Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis, appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3, ymt, pipB2 and pepO, were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen.

Dual Transcriptomics of Host-Pathogen Interaction of Cystic Fibrosis Isolate Pseudomonas aeruginosa PASS1 With Zebrafish.

Pseudomonas aeruginosa is a significant cause of mortality in patients with cystic fibrosis (CF). To explore the interaction of the CF isolate P. aeruginosa PASS1 with the innate immune response, we have used Danio rerio (zebrafish) as an infection model. Confocal laser scanning microscopy (CLSM) enabled visualization of direct interactions between zebrafish macrophages and P. aeruginosa PASS1. Dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the host during P. aeruginosa infection. Following establishment of infection in zebrafish embryos with PASS1, 3 days post infection (dpi), there were 6739 genes found to be significantly differentially expressed in zebrafish and 176 genes in PASS1. A range of virulence genes were upregulated in PASS1, including genes encoding pyoverdine biosynthesis, flagellin, non-hemolytic phospholipase C, proteases, superoxide dismutase and fimbrial subunits. Additionally, iron and phosphate acquisition genes were upregulated in PASS1 cells in the zebrafish. Transcriptional changes in the host immune response genes highlighted phagocytosis as a key response mechanism to PASS1 infection. Transcriptional regulators of neutrophil and macrophage phagocytosis were upregulated alongside transcriptional regulators governing response to tissue injury, infection, and inflammation. The zebrafish host showed significant downregulation of the ribosomal RNAs and other genes involved in translation, suggesting that protein translation in the host is affected by PASS1 infection.

Francisella noatunensis subspecies noatunensis clpB deletion mutant impairs development of francisellosis in a zebrafish model.

BACKGROUND: Francisella noatunensis ssp. noatunensis (F.n.n.) is the causative agent of francisellosis in Atlantic cod and constitutes one of the main challenges for future aquaculture on this species. A facultative intracellular bacterium like F.n.n. exert an immunologic challenge against which live attenuated vaccines in general are most effective. Thus, we constructed a deletion in the F.n.n. clpB gene as ΔclpB mutants are among the most promising vaccine candidates in human pathogenic Francisella. PURPOSE: Characterization of F.n.n. ΔclpB using primary Atlantic cod head kidney leukocytes, the zebrafish embryo and adult zebrafish model with focus on potential attenuation, relevant immune responses and immunogenic potential. MAIN RESULTS: Interleukin 1 beta transcription in Atlantic cod leukocytes was significantly elevated from 24 to 96 h post infection with F.n.n. ΔclpB compared to F.n.n. wild-type (wt). Growth attenuation of the deletion mutant in zebrafish embryos was observed by fluorescence microscopy and confirmed by genome quantification by qPCR. In the immunization experiment, adult zebrafish were immunized with 7 × 106 CFU of F.n.n. ΔclpB before challenge four weeks later with 6 × 108 CFU of F.n.n. wt. One day after challenge, immunized zebrafish responded with significantly lower interleukin 8 levels compared to the non-immunized control. Immunized fish were protected against the acute mortality observed in non-immunized zebrafish after challenge and bacterial genomes quantified by qPCR were reduced to a minimum 28 days post challenge, indicating protective immunity stimulated by F.n.n. ΔclpB. CONCLUSION: Deletion mutation of clpB in F.n.n. causes in vitro and in vivo attenuation and elicits a protective immune response in adult zebrafish against a lethal dose of F.n.n. wt. Taken together, the results presented increases the knowledge on protective immune responses against F.n.n.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

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.

Immunomodulatory properties of Concholepas concholepas hemocyanin against francisellosis in a zebrafish model.

The development of vaccines for aquaculture has been an important milestone in providing a continuous and sustainable production. Most of the vaccines currently on the market for aquaculture include oil as adjuvant. Nevertheless, several studies reported an occurrence of side effects after their use in farmed fish. As a result, there is a need for new and improved adjuvants that can stimulate the immune system while causing as few side-effects as possible. Hemocyanins are versatile macromolecules with strong immunogenic and immunomodulatory properties. Due to these characteristics, hemocyanin from Concholepas concholepas (CCH) has been biochemically characterized and evaluated as vaccine adjuvant in mice and humans. Francisellosis is a chronic granulomatous disease, which can result in high mortality depending on the host. The disease is caused by the facultative intracellular Gram-negative bacteria Francisella noatunensis, which remains an unsolved problem for the aquaculture, as no efficient vaccines are available. The aim of the present work was to investigate the immunoregulatory properties of CCH against francisellosis in an experimental zebrafish model. When immunized with CCH, zebrafish were protected from subsequent challenge with a lethal dose of Francisella noatunensis subsp. orientalis. Subsequently the mRNA expression levels of several immune-related genes were studied, including mhcii, il12a, tnfα and ifng1-1. Taken together, the data report the immunoregulatory properties of CCH and its potential use as a vaccine adjuvant for aquaculture.

Characterization and Vaccine Potential of Membrane Vesicles Produced by Francisella noatunensis subsp. orientalis in an Adult Zebrafish Model.

Vaccine development against extracellular bacteria has been important for the sustainability of the aquaculture industry. In contrast, infections with intracellular pathogens remain largely an unresolved problem. Francisella noatunensis subsp. orientalis is a Gram-negative, facultative intracellular bacterium that causes the disease francisellosis in fish. Francisellosis is commonly characterized as a chronic granulomatous disease with high morbidity and can result in high mortality depending on the host. In this study, we explored the potential of bacterial membrane vesicles (MVs) as a vaccine agent against F. noatunensis subsp. orientalis Bacterial MVs are spherical structures naturally released from the membrane of bacteria and are often enriched with selected bacterial components such as toxins and signaling molecules. MVs were isolated from broth-cultured F. noatunensis subsp. orientalis in the present work, and proteomic analysis by mass spectrometry revealed that MVs contained a variety of immunogenic factors, including the intracellular growth proteins IglC and IglB, known to be part of a Francisella pathogenicity island (FPI), as well as outer membrane protein OmpA, chaperonin GroEL, and chaperone ClpB. By using flow cytometry and electron microscopy, we observed that F. noatunensis subsp. orientalis mainly infects myelomonocytic cells, both in vivo and in vitro Immunization with MVs isolated from F. noatunensis subsp. orientalis protects zebrafish from subsequent challenge with a lethal dose of F. noatunensis subsp. orientalis To determine if MVs induce a typical acute inflammatory response, mRNA expression levels were assessed by quantitative real-time PCR. Expression of tnfa, il1b, and ifng, as well as mhcii, mpeg1.1, and ighm, was upregulated, thus confirming the immunogenic properties of F. noatunensis subsp. orientalis-derived MVs.

Francisella noatunensis ssp. noatunensis iglC deletion mutant protects adult zebrafish challenged with acute mortality dose of wild-type strain.

The intracellular fish pathogen Francisella noatunensis remains an unsolved problem for aquaculture worldwide and an efficient vaccine is needed. In Francisella sp., IglC is an important virulence factor necessary for intracellular growth and escape from phagolysosomes. Deletion of the intracellular growth locus C (iglC) in Francisella sp. causes attenuation, but vaccine potential has only been attributed to ΔiglC from Francisella noatunensis ssp. orientalis, a warm-water fish pathogen. A ΔiglC mutant was constructed in the cold-water fish pathogen F. noatunensis ssp. noatunensis (Fnn), which causes francisellosis in Atlantic cod; the mutant was assessed in primary head kidney leucocytes from Atlantic cod. Fluorescence microscopy revealed reduced growth, while qPCR revealed an initial increase followed by a reduction in mutant genomes. Mutant-infected cod leucocytes presented higher interleukin 1 beta (il1ß) and interleukin 8 (il8) transcription than wild-type (WT)-infected cells. Two doses of mutant and WT were tested in an adult zebrafish model whereupon 3 × 109 CFU caused acute disease and 3 × 107 CFU caused low mortality regardless of strain. However, splenomegaly developed only in the WT-infected zebrafish. Immunization with 7 × 106 CFU of Fnn ΔiglC protected zebrafish against challenge with a lethal dose of Fnn WT, and bacterial load was minimized within 28 d. Immunized fish had lower interleukin 6 (il6) and il8 transcription in kidney and prolonged interferon-gamma (ifng) transcription in spleens after challenge compared with non-immunized fish. Our data suggest an immunogenic potential of Fnn ΔiglC and indicate important cytokines associated with francisellosis pathogenesis and protection.

Comparative Analysis of Membrane Vesicles from Three Piscirickettsia salmonis Isolates Reveals Differences in Vesicle Characteristics.

Membrane vesicles (MVs) are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89), Norway (NVI 5692) and Canada (NVI 5892), respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium's utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species.