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

Lead Research Organisation: Queen Mary University of London
Department Name: Sch of Biological and Chemical Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description A multidisciplinary approach to identify new antibacterial targets within biofilms
Amount £100,000 (GBP)
Funding ID SBF002\1150 
Organisation Academy of Medical Sciences (AMS) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2017 
End 09/2019
 
Description EPSRC
Amount £60,000 (GBP)
Organisation Queen Mary University of London 
Sector Academic/University
Country United Kingdom
Start 09/2016 
End 03/2020
 
Description Structural studies into novel membrane associated virulence factors of Legionella pneumophila
Amount £460,082 (GBP)
Funding ID MRR0176621 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2019 
End 04/2022
 
Description Arianna Fornili 
Organisation Queen Mary University of London
Department School of Biological and Chemical Science QMUL
Country United Kingdom 
Sector Academic/University 
PI Contribution microbiology/structural biology
Collaborator Contribution structural biology (MD)
Impact doi: 10.1371/journal.ppat.1008342 https://doi.org/10.1038/s41522-022-00272-5
Start Year 2015
 
Description Prof. Mike Curtis 
Organisation Queen Mary University of London
Department Barts and The London School of Medicine and Dentistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Expanding our understanding of the type II secretion system to look at other secretion systems. Preliminary structural data for a new grant proposal looking at the type IX secretion system.
Collaborator Contribution Preliminary microbiology data for a new grant proposal looking at the type IX secretion system
Impact None so far - grant application to be written by the end of 2017
Start Year 2016
 
Description Prof. Nick Cianciotto 
Organisation Northwestern University
Department Feinberg School of Medicine
Country United States 
Sector Academic/University 
PI Contribution Structural and biochemical studies of proteins directly related to this grant
Collaborator Contribution Microbiology and mutation analysis to verify data produced in my lab
Impact Paper re currently in preparation but have not yet been submitted
Start Year 2015
 
Description Prof. Ten Feizi 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution protein structure determination
Collaborator Contribution carbohydrate analysis
Impact papers in preparation
Start Year 2015
 
Description Nuffield Research Placement of A-level student in the lab 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 4 week research placement of A-level student from Sir George Monoux College where the student performed cloning, protein purification and interaction studies related to the MRC grant.
My interactions with the School over the last 4 years has now contributed to them being awarded 'STEM Assured' status by the New Engineering Foundation: The Innovation Institute.
Year(s) Of Engagement Activity 2015
 
Description Nuffield Research Placement of A-level students in the lab 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Two A-level students undertook a 4 week research placement in my lab
Year(s) Of Engagement Activity 2017
URL http://www.nuffieldfoundation.org/nuffield-research-placements
 
Description Nuffield Research Placement of A-level students in the lab 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Two A-level students undertook a 4 week research placement in my lab
Year(s) Of Engagement Activity 2016
URL http://www.nuffieldfoundation.org/nuffield-research-placements
 
Description Participation in an open day or visit at my research institution - Nuffield Research Placement of A-level students in the lab 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Nuffeild research project placement
Year(s) Of Engagement Activity 2019
 
Description primary school visit (St Mary Magdalene academy, Islington) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact - outreach activity on antibiotics, bugs and cleanliness. consisted of talking and engagement activities. lasted approx 3 hrs
Year(s) Of Engagement Activity 2019