Immunodetection of rainbow trout IL-8 cleaved-peptide: Tissue bioavailability and potential antibacterial activity in a bacterial infection context.

Chemokines such as IL-8 are part of an important group of proinflammatory response molecules, as well as cell recruitment. However, it has been described in both higher vertebrates and fish that IL-8 has an additional functional role by acting as an antimicrobial effector, either directly or by cleavage of a peptide derived from its C-terminal end. Nevertheless, it is still unknown whether this fragment is released in the context of infection by bacterial pathogens and if it could be immunodetected in tissues of infected salmonids. Therefore, the objective of this research was to demonstrate that the C-terminal end of IL-8 from Oncorhynchus mykiss is cleaved, retaining its antibacterial properties, and that is detectable in tissues of infected rainbow trout. SDS-PAGE and mass spectrometry demonstrated the cleavage of a fragment of about 2 kDa when the recombinant IL-8 was subjected to acidic conditions. By chemical synthesis, it was possible to synthesize this fragment called omIL-8α80-97 peptide, which has antibacterial activity against Gram-negative and Gram-positive bacteria at concentrations over 10 µM. Besides, by fluorescence microscopy, it was possible to locate the omIL-8α80-97 peptide both on the cell surface and in the cytoplasm of the bacteria, as well as inside the monocyte/macrophage-like cell. Finally, by indirect ELISA, Western blot, and mass spectrometry, the presence of the fragment derived from the C-terminal end of IL-8 was detected in the spleen of trout infected with Piscirickettsia salmonis. The results reported in this work present the first evidence about the immunodetection of an antibacterial, and probably cell-penetrating peptide cleaved from the C-terminal end of IL-8 in monocyte/macrophage-like cell and tissue of infected rainbow trout.

New insight to the rol of α-enolase (Eno-1) as immunological marker in rainbow trout fry.

α-Enolase is an enzyme of the glycolytic pathway that has also been involved in vertebrate inflammatory processes through its interaction with plasminogen. However, its participation in the immune response of lower vertebrates during early life development is unknown. Opportunistic pathogens in salmon farming are the principal cause of mortality in the fry stage. For that reason, molecular indicators of their immunological status are required to ensure the success of the large-scale cultivation. Thus, the objective of this work was to analyze if ENO-1 is involved in the immune response of rainbow trout fry. For this purpose, the coding sequence of trout ENO-1 was characterized, identifying the plasminogen-binding domain that has been described for homologs of this enzyme in higher vertebrates. A peptide-epitope of α-enolase was used for producing mice antiserum. The specificity of polyclonal antibodies was confirmed by dot blot, ELISA and Western blot. Then, the antiserum was used to evaluate α-enolase expression in fry between 152 and 264 degree-days post-hatching after 2, 8, and 12 h of challenge with lipopolysaccharide from Pseudomona auroginosa. The expression of α-enolase at both transcriptional (RT-qPCR) and protein (ELISA) levels was significantly increased after 8 h post-challenge with lipopolysaccharide. These results were confirmed by proteomic analysis by 2D-difference gel electrophoresis (DIGE). This work provides the first evidence of the involvement of α-enolase in the early immune response of salmonids. Future research will be required to understand the possible interaction of α-enolase with plasminogen in cells and tissues of the salmonid immune system.