Mitochondrial function implication in the immunological regulation through the inflammasome complex activation in Atlantic salmon, against a viral and bacterial infection

Aquaculture is one of the industries with the highest growth rates in the world, and it is in Chile that it has experienced particularly rapid growth, mainly due to favorable geographic and environmental conditions. However, containment systems and the presence of emerging pathogens increase the risk of infectious disease outbreaks. Two of the diseases that most affect the industry are: Heart and Skeletal Muscle Inflammation (HSMI) and Septicemia Rickettsial Salmonidea (SRS), the first is considered an emerging and high-risk disease caused by Piscine reovirus (PRV) and another disease is caused by the bacterium Piscirickettsia salmonis, generating high mortality rates in salmon farming. Considering the enormous number of salmon introduced into the sea each year and the amount of antibiotics used for the prophylaxis of diseases together with ineffective vaccines, it is imperative to study the main immunological mechanisms that salmonids have against pathogens, for the development of effective therapies. For this reason, this proposal will aim to identify how two pathogens, a bacterium (P.salmonis) and a virus (PRV), are capable of activating and regulating the immune system through the inflammasome in a different way through the mitochondria in Atlantic salmon, generating a different immune response against viral and/or bacterial infections. To justify this project, we present a background where mitochondria act as a control point of the immune system in salmonids. That is why it has been described those mitochondria can be considered the main drivers of inflammation mediated by inflammasomes (NLRP3), which transduce signals detected by specific cytosolic proteins for the proteolytic activation of caspase-1, resulting in cleavage and secretion of cytokines to eliminate the invading agent. According to the background, (i) mitochondria represent a checkpoint for numerous pattern recognition receptors (PRR) towards various pathogens; (ii) mitochondria have been identified as the main site for the generation of damage-associated molecular patterns (DAMPs), which are molecules recognized by the innate immune system that can polarize the fate of the inflammatory response by modulating the energy level of the immune system NLRP3 dependent; and (iii) mtDNA is involved in the activation of the inflammasome (NLRP3), inducing the release of pro-inflammatory cytokines. All these antecedents are still unknown in salmonids. However, as a group we have been working for some time on the characterization of the inflammasome complex in salmonids and we have seen a high homology of sequences and domains of the named components with mammals, in addition to being transcriptionally modulated, for this reason we strongly believe that mitochondria fulfill a regulatory function in the immune system of salmonids and that is dependent on the pathogens it infects (viruses or bacteria), being activated through the inflammasome. Therefore, the objective of this project is: “To determine the role of mitochondria in the regulation of the immune response, activating the inflammasome complex in salmonids against viral and bacterial infection.” To do this, 3 specific objectives are proposed that require integrating appropriate techniques from the fields of cell biology, molecular biology, and biochemistry. In this way it is proposed: (i) Characterize mitochondrial activation in the cell line (SHK-1) and primary erythrocyte and cardiac culture of Atlantic salmon, infected with bacterial and viral pathogens. (ii) To evaluate in vitro the participation of mitochondria in the activation of the inflammasome complex and the regulation of the immune response in cell lines and primary cultures infected with P. salmonis and PRV. (iii) Evaluate in vivo the participation of mitochondria in the innate and non-specific immune response in salmonids infected with P. salmonis and PRV. Methodologically, we intend to cultivate and infect the main cells where they replicate with the pathogens, in addition to evaluating in them the mitochondrial function and activation of the inflammasome complex through different techniques, including RNA-seq, all this will be compared later with an in vivo infection study. All these results will allow us to increase our knowledge of the immune system in this species and the mechanism that leads to the expression of cytokines, helping not only to understand the virulence mechanisms of P. salmonis and PRV, but also the immediate defense mechanism that it possesses. salmonids, which will provide information to develop new therapeutic targets for the design of alternative therapies to antibiotics and vaccines, for the prevention and/or control of these industry pathogens, thus increasing the sustainability of national salmon farming and international