A surface-focused biotinylation procedure identifies the Yersinia pestis catalase KatY as a membrane-associated but non-surface-located protein

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY; Department of Biochemistry and UNC-Duke Michael Hooker Proteomics Core Facility, University of North Carolina, Chapel Hill, NC

^* To whom correspondence should be addressed. Email: scstra01{at}uky.edu.

	Abstract

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 digest, and identification by LC/MS/MS. The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pull-down 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 Yersinia pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis grown at 37^oC. 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 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, it did not protect C57BL/6 mice against bubonic plague.