Surface-exposed proteins of Ehrlichia chaffeensis.
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Journal: 2007/September - Infection and Immunity
ISSN: 0019-9567
Abstract:
The surface proteins of Ehrlichia chaffeensis provide an important interface for pathogen-host interactions. To investigate the surface proteins of E. chaffeensis, membrane-impermeable, cleavable Sulfo-NHS-SS-Biotin was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin-agarose affinity purification. The affinity-captured proteins were separated by electrophoresis, and five relatively abundant protein bands containing immunoreactive proteins were subjected to capillary-liquid chromatography-nanospray tandem mass spectrometry analysis. Nineteen out of 22 OMP-1/P28 family proteins, including P28 (which previously was shown to be surface exposed), were detected in E. chaffeensis cultured in human monocytic leukemia THP-1 cells. For the first time, with the exception of P28 and P28-1, 17 OMP-1/P28 family proteins were demonstrated to be expressed at the protein level. The surface exposure of OMP-1A and OMP-1N was verified by immunofluorescence microscopy. OMP-1B was undetectable either by surface biotinylation or by Western blotting of the whole bacterial lysate, suggesting that it is not expressed by E. chaffeensis cultured in THP-1 cells. Additional E. chaffeensis surface proteins detected were OMP85, hypothetical protein ECH_0525 (here named Esp73), immunodominant surface protein gp47, and 11 other proteins. The identification of E. chaffeensis surface-exposed proteins provides novel insights into the E. chaffeensis surface and lays the foundation for rational studies on pathogen-host interactions and vaccine development.
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Infect Immun 75(8): 3833-3841
PMID: 17517859

Surface-Exposed Proteins of <em>Ehrlichia chaffeensis</em><sup><a href="#fn2" rid="fn2" class=" fn">▿</a></sup> <sup><a href="#fn1" rid="fn1" class=" fn">†</a></sup>

Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210
Corresponding author. Mailing address: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210. Phone: (614) 292-5661. Fax: (614) 292-6473. E-mail: ude.uso@1.asihikir
Received 2007 Feb 5; Revised 2007 Mar 12; Accepted 2007 May 14.
Copyright © 2007, American Society for Microbiology

Abstract

The surface proteins of Ehrlichia chaffeensis provide an important interface for pathogen-host interactions. To investigate the surface proteins of E. chaffeensis, membrane-impermeable, cleavable Sulfo-NHS-SS-Biotin was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin-agarose affinity purification. The affinity-captured proteins were separated by electrophoresis, and five relatively abundant protein bands containing immunoreactive proteins were subjected to capillary-liquid chromatography-nanospray tandem mass spectrometry analysis. Nineteen out of 22 OMP-1/P28 family proteins, including P28 (which previously was shown to be surface exposed), were detected in E. chaffeensis cultured in human monocytic leukemia THP-1 cells. For the first time, with the exception of P28 and P28-1, 17 OMP-1/P28 family proteins were demonstrated to be expressed at the protein level. The surface exposure of OMP-1A and OMP-1N was verified by immunofluorescence microscopy. OMP-1B was undetectable either by surface biotinylation or by Western blotting of the whole bacterial lysate, suggesting that it is not expressed by E. chaffeensis cultured in THP-1 cells. Additional E. chaffeensis surface proteins detected were OMP85, hypothetical protein ECH_0525 (here named Esp73), immunodominant surface protein gp47, and 11 other proteins. The identification of E. chaffeensis surface-exposed proteins provides novel insights into the E. chaffeensis surface and lays the foundation for rational studies on pathogen-host interactions and vaccine development.

Abstract

Human monocytic ehrlichiosis (HME) is an emerging tick-borne zoonosis in the United States (12). It is an acute febrile systemic disease that can cause severe and potentially fatal disease, especially in immunocompromised and elderly people (12, 34). The etiologic agent of HME is Ehrlichia chaffeensis, which belongs to the family Anaplasmataceae (11). In North America, the major vector of E. chaffeensis is the Lone Star tick, Amblyomma americanum, and the white-tailed deer is considered to be the major reservoir of E. chaffeensis (14, 26).

E. chaffeensis is a gram-negative, obligatory intracellular bacterium which has tropism for monocytes/macrophages. The entry and proliferation of E. chaffeensis involve host caveolae, glycosylphosphatidylinositol-anchored proteins, and incorporation of cholesterol into the bacterial membrane (25). After internalization by host monocytes, E. chaffeensis has the ability to subvert the hostile environment by residing in an early endosome-like compartment, which does not fuse with lysosomes (3). As an obligate intracellular bacterium, E. chaffeensis needs to exchange nutrients and metabolites with the host cell cytoplasm. These events are ehrlichial surface related. However, the corresponding bacterial surface components have not been characterized.

Studies on bacteria from the family Anaplasmataceae have revealed an important role for the bacterial outer membrane proteins (OMPs) in the stimulation of the host immune response and protection of the host from infection. Immunization with recombinant P28 (one of the major OMP-1/P28 family members) protected mice from E. chaffeensis challenge (33). Immunization of calves with Anaplasma marginale OMPs induced stronger protection against challenge compared to individual major surface proteins, e.g., MSP-1 and MSP-2 (1, 6, 35, 36, 48). Along this line, efforts have been made to identify the global composition of A. marginale outer membrane immunogens (28).

Polyclonal antibodies against E. chaffeensis or monoclonal antibody against OMP-1g (P28) mediated protection of SCID mice from E. chaffeensis fatal infection (24, 53). While many E. chaffeensis antigenic protein bands have been revealed by Western blotting with whole organisms (7, 8, 42), bacterial surface exposure of E. chaffeensis antigens or proteins other than P28 (33), gp47 (13), and gp120 (39) has not been determined.

Despite the importance of E. chaffeensis surface proteins as a critical interface for pathogen-host interactions, as mentioned above, there has been no systematic investigation of the surface proteins of E. chaffeensis. Therefore, this study focused on the characterization of E. chaffeensis major surface proteins via surface biotinylation with cleavable sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin) labeling (17, 45), streptavidin affinity purification of biotinylated proteins, and identification of the purified proteins by proteomic analysis with the recently published E. chaffeensis genome sequence database (20). Bacterial surface exposure of two proteins was confirmed by immunofluorescence microscopy with surface-exposed-epitope-specific antibodies.

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Acknowledgments

We appreciate the technical assistance of Kari Green-Church in proteomic analysis. We thank Rikihisa laboratory member Haibin Huang for preparing recombinant PET29a-OMP-1B in E. coli BL21(DE3). We also thank Kate Hayes for copy editing of the manuscript.

This work was supported by National Institutes of Health grant R01 AI 30010.

Acknowledgments

Notes

Editor: F. C. Fang

Notes
Editor: F. C. Fang

Footnotes

Published ahead of print on 21 May 2007.

Supplemental material for this article may be found at http://iai.asm.org/.

Footnotes

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