Project description:Piscirickettsia salmonis is an intracellular bacterial fish pathogen that causes piscirickettsiosis, a disease with highly adverse impact in the Chilean salmon farming industry. The development of effective treatment and control methods for piscireckttsiosis is still a challenge. To meet it the number of studies on P. salmonis has grown in the last couple of years but many aspects of the pathogen's biology are still poorly understood. Studies on its metabolism are scarce and only recently a metabolic model for reference strain LF-89 was developed. We present a new genome-scale model for P. salmonis LF-89 with more than twice as many genes as in the previous model and incorporating specific elements of the fish pathogen metabolism. Comparative analysis with models of different bacterial pathogens revealed a lower flexibility in P. salmonis metabolic network. Through constraint-based analysis, we determined essential metabolites required for its growth and showed that it can benefit from different carbon sources tested experimentally in new defined media. We also built an additional model for strain A1-15972, and together with an analysis of P. salmonis pangenome, we identified metabolic features that differentiate two main species clades. Both models constitute a knowledge-base for P. salmonis metabolism and can be used to guide the efficient culture of the pathogen and the identification of specific drug targets.
Project description:Piscirickettsia salmonis, a fastidious Gram-negative intracellular facultative bacterium, is the causative agent o Piscirickettsiosis. P. salmonis has broad host range with a nearly worldwide distribution, causing significant mortality. The molecular regulatory mechanisms of P. salmonis pathogenesis are relatively unknown, mainly due to its difficult in vitro culture and genomic differences between genogroups. Bacterial non-coding RNAs (ncRNAs) are important post-transcriptional regulators of bacterial physiology and virulence that are predominantly transcribed from intergenic regions (trans-acting) or antisense strand of open reading frames (cis-acting). The repertoire of ncRNAs present in the genome of P. salmonis and its possible role in bacterial physiology and pathogenesis are unknown. Here, we predicted and analyzed the core ncRNAs of P. salmonis base on structure and correlate this prediction to RNA sequencing data. We identified a total of 69 ncRNA classes related to tRNAs, rRNA, thermoregulators, antitoxins, ribozymes, riboswitches, miRNAs and antisense-RNAs. Among these ncRNAs, 29 classes of ncRNAs are shared between all P. salmonis genomes, constituting the core ncRNAs of P. salmonis. The ncRNA core of P. salmonis could serve to develop diagnostic tools and explore the role of ncRNA in fish pathogenesis.
Project description:Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.
Project description:Four large cryptic plasmids were identified in the salmon pathogen Piscirickettsia salmonis reference strain LF-89. These plasmids appeared highly novel, with less than 7% nucleotidic identity to the nr plasmid database. Plasmid copy number analysis revealed that they are harbored in chromosome equivalent ratios. In addition to plasmid-related genes (plasmidial autonomous replication, partitioning, maintenance, and mobilization genes), mobile genetic elements such as transposases, integrases, and prophage sequences were also identified in P. salmonis plasmids. However, bacterial lysis was not observed upon the induction of prophages. A total of twelve putative virulence factors (VFs) were identified, in addition to two global transcriptional regulators, the widely conserved CsrA protein and the regulator Crp/Fnr. Eleven of the putative VFs were overexpressed during infection in two salmon-derived cellular infection models, supporting their role as VFs. The ubiquity of these plasmids was also confirmed by sequence similarity in the genomes of other P. salmonis strains. The ontology of P. salmonis plasmids suggests a role in bacterial fitness and adaptation to the environment as they encode proteins related to mobilization, nutrient transport and utilization, and bacterial virulence. Further functional characterization of P. salmonis plasmids may improve our knowledge regarding virulence and mobile elements in this intracellular pathogen.
Project description:The aquaculture industry has confronted severe economic losses due to infectious diseases in the last years. Piscirickettsiosis or Salmonid Rickettsial Septicaemia (SRS) is the bacterial disease caused by Piscirickettsia salmonis. This Gram-negative, non-motile, cellular pathogen has the ability to infect, survive, replicate, and propagate in salmonid monocytes/macrophages generating a systemic infection characterized by the colonization of several organs including kidney, liver, spleen, intestine, brain, ovary and gills. In this study, we attempted to determine whether global gene expression differences can be detected in different genetic groups of Atlantic salmon as a result of Piscirickettsia salmonis infection. Moreover, we sought to characterize the fish transcriptional response in order to reveal the mechanisms that might confer resistance in Atlantic salmon to an infection with Piscirickettsia salmonis. In doing so, after challenging with Piscirickettsia salmonis, we selected the families with the highest (HS) and the lowest (LS) recorded susceptibility for gene expression analysis using 32K cGRASP microarrays. Our results revealed in LS families expression changes are linked to iron depletion, as well as, low contents of iron in kidney cells and low bacterial load, indicated that the iron-withholding strategy of innate immunity is part of the mechanism of resistance against Piscirickettsia salmonis. This information contributes to elucidate the underlying mechanisms of resistance to Piscirickettsia salmonis infection in Atlantic salmon and to identify new candidate genes for selective breeding programmes. Forty full-sibling families of Atlantic salmon (Salmo salar) were infected by intraperitoneal injection with 0.2 mL Piscirickettsia salmonis (PS889, isolated from Oncorhynchus kisutch, 1×104 PFU/mL). After forty days, the fishes were harvested and the cumulative mortality (dead fish / total fish) for each family was calculated. For the second challenge, the six families with the highest cumulative mortality levels were considered of relatively high susceptibility (HS) and the six families with the lowest cumulative mortality levels were considered of relatively low susceptibility (LS) to the infection. Five control and five infected fish from three HS and three LS families were analyzed. For each HS and LS family, pools of RNA from control and infected fish were prepared separately and were reverse transcribed. Four slides were for each used family hybridized including two dye-swaped slides. Labeled samples were hybridized on a 32K cDNA microarray, developed at the Consortium for Genomics Research on All Salmonids Project (cGRASP), GEO accession number: GPL8904.
Project description:Piscirickettsia salmons, the causative agent of piscirickettsiosis, is genetically divided into two genomic groups, named after the reference strains as LF-89-like or EM-90-like. Phenotypic differences have been detected between the P. salmonis genogroups, including antibiotic susceptibilities, host specificities and pathogenicity. In this study, we aimed to develop a rapid, sensitive and cost-effective assay for the differentiation of the P. salmonis genogroups. Using an in silico analysis of the P. salmonis 16S rDNA digestion patterns, we have designed a genogroup-specific assay based on PCR-restriction fragment length polymorphism (RFLP). An experimental validation was carried out by comparing the restriction patterns of 13 P. salmonis strains and 57 field samples obtained from the tissues of dead or moribund fish. When the bacterial composition of a set of field samples, for which we detected mixtures of bacterial DNA, was analyzed by a high-throughput sequencing of the 16S rRNA gene amplicons, a diversity of taxa could be identified, including pathogenic and commensal bacteria. Despite the presence of mixtures of bacterial DNA, the characteristic digestion pattern of the P. salmonis genogroups could be detected in the field samples without the need of a microbiological culture and bacterial isolation.
Project description:Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.
Project description:The attributes of the PCR allowed implementation of an assay for specific detection of Piscirickettsia salmonis from a few microliters of fish serum. This opens the way to less invasive modes of sampling for this microbial pathogen in salmonids.
Project description:Piscirickettsia salmonis is one of the main infectious diseases affecting coho salmon (Oncorhynchus kisutch) farming, and current treatments have been ineffective for the control of this disease. Genetic improvement for P. salmonis resistance has been proposed as a feasible alternative for the control of this infectious disease in farmed fish. Genotyping by sequencing (GBS) strategies allow genotyping of hundreds of individuals with thousands of single nucleotide polymorphisms (SNPs), which can be used to perform genome wide association studies (GWAS) and predict genetic values using genome-wide information. We used double-digest restriction-site associated DNA (ddRAD) sequencing to dissect the genetic architecture of resistance against P. salmonis in a farmed coho salmon population and to identify molecular markers associated with the trait. We also evaluated genomic selection (GS) models in order to determine the potential to accelerate the genetic improvement of this trait by means of using genome-wide molecular information. A total of 764 individuals from 33 full-sib families (17 highly resistant and 16 highly susceptible) were experimentally challenged against P. salmonis and their genotypes were assayed using ddRAD sequencing. A total of 9,389 SNPs markers were identified in the population. These markers were used to test genomic selection models and compare different GWAS methodologies for resistance measured as day of death (DD) and binary survival (BIN). Genomic selection models showed higher accuracies than the traditional pedigree-based best linear unbiased prediction (PBLUP) method, for both DD and BIN. The models showed an improvement of up to 95% and 155% respectively over PBLUP. One SNP related with B-cell development was identified as a potential functional candidate associated with resistance to P. salmonis defined as DD.
Project description: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.