Fernando A. Gómez

Bacterial kidney disease (BKD), caused by the Gram-positive intracellular bacterium Renibacterium salmoninarum , remains a major challenge for salmon aquaculture, particularly in Chile, where the lack of an effective vaccine has led to sustained antibiotic use. In this study, we applied an integrative in silico approach combining comparative genomics, reverse vaccinology, immunoinformatics, structural modeling, molecular docking, and molecular dynamics to design a multiantigenic and multiepitope chimeric protein as a vaccine candidate. Comparative analysis of three genomes revealed a highly conserved proteome, supporting the identification of shared immunogenic targets. From proteins associated with virulence, secretion systems, iron acquisition, chaperones, ribosomal functions, magnesium transport, and carbohydrate uptake, 17 proteins were selected after antigenicity and localization screening. Epitope prediction initially identified 154 candidates; after filtering for antigenicity, toxicity, and host homology, 60 epitopes grouped into 26 antigenic regions derived from 15 proteins were retained. Structural models were evaluated using QMEAN and Ramachandran analysis, and docking against Atlantic salmon MHC class I and II alleles identified the best candidates, with HADDOCK scores ranging from −71.2 to −154.6 (MHC I) and −116.5 to −152.0 (MHC II). Twelve antigenic regions were assembled into a 439-amino-acid chimeric protein using GSGSGS linkers. The construct showed a predicted molecular weight of 43.7 kDa, pI of 9.56, aliphatic index of 87.97, moderate solubility (0.43), and no predicted toxicity. Molecular dynamics (100 ns) supported structural stability, with RMSD convergence at ~85 ns. Surface analysis identified exposed antigenic regions mainly from DnaK, HmuU, Tpl, and DacB. This approach enabled the rational design of a structurally stable and immunologically promising candidate, providing a basis for future experimental validation.

Piscirickettsia salmonis is the causative agent of Rickettsial Septicemia (SRS), a severe disease threatening the sustainability of the growing salmon farming industry in Chile. This pathogen significantly impacts fish throughout their life cycle in the ocean, affecting pathogen-free individuals sourced from certified hatcheries. Systematic outbreaks often lead to catastrophic losses near harvest time, suggesting that primary infections originate from an environmental source that remains largely unidentified. Given the ubiquitous nature of free-living amoebae (FLA), we investigated their potential role as reservoirs and vectors for P. salmonis . We focused on Acanthamoeba castellanii and Vannella sp., two FLA species associated with salmon populations. Using immunofluorescence and quantitative PCR (qPCR), we demonstrated that P. salmonis can be internalized, replicate, and be released by both amoeba species. Furthermore, P. salmonis showed an upregulation on its Dot/Icm secretion system, essential for intracellular replication, during this process. While Vannella sp. exhibited pathogen-induced lysis after 72 h, A. castellanii maintained the bacteria more stably. Our findings indicate that A. castellanii acts as a reservoir and vector for P. salmonis , potentially contributing to the persistence and spread of this pathogen in aquatic environments. This enhanced understanding of the pathogen’s life cycle has implications for developing improved disease control strategies.

Bioluminescence is interesting, among other reasons, for the various technological applications that have been derived from it. Among these applications, developing visualization techniques to record the expression of one or more genes simultaneously in real-time are particularly useful. With this in mind, this study aimed to generate a recombinant Pyrocystis lunula luciferase protein (Luci D2-3 partial CDS). As the main results, i) a fragment of 1467 bp of the luciferase (LCFb) mRNA of the dinoflagellate P. lunula, containing part of domain 2 and all of the domain 3, was cloned in the pET28a vector; ii) the constructed vector was used to transform Escherichia coli to express the recombinant protein and subsequently purify it through an affinity chromatography procedure using a His-Tag; and iii) the purified protein (~50 kDa) was further analyzed by mass spectrometry to confirm its identity. Despite being unable to perform activity tests with the luciferin substrate, the evidence from previous studies indicates that the recombinant protein obtained in this case is enzymatically active. Due to the limited number of currently available luciferases, synthesizing this recombinant protein represents a useful tool, especially in designing expression assays coupled to multiple reporter genes, thus expanding the palette of proteins available for developing this type of biotechnological advances.

Piscirickettsia salmonis, the etiological agent of Piscirickettsiosis, is a Gram-negative and facultative intracellular pathogen that has affected the Chilean salmon industry since 1989. The bacterium is highly aggressive and can survive and replicate within fish macrophages using the Dot/Icm secretion system to evade the host’s immune response and spread systemically. To date, no efficient control measures have been developed for this disease; therefore, the producers use large amounts of antibiotics to control this pathogen. In this frame, this work has focused on evaluating the use of saponins from Quillaja saponaria as a new alternative to control the Piscirickettsiosis. It has been previously reported that purified extract of Q. saponaria (PQSE) displays both antimicrobial activity against pathogenic bacteria and viruses and adjuvant properties. Our results show that PQSE does not present antimicrobial activity against P. salmonis, although it reduces P. salmonis infection in an in vitro model, promoting the phagosome–lysosome fusion. Additionally, we demonstrate that PQSE modulates the expression of IL-12 and IL-10 in infected cells, promoting the immune response against the pathogen and reducing the expression of pathogen virulence genes. These results together strongly argue for specific anti-invasion and anti-intracellular replication effects induced by the PQSE in macrophages.

Small RNA and chaperone proteins form synergistic duos that play pivotal roles in controlling gene expression in bacteria. This is the case for Hfq, a highly pleiotropic pretranslational modulator of general protein expression, which responds to harsh environmental conditions and influences fitness and virulence in a wide range of pathogenic Enterobacteria. Given this relevancy, we evaluated the presence and potential role of Hfq in the fish pathogen Piscirickettsia salmonis, a Gram‐negative bacterium that threatens the sustainability of Chilean salmon production. Using bioinformatics tools were identified and characterized two variants of Hfq, which share the consensus RNA‐binding domains and the active sites described functional Hfq other bacteria. Additionally, we demonstrated that hfq‐1 and hfq‐2 were transcriptionally active when growing in cell‐free media and in infected susceptible fish cell line. Expression of both genes differed under different growth conditions and under stress, suggesting that their roles might be independent and different, depending on the bacterial physiological status. In conclusion, we demonstrate the existence of two different and functional ORF coding for the hfq marker in marine bacteria and a preliminary analysis indicating that these two novel proteins might have relevant roles in the biology and pathogenic potential of P. salmonis.

The Piscirickettsia salmonis genome was screened to evaluate potential flagella-related open reading frames, as well as their genomic organization and eventual expression. A complete and organized set of flagellar genes was found for P. salmonis, although no structural flagellum has ever been reported for this bacterium. To gain further understanding, the hierarchical flagellar cascade described for Legionella pneumophila was used as a reference model for putative analysis in P. salmonis. Specifically, 5 of the most relevant genes from this cascade were chosen, including 3 regulatory genes (fleQ, triggers the cascade; fliA, regulates the σ28-coding gene; and rpoN, an RNA polymerase-dependent gene) and 2 terminal structural genes (flaA and flaB, flagellin and a flagellin-like protein, respectively). Kinetic experiments evaluated gene expressions over time, with P. salmonis assessed in 2 liquid, cell-free media and during infection of the SHK-1 fish cell line. Under all conditions, the 5 target genes were primarily expressed during early growth/infection and were differentially expressed when bacteria encountered environmental stress (i.e. a high-salt concentration). Intriguingly, the flagellin monomer was fully expressed under all growth conditions and was located near the bacterial membrane. While no structural flagellum was detected under any condition, the recombinant flagellin monomer induced a proinflammatory response in SHK-1 cells, suggesting a possible immunomodulatory function. The potential implications of these observations are discussed in the context of P. salmonis biology and pathogenic potential.

Piscirickettsia salmonis is a facultative intracellular bacterium that causes the disease called “salmon rickettsial syndrome”. Attempts to control this disease have been unsuccessful, because existing vaccines have not achieved the expected effectiveness and the antibiotics used fail to completely eradicate the pathogen. This is in part the product of lack of scientific information that still lacks on the mechanisms used by this bacterium to overcome infected–cell responses and survive to induce a productive infection in macrophages. For that, this work was focused in determining if P. salmonis is able to modify the expression and the imbalance of IL-12 and IL-10 using an in vitro model. Additionally, we also evaluated the role the antimicrobial peptide hepcidin had in the control of this pathogen in infected cells. Therefore, the expression of IL-10 and IL-12 was evaluated at earlier stages of infection in the RTS11 cell line derived from Oncorhynchus mykiss macrophages. Simultaneously, the hepcidin expression and location was analyzed in the macrophages infected with the pathogen. Our results suggest that IL-10 is clearly induced at early stages of infection with values peaking at 36 hours post infection. Furthermore, infective P. salmonis downregulates the expression of antimicrobial peptide hepcidin and vesicles containing this peptide were unable to merge with the infective bacteria. Our results suggest that P. salmonis is able to manipulate the behavior of host cytokines and likely might constitute a virulence mechanism that promotes intracellular bacterial replication in leukocytes cells lines of trout and salmon. This mechanism involves the generation of an optimum environment for the microorganism and the downregulation of antimicrobial effectors like hepcidin.