Periodic Reporting for period 1 - chelOMICS (chelOMICS: Application of multi-OMICS approaches to provide a holistic understanding of siderophore mediated host-pathogen interactions and new diagnostic options for aquaculture fish infections)
Periodo di rendicontazione: 2022-09-16 al 2024-09-15
Aquaculture plays a critical role in addressing global food demands as wild fish stocks are declining due to overfishing and environmental changes. However, one of the greatest challenges in fish farming is the outbreak of infectious diseases, such as vibriosis, which lead to significant economics losses that are estimated to exceed 6 billion US dollar annually. Traditional diagnostic methods often lack the speed and precision necessary to prevent large-scale disease outbreaks, highlighting a pressing need for innovative and sustainable disease management strategies.
The overarching objective of this project is to develop an accurate disease diagnostic tool for vibriosis in aquaculture fish using an innovative iron-chelator metabolomics approach, which we termed ‘chelOMICS’. This project aims to identify specific siderophores – iron-chelating metabolites produced by pathogenic bacteria – that can be used as specific disease biomarkers of vibriosis. By integrating modern multi-omics approaches we want to provide a holistic understanding of the contribution of siderophores in host-pathogen interactions in farmed fish during infection.
This project aligns with the global efforts to ensure food security and promoting more sustainable aquaculture practices, which include reducing use of antibiotics and implementing proactive disease management strategies. Our research addresses this need by investigating the molecular mechanisms underlying bacterial infections and providing tools that can lead to the early detection of diseases, thereby promoting healthier fish populations and reducing dependency on antibiotics, which in turn curbs the rise of antibiotic resistance.
The overarching objective of this project is to develop an accurate disease diagnostic tool for vibriosis in aquaculture fish using an innovative iron-chelator metabolomics approach, which we termed ‘chelOMICS’. This project aims to identify specific siderophores – iron-chelating metabolites produced by pathogenic bacteria – that can be used as specific disease biomarkers of vibriosis. By integrating modern multi-omics approaches we want to provide a holistic understanding of the contribution of siderophores in host-pathogen interactions in farmed fish during infection.
This project aligns with the global efforts to ensure food security and promoting more sustainable aquaculture practices, which include reducing use of antibiotics and implementing proactive disease management strategies. Our research addresses this need by investigating the molecular mechanisms underlying bacterial infections and providing tools that can lead to the early detection of diseases, thereby promoting healthier fish populations and reducing dependency on antibiotics, which in turn curbs the rise of antibiotic resistance.
Throughout the project, we successfully implemented a series of experiments aimed at understanding the molecular mechanisms underlying vibriosis in sole (Solea senegalensis) and identifying key biomarkers for early disease detection. Our work involved a combination of metabolomics, proteomics, and microbiome analyses, which were performed across various stages of infection.
We performed laboratory infection experiments in collaboration with the Aquatic One Health Research Centre at the University of Santiago de Compostela (AHRCUS). Initially, an established Vibrio anguillarum virulence assay was conducted via direct intraperitoneal injection and key immune organs, including the spleen and kidney, were sampled. Additionally, an indirect bath-infection model was established to study natural infection dynamics in aquaculture settings. Fish were sampled from various tissues such as mucus, gills, spleen, kidney, and intestine, providing an extensive dataset for downstream molecular analyses.
The virulence assay dataset was used to refine the analytical protocols to optimise the chelOMICS approach. This involved streamlining sample processing, LC-MS/MS method development, and siderophore detection, creating a robust workflow. To complement metabolomics analysis, we incorporated proteomics to gain more insight into the infection’s effect of infection on fish metabolism. We integrated open-source bioinformatics tools such as MZmine, DIA-NN, MetaboAnalyst, clusterProfiler, and MixOmics, to improve reproducibility of data analysis and scalability of the approach.
A key achievement was the successful detection and identification of the Vibrio anguillarum-specific siderophore, piscibactin. As we chelated metabolomics extracts, obtained from sole immune organs, with iron chloride (FeCl3), we were able to detect iron-bound siderophores by searching for the characteristic iron isotopic pattern in LC-MS spectra. Furthermore, the presence of ferri-piscibactin was quantified in high-infection samples using multiple reaction monitoring (MRM).
The integration of multi-omics data revealed significant alterations in metabolite and protein profiles, particularly those related to energy production, nucleotide synthesis, and protein regulation during infection. We identified several metabolite and protein biomarkers, including ferri-piscibactin and the STEAP4-like metalloreductase, emphasising the important role of iron metabolism in infection dynamics. These findings provide novel insights into how Vibrio anguillarum exploits host iron resources to support its virulence.
While the metabolome and microbiome analysis via 16S rRNA sequencing was performed on samples from the bath-infection experiment, the results were inconclusive in linking microbiome alterations to the infection process. Nevertheless, the overall project has significantly advanced our understanding of the molecular responses of Solea senegalensis to vibriosis and has set the stage for future diagnostic tools targeting siderophores as infection markers.
We performed laboratory infection experiments in collaboration with the Aquatic One Health Research Centre at the University of Santiago de Compostela (AHRCUS). Initially, an established Vibrio anguillarum virulence assay was conducted via direct intraperitoneal injection and key immune organs, including the spleen and kidney, were sampled. Additionally, an indirect bath-infection model was established to study natural infection dynamics in aquaculture settings. Fish were sampled from various tissues such as mucus, gills, spleen, kidney, and intestine, providing an extensive dataset for downstream molecular analyses.
The virulence assay dataset was used to refine the analytical protocols to optimise the chelOMICS approach. This involved streamlining sample processing, LC-MS/MS method development, and siderophore detection, creating a robust workflow. To complement metabolomics analysis, we incorporated proteomics to gain more insight into the infection’s effect of infection on fish metabolism. We integrated open-source bioinformatics tools such as MZmine, DIA-NN, MetaboAnalyst, clusterProfiler, and MixOmics, to improve reproducibility of data analysis and scalability of the approach.
A key achievement was the successful detection and identification of the Vibrio anguillarum-specific siderophore, piscibactin. As we chelated metabolomics extracts, obtained from sole immune organs, with iron chloride (FeCl3), we were able to detect iron-bound siderophores by searching for the characteristic iron isotopic pattern in LC-MS spectra. Furthermore, the presence of ferri-piscibactin was quantified in high-infection samples using multiple reaction monitoring (MRM).
The integration of multi-omics data revealed significant alterations in metabolite and protein profiles, particularly those related to energy production, nucleotide synthesis, and protein regulation during infection. We identified several metabolite and protein biomarkers, including ferri-piscibactin and the STEAP4-like metalloreductase, emphasising the important role of iron metabolism in infection dynamics. These findings provide novel insights into how Vibrio anguillarum exploits host iron resources to support its virulence.
While the metabolome and microbiome analysis via 16S rRNA sequencing was performed on samples from the bath-infection experiment, the results were inconclusive in linking microbiome alterations to the infection process. Nevertheless, the overall project has significantly advanced our understanding of the molecular responses of Solea senegalensis to vibriosis and has set the stage for future diagnostic tools targeting siderophores as infection markers.
This project pushes the boundaries of existing knowledge and technologies in aquaculture disease diagnostics through the integration of novel multi-omics approaches. These advances have potential for the development of new disease detection tools, thus contributing to more sustainable and efficient aquaculture practices.
*Novel diagnostic approach using siderophore biomarkers*
The most significant result was the identification of the siderophore piscibactin in fish infected with Vibrio anguillarum. To the best of our knowledge this is the first time that chelOMICS, the detection of iron-chelating metabolites, was used to confirm vibriosis. This siderophore biomarker is highly specific for Vibrio-like pathogens and provides an attractive diagnostics alternative compared to labour-intensive manual clinical observations and isolation of bacterial cultures or costly PCR-based identification or serological tests.
*Comprehensive understanding of iron homeostasis in host-pathogen interactions*
The project provided insights into how Vibrio anguillarum and similar pathogens disrupt iron homeostasis during infection. Via proteomics, we identified highly elevated levels of STEAP4-like metalloreductase in infected sole that aligned with the detection of siderophores, which pathogens use to scavenge iron from the host. This contributes to the scientific knowledge of host-pathogen interactions and reveals new molecular targets for treatments and interventions that do not rely on overuse of current antibiotics.
*Future perspectives*
The chelOMICS workflow has been successfully demonstrated in laboratory conditions. The next step towards broader uptake will require trials in real-world aquaculture settings to further validate the diagnostic efficiency of the approach under commercial fish farming conditions. Further research is also needed to extend the chelOMICS approach to study different fish and shellfish species as well as other bacterial diseases relevant to aquaculture.
*Novel diagnostic approach using siderophore biomarkers*
The most significant result was the identification of the siderophore piscibactin in fish infected with Vibrio anguillarum. To the best of our knowledge this is the first time that chelOMICS, the detection of iron-chelating metabolites, was used to confirm vibriosis. This siderophore biomarker is highly specific for Vibrio-like pathogens and provides an attractive diagnostics alternative compared to labour-intensive manual clinical observations and isolation of bacterial cultures or costly PCR-based identification or serological tests.
*Comprehensive understanding of iron homeostasis in host-pathogen interactions*
The project provided insights into how Vibrio anguillarum and similar pathogens disrupt iron homeostasis during infection. Via proteomics, we identified highly elevated levels of STEAP4-like metalloreductase in infected sole that aligned with the detection of siderophores, which pathogens use to scavenge iron from the host. This contributes to the scientific knowledge of host-pathogen interactions and reveals new molecular targets for treatments and interventions that do not rely on overuse of current antibiotics.
*Future perspectives*
The chelOMICS workflow has been successfully demonstrated in laboratory conditions. The next step towards broader uptake will require trials in real-world aquaculture settings to further validate the diagnostic efficiency of the approach under commercial fish farming conditions. Further research is also needed to extend the chelOMICS approach to study different fish and shellfish species as well as other bacterial diseases relevant to aquaculture.