The project aims to develop and test a novel fluorescent marker that we have been working on. This is called LOV, which stands for 'Light, Oxygen, Voltage'. We work on a form of LOV which emits a strong green fluorescence upon blue light excitation. We believe that LOV might act as an alternative to green fluorescent protein (GFP), used extensively in biology for many applications including the study of protein folding, protein transport, RNA dynamics and responses to infection. In this project we will test to see if LOV can be used to 'tag' proteins that are made by bacteria and then injected into host cells by a dedicated secretion system. The project is strengthened by using expertise from 3 different lead scientists: Dr Chrisitie is a biochemist who has led research on the application of LOV in novel areas of biology, Dr Roe is a microbiologist working on gene regulation and bacterial-host cell interactions and Dr Enninga, a named collaborator, is a specialist in imaging bacterial proteins.

Technical Abstract:
This proposal is focused on the development of a fluorescent reporter than will provide novel insights into the regulation and function of E. coli O157 effector proteins. The application and use of this reporter will aid our study of E. coli O157 and will provide the scientific community with a powerful new reporter system. Recent work has revealed some 39 proteins capable of being exported through the E. coli O157 type three secretion system (T3SS). This raises the question of how bacteria control the expression and secretion of so many proteins to ensure that they are delivered at the correct time for their desired biological effect. The sheer variety of T3SS effectors with diverse roles such as adherence or anti-phagocytosis implies a requirement for secretion hierarchy. Recent work supports a model in which the injection of effectors is a temporally-regulated process to exert precise effects on host cells. Therefore, new tools to monitor effector protein translocation and host cell localisation will allow these temporal processes to be fully examined and understood. The ideal scenario would be to monitor the real-time secretion of effectors from bacterial production to their final localisation within a host cell. To this end, we are working towards a new fluorescent reporter fusion that provides significant advantages over existing technologies. Based on the light-oxygen-voltage sensing (LOV) domain, this reporter is small in size, non-toxic, strongly fluorescent and does not interfere with secretion and function of effector proteins. Our preliminary work has generated improved version of LOV, termed iLOV that form the basis of this application. In this project we will (1) further improve iLOV photostability and fluorescence (2) generate selected effector-iLOV fusions (3) test the real-time secretion and host cell localisation of these effector proteins in collaboration with Dr Enninga at the Insitut Pasteur.

Potential Impact:
Our preliminary work has already shown that LOV fluorescent proteins (LOV-FPs) have the potential to provide a novel method to track bacterial effector proteins in real-time. Bacterial-host cell interactions are studied extensively using numerous different pathogens and models of infection and we feel that the approaches outlined in this proposal could be widely adopted and be of benefit to many researchers in this field. We also feel that the work has the potential for further improving the fluorescent properties and utility of LOV FPs as fluorescent biomarkers for wide-ranging cell biological applications. As outlined in the academic beneficiaries section, there is the potential to apply LOV-FPs to many different fields including biotechnology, pathogenesis and cell biology. Therefore, the development of enhanced, photo-stable LOV proteins may bring many long-term benefits well beyond the immediate scope of this project. For example, our initial studies have demonstrated that LOV can act as a useful marker of protein production and purification which may enhance the large-scale isolation of numerous proteins of biological importance including membrane proteins which are notoriously difficult to isolate in large quantities. Overall, we feel the project will allow further development of novel reagents which will provide a useful tool for the scientific community with many wide-ranging applications.