Lactobacillus casei BL23 Produces Microvesicles Carrying Proteins That Have Been Associated with Its Probiotic Effect

Abstract

Archaea, bacteria, and eukarya secrete membrane microvesicles (MVs) as a mechanism for intercellular communication. We report the isolation and characterization of MVs from the probiotic strain Lactobacillus casei BL23. MVs were characterized using analytical high performance techniques, DLS, AFM and TEM. Similar to what has been described for other Gram-positive bacteria, MVs were on the nanometric size range (30-50 nm). MVs carried cytoplasmic components such as DNA, RNA and proteins. Using a proteomic approach (LC-MS), we identified a total of 103 proteins; 13 exclusively present in the MVs. The MVs content included cell envelope associated and secretory proteins, heat and cold shock proteins, several metabolic enzymes, proteases, structural components of the ribosome, membrane transporters, cell wall-associated hydrolases and phage related proteins. In particular, we identified proteins described as mediators of Lactobacillus' probiotic effects such as p40, p75 and the product of LCABL_31160, annotated as an adhesion protein. The presence of these proteins suggests a role for the MVs in the bacteria-gastrointestinal cells interface. The expression and further encapsulation of proteins into MVs of GRAS (Generally Recognized as Safe) bacteria could represent a scientific novelty, with applications in food, nutraceuticals and clinical therapies.

Keywords: CFSE; Lactobacillus casei BL23; microvesicles; probiotics; proteomics; vesicle size distribution.

FIGURE 1

Schematic representation of the protocol used to isolate L. casei BL23 MVs. Cells were removed from the culture by centrifugation and subsequent filtration through a series of decreasing pore membranes: 0.8, 0.65, and 0.45 μm. Cell free supernatant was then concentrated using a 100 kDa filter membrane. Finally, the concentrated supernatant was spun at 110,000 g to pellet MVs while the soluble proteins remained in the supernatant.

FIGURE 2

Macroscopic aspect of L. casei BL23 and B. subtilis 168 pellets containing MVs.

FIGURE 3

Particle size distribution curves of L. casei BL23 and B. subtilis MVs as determined based on DLS data. Each point represents the mean ± SD of independently isolation processes (n = 4).

FIGURE 4

Atomic force microscopy (AFM) topography of MVs. Phase images and 3D reconstruction of MVs of L. casei BL23 (A) and B. subtilis 168 (B). Size-frequency measurements of MVs expressed as a histogram (C).

FIGURE 5

Transmission electron microscopy (TEM) images of MVs from L. casei BL23 (A) and B. subtilis 168 (B). Size-frequency measurements of MVs were expressed as a histogram (C).

FIGURE 6

Confocal laser scanning microscopy imaging of the formation of MVs in L. casei BL23. (A,B) CFSE was used for bacterial staining to investigate the formation and shedding of MVs. (C,D) To analyze the aspect of MVs from cell culture supernatant by this technique isolated MVs were stained by CFSE.

FIGURE 7

Microvesicles (MVs) are enriched in specific proteins. (A) Differential patterns observed between MV and cell extract proteins from L. casei BL23 in a Coomassie blue stained SDS-PAGE gel (n = 5). (B) Venn diagram of the number of proteins present in the MVs and cell extracts identified by LC-MS/MS. (C) Proteins of MVs identified by LC-MS/MS were grouped in 8 categories according to its function. The function of the 13 proteins exclusively present in the MVs are shown in boxes.