J Korean Med Sci.  2015 Jul;30(7):866-870. 10.3346/jkms.2015.30.7.866.

Identification of Outer Membrane Vesicles Derived from Orientia tsutsugamushi

• Lee SM
• Kwon HY
• Im JH
• Baek JH
• Kang JS
• Lee JS

• KMID: 2164469
• DOI: http://doi.org/10.3346/jkms.2015.30.7.866

Abstract

Orientia tsutsugamushi, a causative pathogen of Scrub typhus, is a gram-negative intracellular bacterium. Outer membrane vesicles (OMVs) are produced from the membrane of bacteria and play many roles related to the survival of the pathogen. However, there have been no reports confirming whether O. tsutsugamushi indeed produce OMVs. O. tsutsugamushi boryong was cultured in ECV-304 cells for the purification of OMVs. Western blot analysis and immunoenrichment using anti-O. tsutsugamushi monoclonal antibody and electron microscopy were employed for identification and characterization of OMVs. We confirm the presence of OMVs derived from O. tsutsugamushi, and also found that those OMVs contain a major surface antigen of 56-kDa protein and variant immunogenic antigens.

Keyword

Orientia tsutsugamushi; Scrub Typhus; Outer Membrane Vesicles; Antigens

MeSH Terms

Antibodies, Monoclonal/*immunology
Antigens, Bacterial/*immunology
Antigens, Surface/*immunology
Cell Line
Cell Membrane/immunology
Humans
Microscopy, Electron
Orientia tsutsugamushi/*immunology/metabolism
Scrub Typhus/diagnosis/microbiology
Secretory Vesicles/*immunology
Antibodies, Monoclonal
Antigens, Bacterial
Antigens, Surface

Figure

• 

  Fig. 1 Transmission electron micrographs of outer membrane vesicles of O. tsutsugamushi. (A) Blebs of vesicles (arrow) before they are liberated from O. tsutsugamushi (arrow). (B) Microvesicles are shown on the surface of purified O. tsutsugamushi. (C) Clusters of purified microvesicles are observed. They vary in size ranging from 50 to 150 nm and are made of monolayer membranes. Scale bars indicate 0.1 µm.

• 

  Fig. 2 Western blot analysis of purified and enriched OMVs derived from O. tsutsugamushi. (A) Blots were revealed with FS15 monoclonal antibody against OMVs and had similar patterns with blots of bacterial lysates. (B) Immunoblot of OMVs with polyclonal antibody showed a prominent 56-kDa protein band. (C) OMVs immunoenriched with FS15 monoclonal antibody revealed major surface antigens. ECV304, lysates of host cells; OT, lysates of purified O. tsutsugamushi; OMVs, outer membrane vesicles; FS15, mouse monoclonal antibody of O. tsutsugamushi; poly Ab, human polyclonal antibody purified from scrub typhus patient`s serum; IP, immunoprecipitation; IB, Immuno blot.

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