Recognition by the type II secretion system and how it enables Legionella pneumophila to thrive

Bacteria are tiny organisms that are present in a wide range of environments on the earth. They can also live within humans and animals where they have both positive and negative benefits to health. In this application I propose a number of experiments to understand certain aspects of how some bacteria cause disease and persist in the environment. Specifically I will be studying Legionella pneumophila, a bacterium that is readily found in both natural and man-made water environments. This bacterium infects the human lungs and causes Legionnaires' disease, an often-fatal type of pneumonia, and Pontiac fever, a milder flu-like disease. Man-made infrastructures that store and distribute water are ubiquitous in Western society and as such Legionella are found in many large buildings (including hospitals and hotels), with the number of Legionnaires' disease cases on the increase. The high prevalence of L. pneumophila within the environment is due to its ability to survive inside biofilms: complex multispecies bacterial masses encased in a defensive layer. Here the inhabitants are protected from the environment, other organisms and antibacterial compounds. However, water-borne amoebae can still graze on these bacteria, although L. pneumophila has developed strategies to survive by going inside them, evading their detection and hiding away from any attack. Unfortunately, some types of mammalian lung cells share similarities with various amoeba strains and this is why Legionella is an opportunistic pathogen that can cause disease when humans come into contact with contaminated water.

Legionella use a 'type II secretion system' (T2SS), a syringe-like mechanism to transport protein substrates into their surroundings. These substrates promote the formation of biofilms and enable L. pneumophila to become fully virulent. They have also been shown to contribute to this bacterium's extensive host range. For example, at least 18 strains of amoeba are the natural hosts of L. pneumophila creating a substantial environmental reservoir of this pathogen. For some of these substrates, however, we do not understand exactly what they target or what their specific roles are, although several have been linked to disease and the ability of L. pneumophila to infect a broad range of amoebae. Analysing which of these proteins are important for these processes will be one aspect of what I shall be investigating but my main objective is to study how L. pneumophila recognizes these substrates before they are secreted. Understanding the details of how this T2SS deploys these substrates will provide the foundations to design compounds that can disarm it. Furthermore, this type of secretion system is also essential for many other human bacterial pathogens, to discharge toxins for example, into the host and cause disease. Therefore in turn, these studies may also reveal a common novel pathway to combat other types of bacterial infections in the future.

Legionella pneumophila is an opportunistic Gram-negative bacterium, ubiquitous in natural and anthropogenic freshwater environments. It is the causative agent of Legionnaires' disease, an often-fatal pneumonia, and Pontiac fever, a milder flu-like disease. Man-made infrastructures that store and distribute water are omnipresent in Western society and these bacteria can be found in many large buildings, including hospitals and hotels. The high prevalence of Legionella within the environment is due to its ability to survive within biofilms or as an intracellular parasite of biofilm associated protozoa. L. pneumophila uses a type II secretion system (T2SS) to translocate substrates across its outer membrane, where they support biofilm formation, replication in the host, dampening of cytokine output and survival in mammalian lungs. A number of these have novel amino acid sequences and have been implicated in promoting broad host tropism and persistence of disease. As these are exclusive to Legionella I have called them Unique Legionella Proteins (ULPs). The T2SS is also used by other human pathogens (e.g. Chlamydia trachomatis, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., Yersinia enterocolitica). Translocation involves a number of recognition events with substrates identified via a 'conformational' motif; but how can a single transport apparatus identify multiple unique substrate structures? I plan to (i) use these ULPs to investigate the molecular details of their recognition in the periplasm prior to export; and (ii) ascertain which ULPs are involved in disease or ecology. Whilst these studies will provide insight into new mechanisms related to Legionella fitness, the major outcome of this research will be to advance of our limited knowledge of T2SS/substrate recognition. In turn this may provide the bedrock for targeted development of sustainable prevention and control strategies for a range of infectious diseases.

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, which 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 translocation through secretion systems, bacterial pathogenesis and biofilm formation.
(b) The outcomes might reveal new insights into protein translocation events across biological membranes, biological processes that increase the fitness of Legionella 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 formation. 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 substrate translocation by the type II secretion system in the development of novel compounds to reduce bacterial virulence and in turn increase the activity of existing antibiotics. 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 technician 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. 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/technician. Members of the general public benefit through outreach events.