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

Yersinia pestis is the causative agent of plague and is thought to have acquired virulence factors and transmission factors to adapt for survival within its mammalian host and transmission by fleabite (7). Investigators in our laboratory are identifying surface proteins and their pathogenic functions in Y. pestis to gain an improved understanding of the physiology of virulence as well as to identify potential prophylactic and therapeutic targets for use against plague. In this study, we evaluated two approaches for characterizing a surface proteome of Y. pestis, with the goal of developing a method that can be applied to small numbers of yersiniae recovered from the lungs of experimental animals with pneumonic plague. Previously, the extraction method of Filip et al. (16), based on the differential solubilities of inner and outer membrane proteins in sodium lauryl sarcosinate (Sarkosyl), has been used in several studies to obtain preparations of outer membrane proteins from gram-negative bacteria, including Y. pestis (e.g., see references 1, 2, 37, and 39). The method is rapid and simple but results in protein profiles comparable to those obtained by the classic isopycnic centrifugation method (35) and is amenable to downscaling to handle small samples (e.g., see references 9 and 39). The outer membrane proteins can then be resolved by two-dimensional (2-D) electrophoresis and analyzed further for surface localization in immunoblots by using antibody probes that bind to the surfaces of intact bacteria (e.g., see reference 14) or by comparing outer membrane protein profiles from intact bacteria subjected to proteolysis to those from mock-treated bacteria (e.g., see reference 40). However, the identification of the entire surface proteome has presented a major challenge. Procedures to obtain outer membrane proteins after bacterial lysis (as in the Sarkosyl method) introduce the serious problem of contamination by abundant cytoplasmic proteins, such as ribosomal proteins and EF-Tu, that confound “blind” identification by mass spectrometry (MS). Further, any method that includes initial resolution of proteins in acrylamide gels has the drawback of missing those proteins that fail to enter the first-dimension gel due to reduced solubility in the sample buffer or to the drift of the alkaline pH extreme of the gel, and few proteins larger than ca. 100 kDa are detected, even though they are predicted potentially to be present (e.g., see reference 33). The outer membranes of gram-negative bacteria present the further challenge that the protein profile is dominated by relatively few proteins, limiting the detection of low-abundance proteins, regardless of the approach taken (see Discussion). We believe that more than one method will need to be applied to obtain a comprehensive picture of the surface proteome of Y. pestis. In this study, we applied two approaches: the identification of immunogenic species in 2-D separations of a Sarkosyl-based preparation and a novel biotinylation method that focuses on the identification of proteins that are exposed to the surface. We validated the methods for proteins with known localization patterns and applied them to evaluate the surface localization of the 79-kDa catalase KatY (encoded by Sanger Centre coding sequence YPO3319 of Y. pestis CO92). Originally discovered by Crumpton and Davies as antigen 5 (13), KatY is a major antigen that is present in Y. pestis cells grown at 37°C and was among the proteins identified by sera from rabbits immunized with live Y. pestis EV76 (27). Garcia et al. and Mehigh and Brubaker (18, 30) characterized it biochemically, reported that it is a weak catalase, and gave it its name. Interestingly, KatY is a major species among a set of virulence-associated proteins that are expressed at 37°C under conditions yersiniae may experience within tissues during bubonic plague (8, 12, 18). Accordingly, KatY is of great interest for vaccine development, because it may be surface located and antibody against it may protect against plague.