Yersinia pestis Proteomics Collaboration

The tasks for Dr. Perry's research team consist of laboratory experiments to be performed at the University of Kentucky (part 1) and advisory services for the PFGRC as it pertains to growing non-virulent Y pestis strains (at TIGR), the performance of cellular separation and analysis procedures as well as proteomic data interpretation (parts 2 and 3). The first part includes laboratory experiments to be performed in a BSL-3 facility according to CDC regulations. Significant costs associated with laboratory work with the human pathogen Yersinia pestis at the BSL-3 level and the delivery of services not measurable in the form of materials or products require a subcontract in this collaboration funded by the PFGRC. Part 1: A non-virulent Ler- Yersinia pestis strain exempt trom the CDC Select Agent List regulations will be sent to TIGR's Pathogen Functional Genomic Resource Center (PFGRC). An avirulent Ler - Y pestis strain will be grown in liquid chemically defined medium (PMH2) under iron-deficient conditions at 26°C and 37°C. Culture supernatants and cells from PMH2 will be harvested. Culture supernatants will be concentrated in Millipore membrane concentration units. Cells will be subjected to a bacteriocidal cocktail of antibiotics, which is a necessity for the fully virulent Yersinia strains (as later described) and frozen at -80°C. The cells (1-2 g of wet cell weight after centrifugation) and the culture supernatant concentrate will be sent to the PFGRC. An avirulent Lcr+ L1pgm Y pestis strain will be grown in PMH2 at 37°C without calcium. Its supernatant will be harvested from a high density liquid culture. This will demonstrate whether PMH2 is suitable for optimum secretion of proteins. A part of the concentrated cell culture supernatant will be sent to the PFGRC for proteomic analysis. Repeats of these two growth conditions may be required during the developmental phase. This task also depends on the success level in the PFGRC laboratory to grow the avirulent Y pestis strain. PMH2 cultures of the virulent Yersinia pestis strain C092 using two different growth conditions (37°C in presence and absence of iron) will be grown in vitro. In each case, cell pellets are harvested and treated according to the bacteriocidal procedure applied for the non-virulent strain and viability tests are conducted to ascertain inactivation of the virulent Y pestis cells. The cell pellets will be frozen .at -80°C. The scale of these cultures is somewhat dependent on the method development results. It is anticipated that 1 g wet cell weight will need to be harvested and inactivated from the liquid culture of virulent Y pestis strain C092. These experiments may need repetition, but they should only be considered a part of the contractual agreement upon further review. PMH2 cultures of the virulent Y. pestis strain C092 using four different growth conditions (26°C and 37°C in presence and absence of calcium) will be grown. For the 37°C conditions, cells will be initially grown at 26°C and then shifted to 37°C (this allows initial unrestricted growth). For the 37°C minus calcium condition, two samples will be taken after the temperature shift. This will allow distinguishing between residual 26°C proteins, 37°C and calcium deficiency induced-proteins from the protein profile due to growth restriction that occurs in the absence of calcium. In each case, cell pellets are treated according to the bacteriocidal procedure applied for the non-virulent strain and viability tests are conducted to ascertain inactivation of the virulent Y. pestis cells. Cell pellet will be frozen at -80°C. The scale of these cultures is somewhat dependent on the method development results, but it is anticipated that 1 g wet cell weight will need to be harvested and inactivated from the liquid culture of virulent Y. pestis strain C092. These experiments may need repetition, but they should only be considered a part of the contractual agreement upon further review. The virulent Y. pestis C092 strain will be grown in PMH2 or in another suitable medium at 37°C without calcium and at 37°C without iron. The supernatants will be harvested from high density cultures. The lack of viable cells in the collected supernatants is to be demonstrated. The supernatants are to be concentrated in membrane concentrators before shipping. In vitro cultures of the virulent Yersinia pseudotuberculosis strain PBl/+, (serotype 0: 1b) using four different liquid culture growth conditions (in presence and absence of iron at 37°C and in the presence and absence of calcium at 37°C) are to be grown. In each case, cell pellets are treated according to the bacteriocidal procedure applied for the Y. pestis strains. Viability tests are to be conducted to ascertain inactivation of the virulent Y. pseudotuberculosis cells (if necessary according to CDC regulations). Cell pellets are frozen at -80°C. The scale of these cultures should be the same as for the Lcr -Yersinia pestis strain (1- 2 g wet cell weight, if necessary). These experiments may need repetition, but they should only be a considered a part of the contractual agreement upon further reVIew. Dr. Perry will send small quantities of antibodies specific to Y. pestis proteins. These antibodies will be used to monitor the quality of subcellular Y. pestis fractionation procedures. Part 2: Dr. Perry will provide protocols and advice as it pertains to growing and maintaining the non-virulent strains in liquid culture and on semi-solid media, to performing and evaluating subcellular fractionation procedures. Part 3: Dr. Perry will assist in the interpretation of the proteomic profiling data. Dr. Perry's wealth of experience in Y. pestis molecular biology and pathogenesis is important to unravel and identify the causes and consequences of proteome abundance data (protein up- or down-regulation processes). The primary purpose of this task is to make such data available to the NIAID as the recipient of this contract research and the secondary purpose of this task is the publication in peerreviewed journals.