;Structural studies into novel membrane associated virulence factors of Legionella pneumophila

Bacteria are tiny organisms that live in a wide range of environments on the earth, including humans and animals, where they have both a positive and negative impact on health. In this application, we propose a number of experiments that will help us to understand certain aspects of how dangerous bacteria are able to persist in the environment and cause disease in humans. We will specifically study the bacterium Legionella pneumophila, which is ubiquitous in aquatic systems (e.g. rivers, reservoirs, hot/cold water supplies, cooling towers) and highly prevalent in large buildings such as hospitals and hotels. L. pneumophila causes Legionnaires' disease (an often-fatal pneumonia), and Pontiac fever, (a milder flu-like disease) and rates of infection are increasing each year, both in the UK and globally. Within the environment L. pneumophila lives within biofilms where it clumps together with other bacteria and is covered in a defensive mesh. This protects it from external factors such as dehydration but also from attack by other organisms and antibacterial compounds. However, single celled organisms called amoebae can still graze on these bacteria and L. pneumophila has developed strategies to survive by going inside them and hiding away from attack. Once inside these hosts, L. pneumophila lives within a membrane-bound compartment (the Legionella containing vacuole; LCV), where it evades detection. Unfortunately, some types of human lung cells share similarities with amoebae and therefore Legionella causes disease when humans come into contact with contaminated water.

L. pneumophila secrete many proteins outside of the bacterium that allow it to sense the outside world, interact with other organisms and also manipulate host amoebae and human cells so that they can survive inside them. For example, the 'type II secretion system' (T2SS) uses a syringe-like mechanism to export proteins that help form biofilms and enables L. pneumophila to become fully virulent. We have identified a unique class of these proteins that once exported are able to either bind to the surface of L. pneumophila, helping it stick to other bacteria in biofilms and recognize host cells; or, when inside a host, bind to the Legionella containing vacuole, helping it to become camouflaged so that it is not detected. Understanding the details of how these proteins function and why they localize to their specific membranes will be crucial to further our knowledge of L. pneumophila infection. Likewise, these studies may also reveal common pathways for infectious disease used by other bacteria, which may in turn help us design compounds which disarm L. pneumophila and other dangerous pathogens.

Legionella pneumophila, is a facultative Gram-negative bacterium, which is ubiquitous in aquatic systems and highly prevalent in large buildings such as hospitals and hotels. In the environment L. pneumophila lives within biofilms or as an intracellular parasite of biofilm-associated protozoa. However, upon entering human lungs via aerosolized droplets, it can also invade and replicate inside resident macrophages and epithelial cells. L. pneumophila causes Legionnaires' disease, an often-fatal pneumonia, and Pontiac fever, a milder flu-like disease; and rates of infection are increasing each year, across the UK and globally. After invasion of eukaryotes, L. pneumophila resides within a membrane bound replication vacuole (the Legionella containing vacuole; LCV) and exports many proteins into the host cytoplasm, which allows it to replicate to large numbers.

L. pneumophila uses a type II secretion system (T2SS) to translocate substrates across its outer membrane. These support biofilm formation, replication in the host, dampening of cytokine output and survival in mammalian lungs. In L. pneumophila, these substrates can be targeted to the bacterial surface during extracellular growth but they are also exported into the host cytoplasm during intracellular infection and can localize to the LCV surface, which represents a new role for the T2SS. These substrates carry out novel functions and are important for both intracellular and extracellular growth of L. pneumophila. We plan to (i) investigate the molecular details of how these substrates function to enable L. pneumophila infection/persistence and (ii) ascertain the atomic mechanisms by which they are targeted to their respective membranes. These studies will provide insight into new mechanisms related to Legionella fitness and disease and may have implications for the development of novel strategies to disperse biofilms and reduce bacterial virulence.

This proposal embraces the scientific aims of the MRC through studying disease-related proteins with a view to basic biological understanding and assisting therapeutic avenues of exploration. This is cutting edge interdisciplinary research and has the potential for substantial economic benefit. It will also maintain the supply of quality research expertise in structural, chemical and microbiology in the UK whilst enabling and promoting knowledge transfer between academia and companies. The following beneficiaries have been identified (a), and methods of how they will benefit are detailed (b).

Beneficiary One
(a) Members of the wider academic community investigating mechanisms of protein targeting, host manipulation, bacterial pathogenesis and biofilm formation.
(b) The outcomes might reveal new insights into protein localization to membranes, biological processes that increase the fitness of bacteria in the environment, novel mechanisms of invading hosts/modulating host responses to infection, promoting intracellular replication within a host and new ways of modulating biofilm growth. This could thereby stimulate research in diverse systems.

Beneficiary Two
(a) Large pharma (e.g. Pfizer, Novartis, GSK), smaller Biotech companies (e.g., Novacta, Biotica) and not-for-profit organisations (e.g. Cystic Fibrosis Foundation, Terrence Higgins Trust, UNICEF, Oxfam).
(b) Outcomes might benefit the commercial sector by providing a new structure-based understanding of biofilm formation and host manipulation by intravacuolar pathogens in the development of novel compounds. Benefits for not-for-profit organisations would be in the form of potential new treatment strategies.

Beneficiary Three
(a) Public sector health professionals.
(b) Clinicians and senior health providers benefit from an improved understanding of where key medicines, such as antibiotics, come from and how they act. Also, outcomes might reveal improved strategies for the HSE to deal with water infrastructure sustainability.

Beneficiary Four
(a) International development.
(b) In the long-term the outcomes might play a significant role in the development of novel treatments for bacterial diseases, which cause suffering and economic harm to a majority of the world's population (including both developing and developed nations).

Beneficiary Five
(a) Skills, training and knowledge economy.
(b) The PDRA and any undergraduate, postgraduate, or part-time students that contribute to the project will be trained with key interdisciplinary skills that will be extremely valuable for UK industry. For example, they will be trained in structural biology techniques and L. pneumophila infection of amoebae/macrophage. The PDRA will present data at national and international conferences/symposia. Along with the PI, they will contribute to the knowledge economy and increase the economic competitiveness of the UK.

Beneficiary Six
(a) Members of the wider general public.
(b) Students and teachers from schools and colleges benefit through engagement with the PI/PDRA. Members of the general public benefit through outreach events.