmonid cultures. The so-far-unidentified nutritional requirements have hindered its axenic culture at laboratory and industrial scales for the formulation of vaccines. The present study describes the development of a defined culture medium for P. salmonis. The culture medium was formulated through rational design involving auxotrophy test and statistical designs of experiments, considering the genome-scale metabolic reconstruction of P. salmonis reported by our group. The whole optimization process allowed for a twofold increase in biomass and a reduction of about 50% of the amino acids added to the culture medium. The final culture medium contains twelve amino acids, where glutamic acid, threonine and arginine were the main carbon and energy sources, supporting 1.65 g/L of biomass using 6.5 g/L of amino acids in the formulation. These results will contribute significantly to the development of new operational strategies to culture this bacterium for the production of vaccines.
Assuntos
Piscirickettsia/crescimento & desenvolvimento , Vacinas/imunologia , Meios de Cultura , Vacinas/metabolismoRESUMO
Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic infection of salmonid fish species. P. salmonis infects and survives in its host cell, a process that correlates with the expression of virulence factors including components of the type IVB secretion system. To gain further insights into the cellular and molecular mechanism behind the adaptive response of P. salmonis during host infection, we established an in vitro model of infection using the SHK-1 cell line from Atlantic salmon head kidney. The results indicated that in comparison to uninfected SHK-1 cells, infection significantly decreased cell viability after 10 days along with a significant increment of P. salmonis genome equivalents. At that time, the intracellular bacteria were localized within a spacious cytoplasmic vacuole. By using a whole-genome microarray of P. salmonis LF-89, the transcriptome of this bacterium was examined during intracellular growth in the SHK-1 cell line and exponential growth in broth. Transcriptome analysis revealed a global shutdown of translation during P. salmonis intracellular growth and suggested an induction of the stringent response. Accordingly, key genes of the stringent response pathway were up-regulated during intracellular growth as well as at stationary phase bacteria, suggesting a role of the stringent response on bacterial virulence. Our results also reinforce the participation of the Dot/Icm type IVB secretion system during P. salmonis infection and reveals many unexplored genes with potential roles in the adaptation to intracellular growth. Finally, we proposed that intracellular P. salmonis alternates between a replicative phase and a stationary phase in which the stringent response is activated.
Assuntos
Macrófagos/microbiologia , Piscirickettsia/metabolismo , Infecções por Piscirickettsiaceae/microbiologia , Salmão/microbiologia , Transcriptoma , Animais , Sistemas de Secreção Bacterianos , Linhagem Celular , Sobrevivência Celular , Citoplasma/microbiologia , Doenças dos Peixes/microbiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Genoma Bacteriano , Rim , Macrófagos/metabolismo , Piscirickettsia/genética , Piscirickettsia/crescimento & desenvolvimento , Piscirickettsia/patogenicidade , Fatores de VirulênciaRESUMO
Piscirickettsia salmonis is an intracellular γ-proteobacteria and the etiological agent of piscirickettsiosis, which causes massive economic losses in the Chilean salmon industry. The type IV pili (T4P) play an important role in adherence to host cell surfaces and bacterial pathogenicity. T4P contains a variable number of components, as predicted in P. salmonis genomes. However, no studies have determined if P. salmonis possesses T4P. The aims of this investigation were to identify T4P components in the P. salmonis type strain LF-89T, evaluate respective transcript expressions, and analyze the main putative T4P proteins using bioinformatics and proteomic approaches. Two main clusters of P. salmonis T4P genes were found. Expression of the pilA gene was upregulated at 4 h post-infection (hpi), while pilQ was upregulated 4 days post-infection. At 16 hpi, pilB and pilD were strongly upregulated. The PilA amino acid sequence analysis showed a conserved N-terminal domain and sequence motifs critical for T4P biosynthesis. MudPIT analysis revealed PilA in the P. salmonis LF-89T proteome, and TEM showed pili-like filamentous structures on the P. salmonis surface. These results strongly suggest the presence of a T4P-like structure in P. salmonis.
Assuntos
Fímbrias Bacterianas/metabolismo , Doenças dos Peixes/microbiologia , Piscirickettsia/metabolismo , Infecções por Piscirickettsiaceae/veterinária , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/genética , Genômica , Piscirickettsia/química , Piscirickettsia/genética , Piscirickettsia/crescimento & desenvolvimento , Infecções por Piscirickettsiaceae/microbiologia , Proteômica , Salmo salar/microbiologia , Alinhamento de SequênciaRESUMO
Piscirickettsia salmonis is a highly aggressive facultative intracellular bacterium that challenges the sustainability of Chilean salmon production. Due to the limited knowledge of its biology, there is a need to identify key molecular markers that could help define the pathogenic potential of this bacterium. We think a model system should be implemented that efficiently evaluates the expression of putative bacterial markers by using validated, stable, and highly specific housekeeping genes to properly select target genes, which could lead to identifying those responsible for infection and disease induction in naturally infected fish. Here, we selected a set of validated reference or housekeeping genes for RT-qPCR expression analyses of P. salmonis under different growth and stress conditions, including an in vitro infection kinetic. After a thorough screening, we selected sdhA as the most reliable housekeeping gene able to represent stable and highly specific host reference genes for RT-qPCR-driven P. salmonis analysis.
Assuntos
Proteínas de Bactérias/genética , Flavoproteínas/genética , Genes Bacterianos , Genes Essenciais , Piscirickettsia/genética , Piscirickettsia/patogenicidade , Reação em Cadeia da Polimerase em Tempo Real/normas , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Chile , DNA Girase/genética , DNA Girase/metabolismo , Primers do DNA/síntese química , Primers do DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Doenças dos Peixes/microbiologia , Doenças dos Peixes/patologia , Flavoproteínas/metabolismo , Expressão Gênica , Macrófagos/microbiologia , Piscirickettsia/crescimento & desenvolvimento , Piscirickettsia/metabolismo , Infecções por Piscirickettsiaceae/microbiologia , Infecções por Piscirickettsiaceae/patologia , Padrões de Referência , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Salmão/microbiologia , Fator sigma/genética , Fator sigma/metabolismoAssuntos
Doenças dos Peixes/microbiologia , Ferro/metabolismo , Piscirickettsia/metabolismo , Sideróforos/biossíntese , Animais , Compostos Férricos/química , Nitratos/química , Oncorhynchus kisutch , Oncorhynchus mykiss , Piscirickettsia/crescimento & desenvolvimento , Infecções por Piscirickettsiaceae , Compostos de Amônio Quaternário/químicaRESUMO
Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, a disease that seriously affects the salmonid industry. Despite efforts to genomically characterize P. salmonis, functional information on the life cycle, pathogenesis mechanisms, diagnosis, treatment, and control of this fish pathogen remain lacking. To address this knowledge gap, the present study conducted an in silico pan-genome analysis of 19 P. salmonis strains from distinct geographic locations and genogroups. Results revealed an expected open pan-genome of 3,463 genes and a core-genome of 1,732 genes. Two marked genogroups were identified, as confirmed by phylogenetic and phylogenomic relationships to the LF-89 and EM-90 reference strains, as well as by assessments of genomic structures. Different structural configurations were found for the six identified copies of the ribosomal operon in the P. salmonis genome, indicating translocation throughout the genetic material. Chromosomal divergences in genomic localization and quantity of genetic cassettes were also found for the Dot/Icm type IVB secretion system. To determine divergences between core-genomes, additional pan-genome descriptions were compiled for the so-termed LF and EM genogroups. Open pan-genomes composed of 2,924 and 2,778 genes and core-genomes composed of 2,170 and 2,228 genes were respectively found for the LF and EM genogroups. The core-genomes were functionally annotated using the Gene Ontology, KEGG, and Virulence Factor databases, revealing the presence of several shared groups of genes related to basic function of intracellular survival and bacterial pathogenesis. Additionally, the specific pan-genomes for the LF and EM genogroups were defined, resulting in the identification of 148 and 273 exclusive proteins, respectively. Notably, specific virulence factors linked to adherence, colonization, invasion factors, and endotoxins were established. The obtained data suggest that these genes could be directly associated with inter-genogroup differences in pathogenesis and host-pathogen interactions, information that could be useful in designing novel strategies for diagnosing and controlling P. salmonis infection.
Assuntos
Genes Bacterianos/genética , Genoma Bacteriano/genética , Genótipo , Piscirickettsia/genética , Animais , Proteínas de Bactérias/genética , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Ontologia Genética , Tamanho do Genoma , Interações Hospedeiro-Patógeno , Cinética , Redes e Vias Metabólicas/genética , Óperon , Filogenia , Piscirickettsia/crescimento & desenvolvimento , Piscirickettsia/isolamento & purificação , Piscirickettsia/patogenicidade , Infecções por Piscirickettsiaceae/microbiologia , Infecções por Piscirickettsiaceae/veterinária , Fatores de Virulência/genética , Sequenciamento Completo do GenomaRESUMO
Piscirickettsia salmonis is an intracellular bacterium and the causative agent of Piscirickettsiosis, a disease responsible for considerable mortalities in the Chilean salmon farming industry. Currently, P. salmonis protein translocation across the membrane and the mechanisms by which virulence factors are delivered to host cells are poorly understood. However, it is known that Gram-negative bacteria possess several mechanisms that transport proteins to the periplasmic and extracellular compartments. The aim of this study was to evaluate the expressional changes of several genes in the P. salmonis Sec-dependent pathway and type 4B secretion system during in vitro infection. Genes homologous and the main proteins belonging to Sec-dependent pathway and Type 4 Dot/Icm secretion system were found in the genome and proteome of P. salmonis AUSTRAL-005 strain. Additionally, several genes of these protein transport mechanisms were overexpressed during in vitro P. salmonis infection in SHK-1 cell line. The obtained data indicate that the Sec-dependent pathway and Type 4B secretion system are biologically active during P. salmonis infection. These mechanisms could contribute to the recycling of proteins into the inner and outer bacterial membrane and in translocate virulence factors to infected cell, which would favor the structural integrity and virulence of this bacterium.
Assuntos
Perfilação da Expressão Gênica , Piscirickettsia/crescimento & desenvolvimento , Piscirickettsia/genética , Sistemas de Secreção Tipo IV/biossíntese , Sistemas de Secreção Tipo IV/genética , Animais , Linhagem Celular , Células Epiteliais/microbiologia , Genômica , Proteômica , SalmãoRESUMO
Piscirickettsia salmonis is the etiological agent of piscirickettsiosis, which, as the main systemic disease in the Chilean salmon industry, causes significant economic losses. This bacterium can produce biofilm as a persistence and survival strategy in adverse conditions. In other bacteria, cheA is a key gene for modulating the onset of bacterial chemotaxis, as well as having a secondary role in biofilm production. Notwithstanding this association, the potential relationships between biofilm formation and genes involved in P. salmonis chemotaxis are poorly understood. This study aimed to determine P. salmonis cheA gene expression when grown in different culture media known to induce biofilm production. Piscirickettsia salmonis AUSTRAL-005 produced moderate/high biofilm levels after 144 h of incubation in the AUSTRAL-SRS and marine broths. In contrast, LF-89 biofilm production was weak/nonexistent in the aforementioned broths. Both assessed P. salmonis strains contained the cheYZA operon. Additionally, AUSTRAL-005 cheA transcripts increased in both culture media. In conclusion, these results suggest potential relationships between biofilm formation and genes related to chemotaxis in the fish pathogen P. salmonis.
Assuntos
Quimiotaxia/genética , Regulação Bacteriana da Expressão Gênica/genética , Óperon/genética , Piscirickettsia/genética , Animais , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Quimiotaxia/fisiologia , Meios de Cultura/química , Doenças dos Peixes/microbiologia , Peixes/microbiologia , Genes Bacterianos/genética , Proteínas Quimiotáticas Aceptoras de Metil/genética , Proteínas Quimiotáticas Aceptoras de Metil/fisiologia , Microscopia Eletrônica de Varredura , Piscirickettsia/crescimento & desenvolvimento , Piscirickettsia/patogenicidade , Infecções por Piscirickettsiaceae/microbiologia , Virulência/genética , Virulência/fisiologiaRESUMO
AIMS: To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally. METHODS AND RESULTS: Specific IgY was produced by immunizing hens with P. salmonis proteins. The IgY was obtained from egg yolks using the ammonium sulphate precipitation method and it was characterized by SDS-PAGE, Western-blot and ELISA, demonstrating that anti-P. salmonis IgY strongly reacted specifically against P. salmonis proteins. In an in vitro neutralization assay, IgY inhibited the growth of P. salmonis in liquid medium at concentrations ranging from 128 to 256 µg ml(-1) in a dose-dependent manner. Interestingly, IgY against P. salmonis also generates a strong protective effect on the infection of P. salmonis in salmon head kidney-1 cells. In addition, the bacteriostatic function of IgY appears to result possibly from agglutination by the interaction of IgY with surface components of the pathogen. Finally, to confirm this IgY as an alternative for salmonid treatment, Atlantic salmon (Salmo salar) specimens were orally inoculated with IgY. The analysis of the sera demonstrates that IgY was effectively transported by fish intestine and that this immunoglobulins maintains its properties and recognizes several proteins of P. salmonis up to 12 h after inoculation of IgY against P. salmonis. CONCLUSIONS: Specific IgY effectively inhibited the growth of P. salmonis and this immunoglobulin can be released in the Atlantic salmon sera when administered orally to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that this specific IgY against this fastidious micro-organism could be a useful strategy for the treatment of piscirickettsiosis.
Assuntos
Antibacterianos/farmacologia , Gema de Ovo/química , Doenças dos Peixes/microbiologia , Imunoglobulinas/farmacologia , Piscirickettsia/efeitos dos fármacos , Infecções por Piscirickettsiaceae/veterinária , Animais , Antibacterianos/isolamento & purificação , Galinhas/imunologia , Eletroforese em Gel de Poliacrilamida , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/imunologia , Imunoglobulinas/isolamento & purificação , Piscirickettsia/crescimento & desenvolvimento , Infecções por Piscirickettsiaceae/tratamento farmacológico , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/microbiologia , Salmo salar/microbiologiaRESUMO
Piscirickettsia salmonis is the bacterium that causes Piscirickettsiosis, a systemic disease of salmonid fish responsible for significant economic losses within the aquaculture industry worldwide. The growth of the bacterium for vaccine formulation has been traditionally accomplished by infecting eukaryotic cell lines, a process that involves high production costs and is time-consuming. Recent research has demonstrated that it is possible to culture pure P. salmonis in a blood containing (cell-free) medium. In the present work we demonstrate the growth of P. salmonis in a liquid medium free from blood and serum components, thus establishing a novel and simplified bacteriological medium. Additionally, the new media reported provides improved growth conditions for P. salmonis, where biomass concentrations of approximately 800 mg cell dry weight L(-1) were obtained, about eight times higher than those reported for the blood containing medium. A 2- level full factorial design was employed to evaluate the significance of the main medium components on cell growth and an optimal temperature range of 23-27°C was determined for the microorganism to grow in the novel liquid media. Therefore, these results represent a breakthrough regarding P. salmonis research in order to optimize pure P. salmonis growth in liquid blood and serum free medium.
Assuntos
Doenças dos Peixes/microbiologia , Piscirickettsia/crescimento & desenvolvimento , Infecções por Piscirickettsiaceae/veterinária , Animais , Linhagem Celular , Meios de Cultura Livres de Soro , Técnicas de Cultura , Concentração de Íons de Hidrogênio , Piscirickettsia/patogenicidade , Infecções por Piscirickettsiaceae/microbiologia , Salmonidae/microbiologia , TemperaturaRESUMO
Piscirickettsia salmonis is a bacterial fish pathogen seriously threatening the sustainability of the Chilean salmon industry. The biology and life cycle of this bacterium is not completely understood and there are no reports explaining how it survives or persists in marine environments. This work provides descriptive data of P. salmonis behavior when it is exposed to stress conditions, producing large cell aggregates closely resembling typical biofilm structures. In order to track this putative biofilm, we used indirect fluorescence and scanning electron microscopy. Complex masses were observed over time; the bacteria appear to be embedded within a matrix which disappears when it is exposed to cellulase, suggesting a polysaccharide nature typical of biofilm formation. Two lectins (ConA and WGA) were used to characterize the matrix. Both lectins showed a strong reaction with the structure, validating the exopolysaccharide nature of the matrix. Recently, several studies have demonstrated a correlation between toxin/anti-toxin system expression at initial stages of biofilm formation. In this report, QRT-PCR analysis was used with the P. salmonis toxin/anti-toxin mazEF operon, showing induction of these genes at early stages of biofilm formation, suggesting that said formation may be an adaptive strategy for survival and persistence under stress conditions in marine environments.
Assuntos
Biofilmes/crescimento & desenvolvimento , Piscirickettsia/fisiologia , Estresse Fisiológico , Toxinas Bacterianas/biossíntese , Celulase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Perfilação da Expressão Gênica , Lectinas/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Dados de Sequência Molecular , Piscirickettsia/crescimento & desenvolvimento , Polissacarídeos Bacterianos/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
This is the first report of a functional toxin-antitoxin (TA) locus in Piscirickettsia salmonis. The P. salmonis TA operon (ps-Tox-Antox) is an autonomous genetic unit containing two genes, a regulatory promoter site and an overlapping putative operator region. The ORFs consist of a toxic ps-Tox gene (P. salmonis toxin) and its upstream partner ps-Antox (P. salmonis antitoxin). The regulatory promoter site contains two inverted repeat motifs between the -10 and -35 regions, which may represent an overlapping operator site, known to mediate transcriptional auto-repression in most TA complexes. The Ps-Tox protein contains a PIN domain, normally found in prokaryote TA operons, especially those of the VapBC and ChpK families. The expression in Escherichia coli of the ps-Tox gene results in growth inhibition of the bacterial host confirming its toxicity, which is neutralized by coexpression of the ps-Antox gene. Additionally, ps-Tox is an endoribonuclease whose activity is inhibited by the antitoxin. The bioinformatic modelling of the two putative novel proteins from P. salmonis matches with their predicted functional activity and confirms that the active site of the Ps-Tox PIN domain is conserved.