Novel protein carrier system based on cyanobacterial nano‐sized extracellular vesicles for application in fish

Abstract

Summary Aquaculture has been one of the fastest‐growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP‐loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

EXCRETE enables deep proteomics of the microbial extracellular environment

“…Proteins secreted by these mechanisms lack recognizable signal peptides or secretion motifs and include, for example, cytoplasmic proteins which can exert a second, “moonlighting”, function outside the cell 60 and proteins secreted via extracellular vesicles 25,79 . Although non-classical secretion remains poorly characterized in cyanobacteria, a recent study has suggested that extracellular vesicles can be responsible for up to 10% of total protein secretion 25 . Taken together, these results suggest that many open questions remain regarding protein secretion in cyanobacteria.…”

“…And T4P are involved in phototaxis, DNA uptake, aggregation, and flotation [17][18][19][20][21][22] . Interestingly, the recent discovery of multiple protein secretion mechanisms that do not involve the aforementioned systems suggests that there may be a myriad of secreted proteins which remain largely unexplored [23][24][25][26] . Mass spectrometry (MS)-based proteomics has become the preferred method to study protein secretion and characterize the exoproteome 27 .…”

LPS-Induced Mortality in Zebrafish: Preliminary Characterisation of Common Fish Pathogens

“…The LPS products were weighted and dissolved in PBS to a final concentration of 10 mg mL −1 . LPS quantification and protein profile was performed as described previously [ 23 ]. Briefly, LPS suspensions were 10-fold diluted, resolved on 16% ( w / v ) SDS-polyacrylamide gel and stained with the Pro-Q ® Emerald 300 Lipopolysaccharide Gel Stain Kit (Life Technologies, Thermo Fisher Scientific, Waltham, MS, USA).…”

Incorporation, fate, and turnover of free fatty acids in cyanobacteria

“…as the result of phospholipase activity of PldA during the process of degrading mislocalized phospholipids in the outer membrane (May and Silhavy 2018 ), it is possible that FFA is loaded into vesicles, and so, released as such from the cells. In fact, accumulation in the periplasm of proteins, peptidoglycan fragments, or even lipopolysaccharides has been shown to induce EV overproduction, likely from an increase in periplasmic pressure (Schwechheimer et al 2014 , Matinha‐Cardoso et al 2022 ). In addition, it is also possible that, upon release, bacterial vesicles are capable of importing FFA available in the environment.…”