O. tsutsugamushi Boryong strain was propagated in ECV-304 cells (CLS, Germany) cultivated in M199 (WelGENE, Korea) with 10% (v/v) fetal bovine serum (Corning Cellgro, USA). Confluency of bacteria in ECV304 was confirmed by immunofluorescence assay (IFA). When ECV-304 cells were heavily infected, they were gathered and used for electron microscopic observation of O. tsutsugamushi in cytosol of host cells. Heavily infected cells were disrupted with glass beads (diameter, 1.0 mm) to release bacteria from the cells and bacteria were purified with 40% percoll density solution utilizing the same method of Tamura et al. (11). Purified bacteria were also observed by electron microscope.

ECV304 cells, heavily infected with O. tsutsugamushi, were centrifuged at 13,000 rpm for 30 min at 4℃, after which the centrifugal media was passed through a 0.22 µm pore size filter system (Corning, MA, USA). The cell free medium was concentrated to 50 mL by ultra-filtration using a QuixStand Benchtop system (GE Healthcare Bio-Sciences, Uppsala, Sweden) with a 100 kDa hollow fiber membrane and harvested by ultracentrifugation at 150,000 g for 3 hr at 4℃. The pelleted OMVs were resuspended in 1 mL of PBS and then purified as described previously with modifications (12). After ultracentrifugation in a sucrose gradient solution, each fraction was analyzed by SDS-PAGE, Coomassie Brilliant Blue staining, and western blotting. The fractions of sucrose density gradient showing protein profiles corresponding to O. tsutsugamushi in immunoblot bands were collected and centrifuged at 150,000 g for 3 hr at 4℃. The resulting pellets of purified OMVs were resuspended in PBS containing protease inhibitor cocktail (Sigma-Aldrich Co., MO, USA). The suspended OMVs were observed using an electron microscope. The purified OMVs were quantified using DC protein assay reagents (Bio-Rad Laboratories Inc., Hercules, CA, USA) and aliquots of the OMVs were stored at -70℃. Purified OMVs were taken for immunoenrichment and immunoblot analysis.

For enrichment of O. tsutsugamushi derived OMVs from a mixed population of vesicles, FS15 mouse monoclonal antibody reacting against 56 kDa protein of O. tsutsugamushi Boryong strain was combined with 10 µL of protein G magnetic beads (NEW ENGLAND BioLabs., MA, USA) and incubated at room temperature for 1 hr while rotating (25). The resultant was washed three times with IP buffer (25 mM Tris pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 0.05% Triton X-100) and then combined with appropriate concentrations of purified OMVs overnight while rotating at 4℃. The mixture was washed four times with IP buffer and the final wash was performed with PBS. Pellets in reducing sample buffer (50 mM Tris-Cl pH 6.8, 100 mM dithiothreitol (DTT), 2% SDS, 0.1% bromophenol blue, 10% glycerol) were solubilized by boiling for 10 min at 100℃. The solubilized samples were loaded on a 10% polyacryl amide gel. The proteins from the OMVs were transferred to a PVDF (Millipore, Darmstadt, Germany) membrane. The membrane was blocked with 5% nonfat dry milk in PBST (0.1% tween20 in PBS) for 1 hr at room temperature and then incubated overnight at 4℃ with O. tsutsugamushi polyclonal antibody. The membrane was washed three times with PBST and incubated with HRP-conjugated secondary antibody (Jackson Immunoresearch Laboratories, PA, USA) for 1 hr at room temperature. The membrane was washed again three times with PBST and developed with enhanced chemiluminescence (ECL) solution (GE Healthcare Life-Sciences, Uppsala, Sweden). Antibody used for the western blot assay, which was purified from the serum of a patient infected with O. tsutsugamushi Boryong, was confirmed by nested PCR amplifying the 56 kDa region. The two pairs of primers used were as follows: outer primers, 1F (5'-ATAATTAATGTATTTTCGAACG-3') and 2R (5'-CCTKCA AAGGACTTTTAGCT-3'), and inner primers, 1Fn (5'-AACACAGTGTTTTATAGATTGTTTA-3'), and 2Rn (5'-RCATTAATTGCTACACCAAGT-3'). The amplified length was 1,562 bp. The PCR product was purified and sequenced by GenoTech Corp. (Daejeon, Korea). The resulting sequence was identified as 56-kDa TSA gene using BLAST (http://ncbi.nlm.nih.gov/blastn).

To observe the blebbing of O. tsutsugamushi in infected ECV304 cells and purified bacteria, each pellet was fixed with 2.5% glutaraldehyde in 0.1 M phosphate (pH 7.4) for 2 hr. The samples were washed and placed in 1% osmium tetroxide for 30 min, dehydrated in ethanol and embedded in epoxy resin (Epon 812, Electron Microscope Sciences, UK) in Beam capsules. Ultrathin sections were cut using an ultramicrotome (UltraE, Reichert-Jung, USA) and stained with uranyl acetate and citrate. For observation of purified OMVs structure, samples were placed on 200 mesh carbon-coated copper grids until settled on the film for 5 min. The grids were stained for 5 min with 1% uranyl acetate. After air drying, the samples were analyzed under a CM200 transmission electron microscope (Phillips, Netherlands).

Electron microscopic examination showed that OMVs were secreted from the bacterial surface membrane in cytosol of host cells (Fig. 1A). Several microvesicles that might have been derived from O. tsutsugamushi were observed near the bacteria. The budding OMVs had a monolayer membrane and were approximately 130 nm in diameter. Microvesicles were observed in the vicinity of purified O. tsutsugamushi (Fig. 1B). Purified OMVs were observed as round or spherical vesicles of relatively diverse size ranging from 50-150 nm in diameter (Fig. 1C).

Western blot analysis of purified OMVs with FS15 monoclonal antibody, specific to O. tsutsugamushi 56-kDa protein showed the presence of proteins corresponding to O. tsutsugamushi (Fig. 2A). In control experiments, ECV 304 cell lysates used for cell culture did not react with either FS-15 monoclonal antibody or polyclonal antibody. This result validates that microvesicles observed in the electron microscopic examination were derived from O. tsutsugamushi. Western blot analysis with O. tsutsugamushi polyclonal antibody showed that a 56-kDa protein band was present as a prominent band (Fig. 2B).

To further purify O. tsutsugamushi derived OMVs from mixed populations with host derived microvesicles, we enriched bacterial OMVs by immunoprecipitation with FS15 monoclonal antibody. Western blot analysis of enriched OMVs showed 42, 46, and 56-kDa protein bands, corresponding to the surface antigens of O. tsutsugamushi (Fig. 2C). As expected, the 56-kDa protein was the most abundant protein band. ECV304 cell lysates used for cell cultures did not have a responding band in the western blot analysis using either FS15 monoclonal or polyclonal antibody. This result shows that OMVs from O. tsutsugamushi could be enriched with immunoprecipitation.