Abstract:

Outer membrane vesicles (OMVs) are nanoparticles with double-phospholipid membrane structures that are secreted by gram-negative bacteria and carry a variety of bioactive substances from parental bacterial cells; consequently, OMVs serve as disease markers. Moreover, bacterial OMVs are potential anticancer- and antibacterial-drug carriers. While the addition of glycine during bacterial culturing promotes the secretion of bacterial OMVs, glycine-induced differences in the lipid compositions of such OMVs have not yet been reported.

In this study, the key plasma membrane components of bacterial OMVs before and after glycine induction were analyzed using lipidomics. Bacterial OMVs were captured from bacterial-culture supernatants using an amphiphilic dendritic polymeric supramolecular probe. Two sets of enriched bacterial OMVs were characterized and their enrichment efficiencies determined, after which the numbers and purities of the OMVs within the samples were determined using a nanofluidic assay. Lipids were extracted using the methyl tert-butyl ether lipid-extraction method when consistent numbers were recorded. The lipid compositions of the bacterial OMVs before and after glycine induction were analyzed using an ultra-performance liquid chromatography-ion mobility spectrometry-quadrupole time-of-flight mass spectrometer (UPLC-IMS-QTOF-MS) and MS-DIAL software. Differential lipid species in the key plasma membranes of the bacterial OMVs following glycine induction were recorded along with their corresponding amounts. Detection was accomplished in positive-ion scanning mode using an ACQUITY UPLC BEH C18 column following UPLC-MS injection, and MS^E mass-spectrometry data-acquisition mode. The lipid components in the two groups were determined by combining mass-spectrometric and software-analysis data, which revealed that the addition of glycine to the E. coli Nissle 1917 culture led to two-to-three-times higher concentrations of OMVs than observed for the untreated group under the same culturing and enrichment conditions. Particle numbers measured for the same volume revealed one-order-of-magnitude more bacterial OMVs after induction than before, with the treated group exhibiting slightly larger particles (on average); however, these particles were better dispersed and less likely to aggregate. The identified lipid components were categorized to determine the amount of each lipid type. Differentially expressed lipids were subsequently screened according to experimental conditions; significantly different expression levels were observed following glycine induction, with 820 lipids identified among the 10165 components detected. The lipid classes were ranked in order of quantity as: glycerolipids (GL), fatty acids (FA), sphingolipids (SP), glycerophospholipids (GP), saccharolipids (SL), and sterol lipids (ST), among which 463 GL lipid fractions (56.4% of all characterized lipids) were recorded. The ST lipid fraction contained the fewest members (10) and qualitative lipids were determined to make up 1.2% of the total. The addition of glycine to the E. coli culture was found to induce significant changes in the surface and internal lipid composition of the E. coli OMVs, with significantly more ceramide (Cer) and lysophosphatidyl choline (LPC), and significantly less bis(monoacylglycerol)phosphate (BMP) expressed. Partial triglyceride (TG) and sphingomyelin (SM) were irregularly expressed following glycine treatment, with equal amounts of up- and down-regulated lipids observed. This study provides a reference for subsequent in-depth studies into the lipid compositions of OMVs and their use as novel drug-delivery carriers. The expression of TGs and SM showed irregular changes, which is worthy of the next step of in-depth exploration of its regulatory mechanism, and the present study provides a certain reference for the subsequent in-depth study of the lipid composition of OMV and its use in the study of novel drug delivery carriers.

Key words: bacterial outer membrane vesicles, ion mobility mass spectrometry, lipidomics, glycine