| Autor: |
Ortiz-Severín J; Laboratorio de Microbiología de Sistemas, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile.; Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago 7830489, Chile.; Center of Integrative Microbiology and Evolution, University of Oslo, 0316 Oslo, Norway., Tandberg JI; Center of Integrative Microbiology and Evolution, University of Oslo, 0316 Oslo, Norway.; Department of Pharmacology and Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, 0316 Oslo, Norway., Winther-Larsen HC; Center of Integrative Microbiology and Evolution, University of Oslo, 0316 Oslo, Norway.; Department of Pharmacology and Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, 0316 Oslo, Norway., Chávez FP; Laboratorio de Microbiología de Sistemas, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago 7800003, Chile., Cambiazo V; Laboratorio de Bioinformática y Expresión Génica, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago 7830489, Chile.; Fondap Center for Genome Regulation, Universidad de Chile, Santiago 8370415, Chile. |
| Abstrakt: |
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 , ym t, 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. |
