Introduction
Mycobacterium marinum is the causative agent of Mycobacteriosis, a fish bacterial disease characterized by granulomatous inflammation in multiple organs, with a mortality rate that can range between 30-100% (1). Mycobacteriosis outbreaks have important effects on commercial fish production, in particular that of European seabass (Dicentrarchus labrax), a fish species of high economic interest. Mycobacteriosis is also an important infectious disease in zebrafish (Danio rerio), associated with severe losses in research facilities. Because no satisfactory vaccine or treatment is available, once a population of fish is infected, the most likely scenario is euthanasia of the entire group. Thus, it is imperative to develop an efficient strategy to prevent mycobacteriosis infections. One possibility is the use of bacterial extracellular vesicles (EVs), which are spherical bilayered structures naturally liberated from the outer membrane of Gram-negative bacteria. Bacterial EVs have been progressively used as carriers of immunogenic antigens (2). An example is the commercially-available EV-based vaccine Bexsero® against Neisseria meningitidis serogroup-B15 in humans (3).
Materials and Methods
First, EVs derived from the non-pathogenic and environmentally friendly cyanobacterium Synechocystis sp. PCC 6803 wild-type and congenic derivatives ΔtolC, ΔtolC/Δspy, ΔfucS (with distinct vesiculation phenotypes), ranging between 50 and 500 μg LPS mL-1, were tested for their effects on 3 days post fertilization (3 dpf) zebrafish larvae survival. LPS isolated from the same bacterial strains were used for comparison while commercial LPS from Pseudomonas aeruginosa (known to induce zebrafish mortality) was used as control. Survival curves were plotted using the Kaplan-Meier method and pairwise comparisons between treatments were performed with nonparametric log-rank test, at 0.05 significance level, in GraphPad Prism version 9. In addition, at days 1 and 5, total RNA was extracted from pools of 5 embryos of each replicate (n=3) from EVs treatments, for analysis of transcript levels of pro-inflammatory cytokines interleukin 1 beta (il1β) and tumor necrosis factor (tnf1α) and of anti-inflammatory cytokine interleukin 10 (il10), by RT-qPCR, using established oligonucleotides. Next, European seabass juveniles were randomly assigned to duplicate groups of 12 fish, and injected with engineered cyanobacterial EVs loaded with the reporter GFP in the following scheme: either one injection with 824 μg EVs containing 16.48 μg GFP; or two injections, separated by 15 days, each with 412 μg EVs containing 8.24 GFP. As controls, one injection of 16.48 μg GFP alone; or PBS were included. At days 15 and 30 after injection, 6 fish from each tank were euthanized for blood collection, plasma extraction and subsequently used for anti-GFP antibodies detection in fish serum, using an indirect ELISA approach.
Results
Neither cyanobacterial EVs from any of the cyanobacterial strains used, nor isolated cyanobacterial LPS, induce significant mortality in zebrafish larvae under the tested conditions. Thus, while 45 µg mL-1 P. aeruginosa LPS triggered severe mortality (>80%) after 24 h, isolated EVs concentrations of up to 500 μg mL-1 from Synechocystis may be used without significantly reducing larvae survival even after 5 days of incubation. Accordingly, and in contrast to LPS from P. aeruginosa that induced significant upregulation of inflammatory markers at sub-lethal concentration of 35 µg mL-1, EVs isolated from Synechocystis strains were shown not to induce significant inflammatory response. Further, we detected anti-GFP-antibodies in the serum of European seabass injected with EVs containing GFP, and no harmful effect could be detected on fish after a 30 days trial.
Conclusions
Our results support that Synechocystis EVs structural components are biocompatible with fish even at high concentrations. Cyanobacterial EVs are thus good candidates to be used as carriers of immunogenic antigens in zebrafish and with potential to be used in aquaculture species such as the European seabass. Future work will engineer EVs with heterologous M. marinum antigens, and evaluate these as delivery vehicles to immunize zebrafish and European seabass against M. marinum.
Acknowledgements
The authors acknowledge the support of the i3S Scientific Platforms “Histology and Electron Microscopy” and “Advanced Ligh Microscopy”, members of the national infrastructure Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). This work was partly funded by the Strategic Funding to UID/Multi/04423/2019 (POCI-01-0145-FEDER-007621), financed by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-029540 (PTDC/BIA-OUT/29540/2017). Fundação para a Ciência e a Tecnologia is also greatly acknowledged for the PhD fellowship SFRH/BD/130478/2017 (SL) and FCT Investigator grant IF/00256/2015 (PO).
References
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