Incorporation of recombinant proteins into extracellular vesicles by Lactococcus cremoris

Abstract

Extracellular vesicles (EVs) are nanosized lipid bilayer particles released by various cellular organisms that carry an array of bioactive molecules. EVs have diagnostic potential, as they play a role in intercellular interspecies communication, and could be applied in drug delivery. In contrast to mammalian cell-derived EVs, the study of EVs from bacteria, particularly Gram-positive bacteria, received less research attention. This study aimed to investigate the production of EVs by lactic acid bacterium Lactococcus cremoris NZ9000 and to examine the impact of recombinant protein expression on their formation and protein content. Four different recombinant proteins were expressed in L. cremoris NZ9000, in different forms of expression and combinations, and the produced EVs were isolated using the standard ultracentrifugation method. The presence of vesicular structures (50-200 nm) in the samples was confirmed by transmission electron microscopy and by flow cytometry using membrane-specific stain. Mass spectrometry analyses confirmed the presence of recombinant proteins in the EVs fraction, with amounts ranging from 13.17 to 100%, highlighting their significant incorporation into the vesicles, together with intrinsic L. cremoris NZ9000 proteins that were either more abundant in the cytoplasm (ribosomal proteins, metabolic enzymes) or present in the membrane. The presence of the most abundant lactococcal proteins in EVs fraction suggests that protein cargo-loading of EVs in L. cremoris NZ9000 is not regulated. However, our data suggests that L. cremoris NZ9000 genetically engineered to express recombinant proteins can produce EVs containing these proteins in scalable manner. As L. cremoris NZ9000 is considered safe bacterium, EVs from L. cremoris NZ9000 could have several advantages over EVs from other bacteria, implying possible biotechnological applications, e.g. in therapeutic protein delivery.

Keywords: Lactococcus cremoris; Delivery vehicle; Extracellular vesicles; Recombinant protein.

Fig. 1

TEM analysis of L. cremoris NZ9000 ultracentrifugation samples reveals the presence of EVs. (a) : EVs from L. cremoris NZ9000/pNZ8148, (b): EVs from L. cremoris NZ9000/pSD-fZIL6, (c): EVs from L. cremoris NZ9000/pSD-fARS019. Scale bars indicate 50–100 nm.

Fig. 2

Flow cytometry analysis of the EVs from L. cremoris NZ9000 expressing recombinant proteins carrying plasmids pNZ8148 (a), pSD-fZIL6 (b), pSD-fARS019 (c), p-mFyn (d), p-fARS019-mFyn (e), pSC-fARS019 (f), p-Cher (g). Each sample was analyzed unstained, stained with the membrane dye, and treated with detergent TX100 after staining (a-g). Respective controls were included (h, buffer PBS and i, sterile bacterial growth medium). Scatterplots show the proportions (%) of EVs in each quadrant. j: Absolute number of green fluorescently labelled particles in the upper right quadrant.

Fig. 3

Flow cytometry analysis of the EVs from L. cremoris NZ9000 expressing red fluorescent protein mCherry. Respective controls were included (b, EVs from L. cremoris NZ9000 expressing proteins from plasmids pNZ8148, c, buffer PBS and d, sterile bacterial growth medium). Scatterplots show the proportions (%) of EVs in each quadrant. e: Absolute number of EVs in the upper right quadrant.

Fig. 4

Proteomic analysis of the EVs with SDS-PAGE and silver staining (a) or representative images of Western blot (b,c). Full-length Western blots are included in Supplementary data as Fig. S3a and b. EVs containing proteins from L. cremoris NZ9000/pNZ8148 (1), pSD-fZIL6 (2), pSD-fARS019 (3), p-mFyn (4), p-fARS019-mFyn (5), p-Cher (6), pSC-fARS019 (7) were analyzed using anti-flag antibodies (b) or anti-myc antibodies (c). f represents inclusion of FLAG tag and m represents myc tag.

Fig. 5

Mass spectrometry analysis of EVs from L. cremoris NZ9000. Individual proteins identified in EV samples were arranged according to their abundance in the cell lysate (determined as NSAF) and the number of EV samples in which they were identified is depicted (a). Gene and protein names of proteins identified in EV samples and their simplified genetic ontology. Of those, names of EV proteins that were not found in cell lysates are highlighted in gray (b). Occurrence of recombinant proteins in the EV samples and their percentage of total protein NSAF (c).