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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 t...

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Autores principales: Ortiz-Severín, Javiera, Tandberg, Julia I., Winther-Larsen, Hanne C., Chávez, Francisco P., Cambiazo, Verónica
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8706985/
https://www.ncbi.nlm.nih.gov/pubmed/34946119
http://dx.doi.org/10.3390/microorganisms9122516
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author Ortiz-Severín, Javiera
Tandberg, Julia I.
Winther-Larsen, Hanne C.
Chávez, Francisco P.
Cambiazo, Verónica
author_facet Ortiz-Severín, Javiera
Tandberg, Julia I.
Winther-Larsen, Hanne C.
Chávez, Francisco P.
Cambiazo, Verónica
author_sort Ortiz-Severín, Javiera
collection PubMed
description 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.
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spelling pubmed-87069852021-12-25 Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis Ortiz-Severín, Javiera Tandberg, Julia I. Winther-Larsen, Hanne C. Chávez, Francisco P. Cambiazo, Verónica Microorganisms Article 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. MDPI 2021-12-06 /pmc/articles/PMC8706985/ /pubmed/34946119 http://dx.doi.org/10.3390/microorganisms9122516 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ortiz-Severín, Javiera
Tandberg, Julia I.
Winther-Larsen, Hanne C.
Chávez, Francisco P.
Cambiazo, Verónica
Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis
title Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis
title_full Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis
title_fullStr Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis
title_full_unstemmed Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis
title_short Comparative Analysis of Salmon Cell Lines and Zebrafish Primary Cell Cultures Infection with the Fish Pathogen Piscirickettsia salmonis
title_sort comparative analysis of salmon cell lines and zebrafish primary cell cultures infection with the fish pathogen piscirickettsia salmonis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8706985/
https://www.ncbi.nlm.nih.gov/pubmed/34946119
http://dx.doi.org/10.3390/microorganisms9122516
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