Kim et al. Clin Proteom (2016) 13:6 DOI 10.1186/s12014-016-9107-z Clinical Proteomics RESEARCH Open Access Extracellular vesicle proteomes reflect developmental phases of Bacillus subtilis Yeji Kim1, Nathan Edwards2 and Catherine Fenselau1* Abstract Background: Extracellular vesicles (EV) are spherical membrane-bound vesicles with nano-scale diameters, which are shed to the extracellular region by most eukaryotic and prokaryotic cells. Bacterial EV are proposed to contribute to intercellular communication, bacterial survival and human pathogenesis as a novel secretion system. EV have been characterized from many Gram-negative species and, more recently, from several vegetative Gram-positive bacteria. Further characterization of EV and their molecular cargos will contribute to understanding bacterial physiology and to developing therapeutic approaches. Results: Bacillus subtilis were observed to release EV to a similar extent during sporulation as during the vegetative growth phase. However, the two vesicular cargos show qualitatively and quantitatively different proteomes. Among 193 total proteins identified across both samples, 61 were shown to be significantly more abundant in EV shed by sporulating cells, with (log) ratio of spectral counts RSC > 1 and Fisher-exact test FDR < 5 %. Sixty-two proteins were found to be significantly more abundant in EV shed by vegetative cells. Membrane fusion was shown to take place between these EVs and Gram-positive cells. Conclusion: Biogenesis of EV is a continuous process over the entire life cycle of this sporulating bacterium. The formation of EV during sporulation is strongly supported by the delineation of protein content that differs from the proteome of EV formed by vegetative spores. Keywords: Label-free quantification, Bacillus subtilis, Secretome, Sporulation, Extracellular vesicles Background Gram-positive bacteria were thought not to produce EV Extracellular vesicles (EV) are universally produced because they lack outer membranes. However, Gram- from diverse eukaryotes and prokaryotes [1, 2]. They are positive bacterial EV were microscopically observed spherical and membranous vesicles shed to the extra- in 1990 [7], and now EV have been characterized from cellular region, and enclosed by a lipid bilayer with a several infectious Gram-positive bacteria, including S. nano-scale diameter between 20 and 1000 nm depend- aureus [8], B. anthracis [9], Listeria monocytogenes [10], ing on the organism [3, 4]. As in the case with EV shed Bacillus subtilis [11], and Clostridium perfringens [12]. by multicellular organisms, Gram-negative bacterial EV Gram-negative bacterial EV are already under develop- carry diverse cell-derived components, including numer- ment as vaccines and antibiotics, however the characteri- ous proteins, lipids, genetic materials, toxins, commu- zation of Gram-positive bacterial EV is still in the early nication signals, and immunomodulatory compounds stage. Further investigations of components and biologi- [4–6]. EV have been proposed to contribute to intercellu- cal activities of Gram-positive bacterial EV are needed lar communication, bacterial survival, and human patho- to better understand bacterial physiology and to develop genesis as a novel secretion system [1, 4–6]. Initially, therapeutic targets and applications. One characteristic that distinguishes several Gram-positive species is the *Correspondence: [email protected] propensity to form spores [13, 14] when nutrient levels 1 Department of Chemistry and Biochemistry, University of Maryland, fall. In the context of looking for mechanisms to control College Park, MD 20742, USA Full list of author information is available at the end of the article © 2016 Kim et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Clin Proteom (2016) 13:6 Page 2 of 8 bacterial growth, it is of interest to ask if EV are formed ultracentrifuge with 70Yi rotor). Each pellet was resus- during sporulation and what kind of cargo they carry. pended in PBS. Finally the EV were washed three times High resolution microscopy and proteomics strategies with PBS on a 100 kDa Amicon filter. The resulting EV are the major techniques used to characterize EV. Elec- were stored at −80 °C until further use. Protein con- tron microscopy has been utilized to confirm the pres- centrations in purified EV were determined with a BCA ence and purity of EV and also to visualize the shedding protein assay kit (Pierce) according to the supplier’s of EV from cells. MS-based proteomics and bioinformat- instructions. To estimate lipid content, EV were stained ics allow qualitative and quantitative characterization of with 5 µM DiO lipophilic dye (Invitrogen, Carlsbad, CA) the proteins of EV, which may in turn suggest biological in PBS for 1 h. Relative amounts of lipids were deter- activity and function [2]. In addition, fluorescence probes mined as a function of CFUs, using a F-4500 fluorescence have recently emerged to analyze diverse interactions of spectrophotometer (Hitachi, Tokyo, Japan) at 498 nm EV with cells and organelles [2, 15]. We have used these excitation and 510 nm emission. Total intensities of solu- techniques to ask whether B. subtilis shed EV during tions and unstained EV were used for background sub- sporulation, if and how the EV protein content differs traction [19]. The EV were then characterized by electron based on cellular stages, and how EV may interact with microscopy. The EV population from the culture contain- other B. subtilis cells. We have qualitatively and quantita- ing vegetative cells is referred to herein as vegetative EV, tively examined the protein cargos of EV from vegetative while that from the culture containing mostly sporulating and sporulating B. subtilis cells, seeking correlations with cells is called sporulating EV. Three biological replicates the distinctive process of sporulation. of EV were prepared from vegetative and three from sporulating cells. These were processed and analyzed Methods separately, and peptide identifications were combined Cell culture within each phase type during the bioinformatic analysis. To obtain vegetative cells, B. subtilis 168 cells (ATCC #23857) were grown in brain heart infusion (BHI) (Bec- Transmission electron microscopy ton–Dickinson, Franklin Lakes, NJ). A sub-culture from For negative staining TEM, EV solutions (tenfold diluted) a colony on BHI agar was inoculated into 500 mL BHI were applied to a carbon-coated formvar grid, which was and incubated for 12 h (720 min) at 37 °C. Phase contrast covered by bacitracin (1 %) to spread the EV. The samples microscopy indicated that sporulation had begun at 17 h, on the grids were washed and stained by 2 % uranyl ace- but not at 12 h. Cells were pelleted and supernatants that tate for 30 s. Images were obtained at 80 keV on a Zeiss contained the EVs were collected at 12 h, as found previ- EM10CA TEM (LEO Electron Microscopy, Thornwood, ously to be optimal [11]. Three biological replicates were NY). Diameters of EV were sorted using Image J [20]. collected. To induce sporulation, vegetative cell pellets were Lysis of EV washed with PBS and resuspended in BHI-based sporula- Aliquots of vegetative and sporulating EV were lysed tion medium (6 g BHI, 12 mg MnCl2, 4.8 g MgSO4, and by three cycles of sonication on ice for 40 s in 50 mM 0.2 g CaCl2 in 500 mL) [16]. A culture time of 12 h was NH4HCO3, pH 7.8, containing 8 M urea and 1× pro- selected to obtain an optimal amount of EV. Cells in this tease inhibitor cocktail (Sigma-Aldrich). The urea was medium were pelleted using the same conditions as for then diluted to about 8 mM with 50 mM NH4HCO3. vegetative cells. Schaeffer-Fulton staining phase con- Protein concentrations were determined with a BCA pro- trast microscopy [17] confirmed endospore formation in tein assay kit (Thermo Fisher Scientific, Rockford, IL). about 70 % of cells. The amounts of vegetative and sporu- Each lysate was reduced in 20 mM DL-dithiothreitol for lating cells were quantified as CFUs. Three biological rep- 30 min at 56 °C and alkylated with 40 mM iodoacetamide licates were prepared. in the dark for 30 min at room temperature. The sam- ples were digested using Sequencing Grade Trypsin Gold Isolation and characterization of EV (Promega) for 16 h at 37 °C with an enzyme to protein EV were isolated from the supernatants of vegetative and ratio of 1:30. sporulating cultures after 12 h, following methods pub- lished for Gram-positive bacterial EV with slight modi- HPLC–MS/MS fications [8, 9, 18]. Briefly, the supernatants were filtered Tryptic peptides were analyzed on a Shimadzu Promi- through a 0.22 μm bottle-top vacuum filter (Corning) nence nano-HPLC (Shimadzu BioSciences, Columbia, to remove remaining cell debris. EV were pelleted by MD) in-line with a LTQ Orbitrap XL (Thermo Fisher ultracentrifugation at 150,000g for 90 min at 4 °C and Scientific). Peptides prepared from 1 µg protein were washed with PBS (Beckman Coulter OptimaLE-80K loaded onto an Acclaim PepMap 300 C18 precolumn Kim et al. Clin Proteom (2016) 13:6 Page 3 of 8 (Dionex, Sunnyvale, CA) with 95 % solvent A (97.5 % each protein from vegetative EV versus sporulating EV. H2O, 2.5 % acetonitrile, and 0.1 % formic acid) and 5 % Proteins with Rsc exceeding +1 or −1 and Fisher FDR solvent B (97.5 % acetonitrile, 2.5 % H2O, and 0.1 % for- values <0.05 were considered to have significantly greater mic acid) for 10 min. Peptides were fractionated on a abundances in their respective sample (>twofold).