Reducing the degree of crosslinking of peptidoglycan in Listeria monocytogenes promoted the secretion of membrane vesicles

Abstract Listeria monocytogenes (LM) is a Gram‐positive (G + ) bacterium that secretes nanoscale membrane vesicles (MVs). LM MVs comprise various bacterial components and may have potential as an antigen or drug‐delivery vehicle; however, the low yield of the LM MVs limits related research. G + ‐bacterial MVs germinate from the bacterial plasma membrane and must pass through a thick crosslinked peptidoglycan layer for release. Herein, we aimed to increase the release of MVs by reducing the degree of crosslinking of peptidoglycan. We knocked out two genes related to the longitudinal crosslinking of peptidoglycan, dal and dat , and supplemented the knocked‐out dal gene through plasmid expression to obtain a stably inherited recombinant strain LMΔ dd ::pCW633. The structure, particle size, and main protein components of MVs secreted by this recombinant strain were consistent with those secreted from the wild strain, but the yield of MVs was considerably increased ( p < 0.05). Furthermore, Listeria ivanovii (LI) was found to secrete MVs that differed in the composition of the main proteins compared with those of LM MVs. The abovementioned method was also feasible for promoting the secretion of MVs from the attenuated LM strain and LI wild‐type and attenuated strains. Our study provides a new method to increase the secretion of MVs derived from Listeria that could be extended to other G + bacteria.