Glycine significantly enhances bacterial membrane vesicle production: a powerful approach for isolation of LPS‐reduced membrane vesicles of probiotic Escherichia coli

Abstract

Bacterial membrane vesicles (MVs) have attracted strong interest in recent years as novel nanoparticle delivery platforms. Glycine is known to induce morphological changes in the outer layer of bacteria. We report here that glycine dramatically facilitates MV production in a flagella-deficient mutant of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. Supplementation of culture medium with 1.0% glycine induced cell deformation at the early exponential phase, eventually followed by quasi-lysis during the late exponential to stationary phase. Glycine supplementation also significantly increased the number of MVs with enlarged particle size and altered the protein profile with an increase in the inner membrane and cytoplasmic 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 noninduced 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 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.

Membrane Vesicle Production as a Bacterial Defense Against Stress

“…Glycine can disrupt the transpeptidation step of bacterial cell wall synthesis by replacing D -alanine; thus, the bacterial cell wall cannot be properly formed in the presence of glycine ( Hishinuma et al, 1969 ). Moreover, accumulation of the building blocks of the bacterial cell wall—i.e., N -acetyl glucosamine and N -acetylmuramic acid—and parts of the incomplete cell wall perturb the balance between intra- and extracellular cell wall components, which induces membrane vesicle formation ( Hirayama and Nakao, 2020 ). Hirayama et al used glycine to induce membrane vesiculation in the probiotic E. coli strain Nissle 1917 and observed deformity and quasi-lysis during logarithmic and stationary growth phases, respectively, resulting in a 6–8-times higher rate of vesicle expulsion; moreover, membrane vesicles produced in the stationary phase were larger and more deformed than those in the exponential phase.…”

“…Hirayama et al used glycine to induce membrane vesiculation in the probiotic E. coli strain Nissle 1917 and observed deformity and quasi-lysis during logarithmic and stationary growth phases, respectively, resulting in a 6–8-times higher rate of vesicle expulsion; moreover, membrane vesicles produced in the stationary phase were larger and more deformed than those in the exponential phase. The authors hypothesized that vesicles in the exponential phase originate from membrane blebbing whereas those in the stationary phase mainly arise from explosive cell lysis ( Deatherage et al, 2009 ; Hirayama and Nakao, 2020 ).…”

Incorporation of Plasmid DNA Into Bacterial Membrane Vesicles by Peptidoglycan Defects in Escherichia coli

“…To understand the relationship between defects in peptide crosslinks in PG and the association of DNA with OMVs, we investigated the impact of glycine on OMV formation. Glycine inhibits PG synthesis by substituting glycine for the D - or L -alanine of PG during growth in E. coli ( Hammes et al, 1973 ), and the addition of glycine significantly enhances OMV formation in E. coli Nissle 1917 ( Hirayama and Nakao, 2020 ). As described in a previous report, the addition of a final concentration of 1% (w/v) glycine enhanced OMV formation in E. coli BW25113 harboring pUC19 ( Figure 3A ).…”

“…To further corroborate if PG defect-based OMV production enhances the encapsulation of plasmid DNA into OMVs, the influence of 1.0% glycine supplementation on plasmid copy numbers within OMVs was investigated. Glycine substitutes for D - and L -alanine of PGs during growth ( Hammes et al, 1973 ; Jonge et al, 1996 ), and it also induces OMV production ( Hirayama and Nakao, 2020 ). The mechanism for OMV induction by glycine is considered to be a similar phenomenon to that observed in the depletion of nlpI , where the loss of the OM-PG bridge (i.e., Braun’s lipoprotein Lpp) increases OM looseness and the accumulation of PG fragments increases OM protrusion, thus resulting in OMV production ( Figures 8A,B ).…”

Outer membrane vesicles (OMVs) enabled bio‐applications: A critical review

“…Weakened bacteria was used to treat cancer patients in the early of 1890s (Patyar et al, 2010). However, the safety issue associated with bacterial components has been a great concern that limits the clinical practice of bacteria mediated cancer therapy (Hirayama & Nakao, 2020). In 1997, Bermudes's group demonstrated that Salmonella can be used as a novel anticancer vector with tumor targeting function (Pawelek et al, 1997).…”