The Salmonella SPI-2 type three secretion switch

Salmonella is a bacterial pathogen that causes many important diseases in man (including typhoid fever) and veterinary livestock. The bacteria replicate inside host cells, in a membrane-bound compartment called a vacuole. In order to replicate, bacteria must interfere with normal cellular processes. They accomplish this by assembling a needle-like structure on their cell surface, through which virulence proteins can be discharged across the vacuole membrane into the host cell. But before this happens, some other bacterial proteins pass through the needle and make a small hole or 'pore' in the vacuole membrane. During this time, virulence protein secretion is held in check by a specific 'safety catch', within the bacterial cell. We found recently that once the pore is assembled, the bacteria recognize the mild pH of the host cytoplasm and exploit it as a signal to trigger disassembly of the safety catch, which then allows virulence proteins to be delivered. This process is absolutely crucial for the bacteria, because if they cannot deliver these proteins they cannot replicate. Therefore the purpose of this proposal is to study, at a detailed molecular level, the way in which the safety catch responds to the pH signal. The approaches that we will take to tackle this question are well established in my laboratory, and the results may have potential applications in the design of vaccines against Salmonella and also for new antibiotics that interfere with the ability of the bacteria to respond to the pH signal.

The purpose of this proposal is to increase our understanding of the mechanism underlying secretion of translocon and effector proteins by the SPI-2 type III secretion system, and the switch from translocon to effector secretion, which is controlled by the SpiC/SsaM/SsaL regulatory complex. The major objectives are (1) to understand how translocon proteins are recognized for secretion by the complex, (2) to identify the pH sensor and (3) to understand how the signal is transduced to cause regulatory complex disassembly and effector secretion. We can induce the secretion switch artificially in bacteria exposed to media of different pH, and this system will be exploited using bacterial strains expressing mutant and epitope-tagged versions of relevant proteins, cellular fractionation, immune precipitation, conventional and structural biochemistry, microscopy, and genetic screens.

Beneficiaries: Key potential beneficiaries of the proposed research are: Academics - who will be the main short to medium term beneficiary, as the research will provide knowledge, reagents and new methods to study type III secretion systems of bacterial pathogens. Type III secretion by Salmonella is a very active area of research with many international groups involved; we receive requests for strains and reagents on a regular basis. The project will provide opportunities for career development and training of researchers. The research will enhance the career development of Dr. Yu (named Research Co-Investigator), who designed, executed and interpreted a significant amount of the background research, and who made a substantial contribution to the formulation of this proposal. It is hoped that he will eventually gain an independent position within a UK University, or in the biotechnology/pharmaceutical industry. He provides invaluable guidance and advice to junior researchers, overseas interns, Undergrads, Masters and PhD students. Dr Yu will present the results of his work at national and international conferences. Industry - Holden co-founded the biotech company Microscience. This grew to employ approximately 120 people before it was acquired in 2005 by Emergent Biosolutions, which has approximately 75 employees at the former Microscience site. These companies exploited research from Holden's group to develop a live attenuated SPI-2-based Salmonella typhi strain as a typhoid vaccine. The work described in this proposal is fundamental research into the molecular mechanisms that underlie the delivery of bacterial virulence proteins into the host cell. As such it is not reasonable to expect an immediate impact in terms of a new drug or vaccine to combat bacterial disease. It is possible that future work on the structure of proteins that control this process will enable the design of small molecule inhibitors. These could be tested to see if they can inhibit disease progression in infection models, and promising candidates could be patented. IC has a Drug Discovery Centre whose remit is to translate early research into drug discovery projects. Holden is a named inventor on several patents, is familiar with the processes of licensing materials and intellectual property. Through its technology transfer company, IC has all the support and expertise for patent applications and contract negotiations. Communications and Engagement: Knowledge of value to the academic sector will be communicated in a timely manner by publication in peer-reviewed journals, oral and poster presentations at conferences and via invited lectures at Universities and other research institutions. IC has a press office responsible for the dissemination of science through the media and with grant giving agencies. Newsworthy publications are given prominent coverage on the IC website. The CMMI (the bacterial infection biology Centre at IC of which Holden's group are members) also has an open-access website which is updated regularly with research highlights. In the past, publications from Holden's lab have received coverage from national and international newspapers, and he has been interviewed on the BBC radio 4 Today programme. Collaboration: The main collaborative interaction will be with Profs Matthews and Freemont of the Centre for Structural Biology, IC. We have already co-authored papers with Freemont (Cell Microbiol. 2008 10:20-30; Proc Natl Acad Sci U S A. 2007 104:3502-7). We continue to have an excellent collaboration with Meresse and his group at the CIML, Marseille, France, as well as many other laboratories around the world. We share reagents and ideas freely with these groups and there are regular visits by students and post-docs back and forth between laboratories. Resources for the activity: Funding for conference attendance and publication costs will be required, and is detailed in the justification of resources section.