Characterization of commensal enterococcal membrane vesicles and their cytotoxic effect on various host cells

Abstract

Enterococcus are a group of bacteria growing on different environmental condition behaving as a commensal as well as opportunistic pathogen. Among the enterococci, Enterococcus faecium is recently emerged as a nosocomial multi-resistance pathogen especially in the severely ill and immunocompromised patients causing a wide range of diseases like endocarditis, bacteraemia and urinary tract infections. Membrane vesicles are released by these bacteria and are the membranous structure carrying different proteins and some virulence factors. This present study focuses on the isolation of membrane vesicles released by the commensal E. faecium E1007, to study the morphological characteristics and measure the host responses in different cells. First in preliminary observations, E. faecium E1007 was grown on different cultural plates and their colony characteristics were observed. Gram staining from blood agar and growth curve of E. faecium in BHI medium was monitored. E. faecium grown on BHI from both exponential and stationary growth phase releases membrane vesicles which were further purified using the density gradient centrifuge. The purified vesicles were analysed by Transmission electron microscope that showed their broad size range from 30-182 nm and 20-174 nm in stationary and exponential phases respectively and they look small circular structures. Dynamic light scattering shows an average size of 55.1 nm in crude and 100.1 nm in OptiPrep samples exponential growth phase whereas 73.7 nm in crude and 114 nm in OptiPrep samples from Stationary growth phase. The proteomic analysis shows a total of 1160, 1012, 497 proteins from Stationary growth phase OptiPrep fractions 1, 2 and 3 respectively, 425 proteins from Stationary growth phase crude samples, 362 proteins from exponential phase OptiPrep and 1044 proteins from exponential phase crude samples. Membrane vesicles were inoculated in HaCaT cells, CaCo-2 cells and neutrophil cells at different concentration and cytotoxicity in these cells were observed. Membrane vesicles from E. faecium E1007 showed high toxic to HaCaT cells and Neutrophil cells but not cytotoxic to CaCo-2 cells. This study provides a better understanding of Gram-positive E. faecium membrane vesicles and their cytotoxic nature to different host cells.