Extracellular vesicles of Lactiplantibacillus plantarum PCM 2675 and Lacticaseibacillus rhamnosus PCM 489: an introductory characteristic

Abstract

Aim: Extracellular vesicles (EVs) are involved in intercellular and interkingdom communication in the complex communities that constitute the niche-specific microbiome of the colonized host. Therefore, studying the structure and content of EVs produced by resident bacteria is crucial to understanding their functionality and impact on the host and other microorganisms. Methods: Bacterial EVs were isolated by differential centrifugation, their size and concentration were measured by transmission electron microscopy and nanoparticle tracking analysis, and the cargo proteins were identified by liquid chromatography coupled to tandem mass spectrometry. The cytotoxicity of bacterial EVs was tested using the human epithelial cell line A549 and an in vivo model of Galleria mellonella larvae. Results: The isolation and preliminary characteristics of EVs from two strains of lactic acid bacteria - Lactiplantibacillus plantarum PCM 2675 and Lacticaseibacillus rhamnosus PCM 489 - were presented, confirming the production of vesicular structures with sizes in the range of 50-170 nm for L. plantarum and 80-250 nm for L. rhamnosus. In addition, various proteins were identified within EVs cargo, with distinct locations of origin, including membrane, cytoplasmic and extracellular proteins, and with diverse functions, including enzymes with confirmed proteolytic activity. Furthermore, bacterial EVs did not show statistically significant cytotoxicity to the host under the tested conditions. Conclusions: A better understanding of the composition and functionality of bacterial EVs may contribute to their future effective use in supporting human health.

Keywords: EVs; Extracellular vesicles; lactic acid bacteria; postbiotics; probiotics.

Figure 1

Step-by-step procedure for isolation of bacterial EVs from cells grown on MRS solid media. The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 4.0 unported license. EVs: Extracellular vesicles; MRS: De Man Rogosa and Sharp.

Figure 2

Characteristics of EVs produced by L. plantarum PCM 2675. (A) NTA-based particle size distribution analysis; a representative histogram of the average size distribution from three measurements of a single sample (black line) is presented. The presented blue numbers indicate the maxima of peaks, and the red areas show the SD between measurements. The size parameters of EVs are included. Factors D10, D50, and D90 denote that 10%, 50%, and 90% of the EV population had a diameter of less than or equal to the presented value; (B) TEM images of LpEVs. White arrows point to the individual particles. Scale bars included in individual panels. EVs: Extracellular vesicles; NTA: nanoparticle tracking analysis; TEM: transmission electron microscopy; SD: standard deviation.

Figure 3

Characteristics of EVs from L. rhamnosus PCM 489. (A) NTA-based particle size distribution analysis; a representative histogram of the average size distribution from three measurements of a single sample (black line) is presented. The presented blue numbers indicate the maxima of peaks, and the red areas show the SD between measurements. The size parameters of EVs are included. Factors D10, D50, and D90 denote that 10%, 50%, and 90% of the EV population had a diameter of less than or equal to the presented value; (B) TEM images of LrEVs. White arrows point to the individual particles. Scale bars included in individual panels. EVs: Extracellular vesicles; NTA: nanoparticle tracking analysis; TEM: transmission electron microscopy; SD: standard deviation.

Figure 4

Characteristics of proteins identified in LpEVs and LrEVs. (A) Proteins grouped by their location; (B) Proteins grouped by their function.

Figure 5

The effect of bacterial EVs on the host. (A) EVs-related proteinase activity measured with BODIPY FL casein as a substrate. The control sample was DPBS with substrate, without EVs. Statistical significance levels versus control are marked as ^**** for P < 0.0001, and ns when not significant; (B) The survival curve of Galleria mellonella larvae after the injection of EVs produced by L. plantarum (LpEV) and L. rhamnosus (LrEV). Injection with DPBS served as a control; (C) The metabolic activity and (D) LDH release by A549 epithelial cells following treatment with 5 × 10⁸ EVs produced by L. plantarum (LpEV) and L. rhamnosus (LrEV). Untreated epithelial cells and lysed cells served as negative and positive controls, respectively. Statistical significance levels versus control: ns when not significant. Evs: Extracellular vesicles; DPBS: Dulbecco’s phosphate-buffered saline.