Eun-Young Lee^1,†, Do-Young Choi^2,†, Dae-Kyum Kim¹, Jung-Wook Kim¹, Jung Ok Park², Sungjee Kim³, Sang-Hyun Kim⁴, Dominic M. Desiderio⁵, Yoon-Keun Kim¹, Kwang-Pyo Kim^2,* and Yong Song Gho<sup>1,*

1</sup> Department of Life Science and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea
² Institute of Biomedical Science and Technology, Department of Molecular Biotechnology, Konkuk University, Seoul, Republic of Korea
³ Department of Chemistry, Pohang University of Science and Technology, Pohang, Republic of Korea
⁴ The National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Republic of Korea
⁵ Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, USA

^† These authors contributed equally to this work

^*Corresponding authors

Abstract
Although archaea, Gram-negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell-free intercellular communication, this cellular process has been overlooked in Gram-positive bacteria. Here, we found for the first time that Gram-positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus-derived MVs are both similar to those of Gram-negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus-derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram-positive bacteria.

Keywords: Gram-positive bacteria; IgG-binding protein; Membrane vesicles; Microbiology; Microvesicles; Staphylococcus aureus