Effectors of the Salmonella SPI-2 Type III secretion system

Salmonella species are important bacterial pathogens causing a variety of human diseases, such as gastroenteritis and typhoid fever. Salmonella can invade and grow inside the cells of its host, including specialised white blood cells (macrophages) whose function is to destroy potential pathogens. Having entered host cells, Salmonella resides within a membranous sac (vacuole), and transfers through it a large number of virulence proteins. We know that several of these proteins enable bacterial growth inside the vacuole by detoxifying host defences and by acquiring nutrients. However other proteins, about which we know very little, block the ability of host cells to send alarm signals to other cells to mount an immune response. The proposed research involves detailed investigations into the functions of these 'anti-immune' virulence proteins. A key objective is to identify the cellular 'targets' with which they must interact to interfere with the host. To do this we will take advantage of very powerful modern techniques and instruments that can detect very small changes in the very complex environment of the infected cell. Through this research we are likely to discover new processes of pathogen and host cell biology, which could have implications for other important pathogens that propagate within our cells. Our work is also likely to provide valuable information for designing vaccines, which are still needed to provide effective long-term protection against Salmonella and other bacterial pathogens.

Serovars of Salmonella enterica are important intracellular bacterial pathogens that cause a variety of human diseases, including gastroenteritis and typhoid fever. After internalisation by host cells into a vacuole, S. enterica produces a surface exposed type III secretion system (T3SS) encoded by the SPI-2 pathogenicity island; this needle-like structure and its translocon transfer approximately 30 different effectors across the vacuolar membrane into host cells. A major function of the SPI-2 T3SS is to enable intracellular bacterial replication and we have made significant progress on understanding the biochemical activities of some of the associated effectors. Other effectors seem to be involved in processes other than intracellular bacterial replication. The purpose of this proposal is to gain an understanding of their functions, through four specific objectives. First, we will characterize the mechanism of action of SpvD, an effector that appears to inhibit the host cell NF-kB pathway in a novel way. Second, we will identify effector(s) that interfere with presentation of MHC class II molecules and then study their mode(s) of action. Third, we will conduct host cell RNA-seq screens in macrophages infected with mutant strains to identify changes in mRNA levels, to provide information on effector function. Fourth, we will use state of the art proteomics techniques to identify host proteins that interact with effectors following their delivery into cells via the physiological route, in the context of infection. Further analysis of effector function will involve microbiology, molecular cell biology, biochemistry, and in vivo imaging. We will be aided by collaborations with experts in RNA-seq and mass spectrometry. Through this research we will gain a deeper understanding of the virulence mechanisms of an important intracellular pathogen. It could also help in the design of improved vaccines against Salmonella infections and possibly other diseases.

Academic beneficiaries are described in another section. Other beneficiaries could include the private sector companies who are interested in developing Salmonella-based vaccines, both against typhoid fever and other diseases. In the UK, gastroenteritis caused by Salmonella remains a significant public health problem. Worldwide, it is estimated that hundreds of thousands of deaths occur each year due to typhoid fever and similar levels of mortality result from non-typhoidal Salmonella infections. In both cases, multi-drug resistant bacterial strains have become a significant problem. Current typhoid vaccines offer only partial efficacy rates. A World Health Organization position paper on typhoid states "improved vaccines against typhoid fever are desirable. Such vaccines should confer higher levels and more durable protective immunity in all age groups, including those aged <2 years, preferably without the need for booster doses."'.

Our work has the potential to improve quality of life (enhancing health quality in the developing world) as we expect that it will have applications in vaccine design. Work on which this application is originally based led to the formation of the UK-based vaccine company Microscience, of which I was a co-founder. Microscience grew from being a very small University spin-out company to employ over 70 people at their site in Berkshire, before being acquired in 2005 by Emergent BioSolutions, a USA-based vaccine/therapeutics company. It therefore had an economic benefit in helping to create jobs and company value. Microscience developed a single oral dose vaccine candidate against typhoid, based on a Salmonella typhi SPI-2 mutant strain. Several successful clinical trials have been conducted using this strain. Rights to its use were acquired recently by Prokaryium (http://prokarium.com). They are exploiting it to develop a dual oral vaccine against typhoid and enteropathogenic E. coli. Greater knowledge about the functions of the SPI-2 effectors will be gained from the planned research and could be invaluable in optimising Salmonella-based vaccines. The applicant has extensive experience in patenting and licensing of intellectual property. It is not possible to predict with certainty if valuable intellectual property will emerge from the planned work, but if commercially exploitable discoveries are made, I and Imperial College's technology transfer company Innovations are well placed to ensure that it is protected properly by patents, and exploited along the lines indicated above.