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

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 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. 10 9 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.

Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis

“…YapE expression was induced with 80 ng of anhydrous tetracycline ml −1 (Sigma) for an additional 3 h and 32 OD600 equivalents of bacteria were harvested. Outer membrane proteins were isolated using sarkosyl enrichment as described by Myers-Morales et al (2007) and separated by SDS-PAGE. The gel was stained with Flamingo fluorescent stain (Bio-Rad) and two peptides present in the YapE sample, but absent in the vector control sample, were confirmed as being YapE by mass spectrometry (performed by the University of Louisville Protein Core).…”

Temperature and growth phase influence the outer-membrane proteome and the expression of a type VI secretion system in Yersinia pestis

“…A different study reported that complementdependent killing and human serum resistance of Y. pestis was mediated by Ail (Bartra et al, 2008). Cell surface localization was also reported for the OM proteins Pla, Lpp, Ail and Pcp (Myers-Morales et al, 2007).…”

Outer Membrane Protein X (Ail) Contributes to Yersinia pestis Virulence in Pneumonic Plague and Its Activity Is Dependent on the Lipopolysaccharide Core Length

“…OmpX is required for efficient delivery of Yersinia outer proteins (Yops) into human epithelial and monocyte cell lines (14). Additionally, OmpX is one of the most abundant proteins found in the Y. pestis outer membrane (OM) (37), and as such, is likely an important part of that structure.…”