Supplementary Materials Figure S1. protein contents as compared to non\induced MVs. Of note, the endotoxin activity of glycine\induced MVs was approximately eightfold or sixfold lower than that of non\induced MVs when compared at equal protein or lipid concentrations respectively. Nevertheless, glycine\induced MVs efficiently induced both immune responses in a mouse macrophage\like cell line and adjuvanticity in an intranasal vaccine mouse model, comparable to those of non\induced MVs. We propose NU6300 that the present method of inducing MV production with glycine can be used for emerging biotechnological applications of MVs that have immunomodulatory activities, while dramatically reducing the presence of endotoxins. Abstract Physiologically excessive amount of glycine significantly enhanced MV production from NU6300 a flagella\deficient clone of probiotic strain Nissle 1917. The glycine\induced MVs could elicit strong immune responses, while dramatically reducing the presence of endotoxins. Introduction During various phases of growth, bacteria produce membrane vesicles (MVs) that contain much of the biological content derived from their parent bacterial cells, such as phospholipids, lipopolysaccharides (LPS), proteins, enzymes, toxins, DNA and RNA (Kaparakis\Liaskos and Ferrero, 2015). MVs are anticipated to have a wide range of applications in medical and healthcare settings (Toyofuku Nissle 1917 (EcN) is the most commonly used Gram\unfavorable probiotic strain and is marketed in several countries as the drug product Mutaflor (Ardeypharm, Herdecke, Germany) (Behnsen (McBroom deletion mutant (EcNdid not express any flagellin FliC (Fig. S1), and thus, flagella\free real MVs were successfully isolated using a simple, one\step approach by ultracentrifugation of the culture supernatant (Fig.?1A and B).?We did not observe significant differences regarding MV morphology between the EcN and isogenic EcNstrains (Fig.?1A). Furthermore, SDS\PAGE analysis results revealed no obvious difference in the proteins profile from the MVs between these strains, aside from the lack of a sharpened NU6300 band matching to FliC in the MV arrangements from the EcNmutant. A. TEM pictures of MV fractions of EcN outrageous type (WT) as well as the mutant (mutation. We’ve also discovered some filamentous buildings thinner compared to the flagella in the MV arrangements from both EcN and EcNstrains (Fig.?1A, white arrowheads), which is within good agreement using a prior proteomic research of MV preparations from EcN strain, in which some fimbrial proteins were detected (Aguilera strain. The EcNwas aerobically cultured in LuriaCBertani (LB) broth supplemented with or without glycine at different concentrations (Fig.?2A). Exponential\phase growth was inhibited in a dose\dependent manner by glycine supplemented at final concentrations of 1 1.2%, 1.5% and 2.0%, but not by lower glycine concentrations of 0.5% and 1.0% (Fig.?2A). However, the OD values significantly decreased at the stationary phase over time, when 1.0% glycine was used (Fig.?2A). Morphologically, all EcNcells were deformed when cultured in the presence of 1.0% glycine for at least 3?h (Fig.?2B). The whole cells of EcNcultured in 1.0% glycine\supplemented LB broth appeared as spindle\like, distorted rods at 3?h NU6300 (Fig.?2B). The morphological abnormality caused by 1.0% glycine further progressed NU6300 with the longer incubation (6?h) (Fig.?2B). Some cells were finally broken at 16?h (Fig.?2B), whereas no broken or deformed cells were found under non\induced conditions at the same time points (Fig.?2B). These data clearly indicated that supplementation with 1.0% glycine deformed the cell structure despite Rabbit Polyclonal to MLKL a normal exponential growth curve, which was followed by quasi\lysis during the stationary growth phase. Open in a separate window Fig. 2 Growth and morphology of whole cells and MVs of EcNstrain in the absence or presence of glycine. A. Growth curves of the EcNin the absence or presence of glycine at different concentrations. Error bars represent standard error of the mean of three impartial experiments. The growth curves were generated by sequential measurement of OD at 660?nm. The cells tended to aggregate when.