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A Surface-Focused Biotinylation Procedure Identifies the Yersinia pestis Catalase KatY as a Membrane-Associated but Non-Surface-Located Protein

Tanya Myers-Morales,¹ Clarissa Cowan,¹ Michael E. Gray,^1, Christine R. Wulff,^1, Carol E. Parker,² Christoph H. Borchers,^2, and Susan C. Straley^1*

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky,¹ Department of Biochemistry and University of North Carolina-Duke Michael Hooker Proteomics Core Facility, University of North Carolina, Chapel Hill, North Carolina²

Received 21 December 2006/ Accepted 28 June 2007

This study identified major surface proteins of the plague bacterium Yersinia pestis. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium Y. pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis cells grown at 37°C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10⁹ cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the Y. pestis pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.

Published ahead of print on 20 July 2007.

Present address: Hospital, A. B. Chandler Medical Center, Lexington, KY 40536-0293.

Present address: Lutheran Theological Southern Seminary, Columbia, SC.

Present address: Genome British Columbia Proteomics Centre, University of Victoria, Victoria, British Columbia, Canada.