Comparative Lipidomic Analysis of Extracellular Vesicles Derived from Lactobacillus plantarum APsulloc 331261 Living in Green Tea Leaves Using Liquid Chromatography-Mass Spectrometry

Abstract

Lactobacillus plantarum is a popular probiotic species due to its safe and beneficial effects on humans; therefore, novel L. plantarum strains have been isolated and identified from various dietary products. Given that bacteria-derived extracellular vesicles (EVs) have been considered as efficient carriers of bioactive materials and shown to evoke cellular responses effectively, L. plantarum-derived EVs are expected to efficiently elicit health benefits. Herein, we identified L. plantarum APsulloc 331261 living in green tea leaves and isolated EVs from the culture medium. We performed quantitative lipidomic analysis of L. plantarum APsulloc 331261 derived EVs (LEVs) using liquid chromatography-mass spectrometry. In comparison to L. plantarum APsulloc 331261, in LEVs, 67 of 320 identified lipid species were significantly increased and 19 species were decreased. In particular, lysophosphatidylserine(18:4) and phosphatidylcholine(32:2) were critically increased, showing over 21-fold enrichment in LEVs. In addition, there was a notable difference between LEVs and the parent cells in the composition of phospholipids. Our results suggest that the lipidomic profile of bacteria-derived EVs is different from that of the parent cells in phospholipid content and composition. Given that lipids are important components of EVs, quantitative and comparative analyses of EV lipids may improve our understanding of vesicle biogenesis and lipid-mediated intercellular communication within or between living organisms.

Keywords: Lactobacillus plantarum APsulloc 331261; extracellular vesicles; green tea leaf; intercellular communication; lipidomic analysis; liquid chromatography-mass spectrometry.

Figure 1

Lactobacillus plantarum APsulloc 331261 spontaneously releases extracellular vesicles. (A) Scheme for L. plantarum APsulloc derived extracellular vesicle (LEV) purification. (B) Size distribution, dispersity, mean diameter/mode diameters, and concentration of purified LEVs based on tunable resistive pulse sensing (TRPS) analysis using qNano-Gold. (C) Cryo-TEM image of LEVs (left). The outlined LEV images are enlarged and the lipid bilayer indicated by black and white arrows (middle). The size distribution of LEVs was automatically analyzed on cryo-TEM images (right). Scale bars, 200 nm (low) and 100 nm (high magnification).

Figure 2

The discriminative lipidomic profile of L. plantarum-derived extracellular vesicles. Principal component analysis (PCA) of lipidomic information was performed based on the mass spectrometry (MS) data of LEVs and L. plantarum. Lipidomic data were analyzed statically using the package in MetaboAnalyst (n = 9 per group; three independent biological replicates and three technical replicates). X axis: principal component 1 (PC1). Y axis: principal component 2 (PC2), Z axis: principal component 3 (PC3).

Figure 3

Relative expression of total lipids in LEVs and L. plantarum. The expression levels of total lipids were determined by summing the amounts of identified lipid species in LEVs and L. plantarum, respectively. The value of fold change is shown as mean ± S.D. (n = 9 per group; three independent biological replicates and three technical replicates). Statistical significance was analyzed by Student’s t-tests for two groups (** p < 0.01).

Figure 4

Differentially enriched lipid species in LEVs versus L. plantarum. (A) The 320 identified lipids in LEVs are listed in a volcano plot according to their statistical p-value and fold changes compared to those in L. plantarum. Red lines at left and right sides represent distinct boundaries for fold-decreased (<0.5) or -increased (>2.0) lipid species, respectively, in LEVs compared to L. plantarum and the horizontal red line represents a boundary for the significance (p < 0.01). Statistical significance was analyzed by Student’s t-tests for two groups. (B) Hierarchical clustering of differentially enriched lipid species in LEVs versus L. plantarum (fold change > 2.0, <0.5, p < 0.01). A heat map shows graphically the differential fold changes of 86 lipid species in LEVs compared to L. plantarum. The relative fold difference is encoded by color intensity. Red: increased; green: decreased.

Figure 5

Lipid composition and proportion in L. plantarum versus LEVs. In pie diagrams for L. plantarum (A) and LEVs (B), the composition by lipid class (glycerolipids, sphingolipids, sterol lipids, phospholipids, and lyso-phospholipids) is expressed as a percentage of lipid category (mol% of lipid category) which was defined as the sum of the amount of identified lipid species (nmol/mg proteins). The markedly changed lipid classes in LEVs compared to L. plantarum are designated by red (increased) or green (decreased).