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Growth phase matters: Boosting immunity via Lacticasebacillus-derived membrane vesicles and their interactions with
Miriam Sandanusova1,2, Kristyna Turkova2, Eva Pechackova3
1Faculty of Science, Department of Experimental Biology Masaryk University Brno Czech Republic.
Abstract:
Lipid bi-layered particles known as membrane vesicles (MVs), produced by Gram-positive bacteria are a communication tool throughout the entire bacterial growth. However, the MVs characteristics may vary across all stages of maternal culture growth, leading to inconsistencies in MVs research. This, in turn, hinders their employment as nanocarriers, vaccines and other medical applications. In this study, we aimed to comprehensively characterize MVs derived from Lacticaseibacillus rhamnosus CCM7091 isolated at different growth stages: early exponential (6 h, MV6), late exponential (12 h, MV12) and late stationary phase (48 h, MV48). We observed significant differences in protein content between MV6 and MV48 (data are available via ProteomeXchange with identifier PXD041580), likely contributing to their different immunomodulatory capacities. In vitro analysis demonstrated that MV48 uptake rate by epithelial Caco-2 cells is significantly higher and they stimulate an immune response in murine macrophages RAW 264.7 (elevated production of TNFα, IL-6, IL-10, NO). This correlated with increased expression of lipoteichoic acid (LTA) and enhanced TLR2 signalling in MV48, suggesting that LTA contributes to the immunomodulation. In conclusion, we showed that Lacticaseibacillus rhamnosus CCM7091-derived MVs from the late stationary phase boost the immune response the most effectively, which pre-destines them for therapeutical application as nanocarriers.
Insights
Membrane vesicles (MVs) from Lacticaseibacillus rhamnosus CCM7091, particularly those from the late stationary phase (MV48), show enhanced immune stimulation. These MVs are promising for therapeutic nanocarrier applications.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Membrane vesicles (MVs) are bacterial communication tools, but their characteristics vary with growth stage, impacting research and applications.
- Inconsistencies in MV properties hinder their use as nanocarriers and in vaccines.
- Understanding MV heterogeneity is crucial for optimizing their therapeutic potential.
Purpose of the Study:
- To comprehensively characterize MVs from Lacticaseibacillus rhamnosus CCM7091 at different growth phases (early exponential, late exponential, late stationary).
- To evaluate the immunomodulatory capacity and cellular uptake of these distinct MVs.
- To identify factors contributing to differential immunomodulation for potential therapeutic applications.
Main Methods:
- Isolation and characterization of MVs from Lacticaseibacillus rhamnosus CCM7091 at 6h (MV6), 12h (MV12), and 48h (MV48).
- Proteomic analysis to compare protein content differences.
- In vitro assays using Caco-2 cells for uptake and RAW 264.7 macrophages to assess immune response (cytokine production, nitric oxide release).
- Analysis of lipoteichoic acid (LTA) expression and Toll-like receptor 2 (TLR2) signaling.
Main Results:
- Significant differences in protein content were observed between early (MV6) and late stationary phase (MV48) MVs.
- MV48 exhibited a significantly higher uptake rate by Caco-2 cells compared to other MVs.
- MV48 strongly stimulated immune responses in macrophages, including elevated TNFα, IL-6, IL-10, and NO production.
- Increased lipoteichoic acid (LTA) expression and enhanced TLR2 signaling were associated with MV48, suggesting LTA's role in immunomodulation.
Conclusions:
- Lacticaseibacillus rhamnosus CCM7091-derived MVs vary significantly based on bacterial culture growth stage.
- Late stationary phase MVs (MV48) demonstrate superior immunomodulatory effects and cellular uptake.
- The enhanced immune response induced by MV48 is linked to increased LTA expression and TLR2 signaling, positioning them as promising candidates for nanocarrier-based therapeutics.
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