Formation of intracellular vesicles within the Gram+ Lactococcus lactis induced by the overexpression of Caveolin-1β

Abstract

Background: Caveolae are invaginated plasma membrane domains of 50-100 nm in diameter involved in many important physiological functions in cells. They are composed of different proteins, including the membrane-embedded caveolins and the peripheric cavins. Caveolin-1 has already been expressed in various expression systems (E. coli, insect cells, Toxoplasma gondii, cell-free system), generating intracellular caveolin-enriched vesicles in E. coli, insect cells and T. gondii. These systems helped to understand the protein insertion within the membrane and its oligomerization. There is still need for fundamental insights into the formation of specific domains on membrane, deformation of a biological membrane driven by caveolin-1, the organization of a caveolar coat, and the requirement of specific lipids and proteins during the process. The aim of this study was to test whether the heterologously expressed caveolin-1β was able to induce the formation of intracellular vesicles within a Gram+ bacterium, Lactococcus lactis, since it displays a specific lipid composition different from E. coli and appears to emerge as a good alternative to E. coli for efficient overexpression of various membrane proteins.Results: Recombinant bacteria transformed with the plasmid pNZ-HTC coding for the canine isoform of caveolin-1β has been shown to produce caveolin-1β, under its functional oligomeric form, at a high expression level unexpected for an eukaryotic membrane protein. Electron microscopy revealed several intracellular vesicles from 30 to 60 nm, a size comparable to E. coli h-caveolae, beneath the plasma membrane of the overexpressing bacteria, showing that caveolin-1β is sufficient to induce membrane vesiculation. Immunolabelling studies showed antibodies on such neo-formed intracellular vesicles, but none on plasma membrane. Density gradient fractionation allowed the correlation between detection of oligomers on Western blotting and appearance of vesicles measurable by DLS, showing the requirement of caveolin-1β oligomerization for vesicle formation. Conclusion: These caveolin-1β enriched intracellular neo-formed vesicles might be useful for potential co-expression of membrane proteins of pharmaceutical interest for their simplified functional characterization.