Dissecting the modulation of nucleocytoplasmic signalling, host cell cycle and differentiation by a new family of Legionella protease effectors

Lead Research Organisation: Queen's University of Belfast
Department Name: Medicine Dentistry and Biomedical Sci

Abstract

Legionella pneumophila and related bacteria are environmental microbes, which can contaminate man-made water systems, such as air conditioning systems or spa-pools. Accidental inhalation of contaminated water by humans can result in a severe lung infection called Legionnaires' disease. Infections can be limited to individual persons, but, if contaminated water droplets are distributed over wide areas with airflow, can also elicit outbreaks with dozens or hundreds of cases (e.g. New York, USA in 2015, and Spain, 2016).

The elderly, immunocompromised people and persons with underlying respiratory conditions are at the most risk, but the exact defects in the human body that predispose to infection, and the weapons bacteria employ to overcome human immune defences are not well defined.

L. pneumophila uses a sophisticated transport machinery to inject a large number of proteins, so called effectors, into human macrophages; cells of the immune system, which are dedicated to kill bacterial intruders. These effectors manipulate the normal functions of macrophages allowing bacteria to persist and multiply inside the cell until the resources are exhausted and a large number of bacteria are released, amplifying the infection and causing damage to the lung. Understanding the function of effectors and finding the weak points of human macrophages is key to develop new, much needed, drugs to treat infections with Legionella and other bacteria that exploit macrophages to cause disease.

Our research focusses on elucidating the role of a new family of effectors that manipulate human cells. We found that these effectors have the powerful ability to cleave human proteins, inactivating them or changing their function. Our preliminary experiments reveal that two of these effectors target either an important signalling relay, which allows human cells to adjust and respond to signals from other cells, or the machinery controlling cell division and growth. Both of these processes are central to cell homeostasis and communication. In this project, we aim to reveal the detailed mechanisms and consequences of this manipulation for the function and defence responses of the human macrophages and for the potential of the bacteria to cause disease; promising to unlock new ways to combat bacterial infections.

Technical Summary

We identified a new family of Legionella Dot/Icm type IV secretion system effectors with a protease and an Ankyrin-repeat domain, which are widely distributed across the Legionella genus. We hypothesise that these effectors play an important role in the interaction of Legionella spp. with phagocytic cells. Substrate trapping experiments revealed that one of them targets nuclear transport and a receptor involved in Wnt-signalling, while the other effector cleaves itself, localises to the nucleus and seems to interfere with the cell cycle. This project aims to reveal the function of these proteases and their targets during infection. We will:
1. Define the cleavage specificity of the proteases by affinity purification and N-terminal protein sequencing or intact protein by mass spectrometry of the cleavage products.
2. Generate stable, inducible cell lines for the effectors and survey protease substrates at the proteome level using ProtoMAP and Terminal amine isotopic labelling of substrates (TAILS).
3. Validate binding of inactive proteases to known and newly identified substrates by yeast-two hybrid assay, ELISA or co-immunoprecipitation.
4. Investigate the fate of cleaved substrates during infection using Western Blot and follow their localisation by immunofluorescence microscopy (IF) and live cell imaging.
5. Create L. pneumophila protease mutants or deplete the protease substrates by gene-editing or RNA interference and study the effect on intracellular growth and trafficking, NF-kB activation, nuclear and whole cell proteome and cytokine response using microplate assays, IF, qRT-PCR, ELISA and quantitative proteomics.
6. Assess how Legionella modulates Wnt-signal transduction after stimulation of infected cells or cells ectopically-expressing the effectors with Wnt-ligands using qRT-PCR, luciferase reporters and Western Blot.
7. Dissect the effect of Legionella and the protease effector on the host cell cycle using IF, Western Blotting and flow cytometry.

Planned Impact

Our project aims to reveal the molecular processes that enable the bacterial pathogen Legionella pneumophila to subvert environmental protozoan hosts and human pulmonary macrophages, which causes Legionnaires' disease, a severe, potentially fatal pneumonia. Legionella spp. cause sporadic infections and are associated with app. 4% of severe community and hospital acquired infections, but can also cause outbreaks with dozens or hundreds of victims. Transmission occurs via aerosols from contaminated water systems, such as air conditioners or cooling towers. The elderly, immunocompromised or patients with underlying respiratory conditions are at the most risk of infection.

In the short-term, the findings of this fundamental research, which we will disseminate through publication in peer-reviewed journals, presentation at conferences and the media, will primarily generate impact on the scientific community and the UK knowledge-based economy by
1. advancing the field of Legionella and macrophage research and, as Legionella stands exemplary for many other bacteria, such as Salmonella and Mycobacterium tuberculosis, which exploit macrophages to cause disease, the field of microbial pathogenesis research in general.
2. building capacity e.g. at Queen's University Belfast and in Northern Ireland in proteomics methodology and cellular microbiology research.
3. providing the PDR, who will be recruited for this project, as well as PhD and project students in the Schroeder lab with training and career development opportunities.

Moreover, although not expected at this stage of the research, the project could uncover e.g. diagnostic markers for Legionella infections or drug targets with development and commercial potential. In this case, we will liaise with the QUB research and development office, the biopharmaceutical industry and healthcare providers to maximise the socio-economic benefit.

We are convinced that this project will generate important knowledge, which will lay the foundation for further discoveries in the mid and long-term that will pave the way to new host-directed therapies to treat Legionella and other bacterial infections. These new treatments will be an important component in the global fight against antimicrobial resistance and as such, will have substantial impact on health service policies and clinical practice, improving the quality of life and life expectancy of the Legionella risk groups and the general population.

Publications

10 25 50
 
Description Dissecting the molecular pathogenesis of Legionella spp. in human lung models - OPTIMISE
Amount € 212,934 (EUR)
Funding ID MSCA-IF-2018 - Individual Fellowships - Grant agreement ID: 844197 
Organisation European Commission H2020 
Sector Public
Country Belgium
Start 08/2019 
End 07/2021
 
Description MSci in Biochemistry - Student Research Project
Amount £2,000 (GBP)
Organisation Queen's University Belfast 
Sector Academic/University
Country United Kingdom
Start 10/2020 
End 04/2021
 
Description MSci in Biochemistry - Student Research Project
Amount £2,000 (GBP)
Organisation Queen's University Belfast 
Sector Academic/University
Country United Kingdom
Start 10/2019 
End 04/2020
 
Title Reporter Celllines for Intracellular Trafficking of Pathogens 
Description We have generated stables cell lines, e.g. A549 lung epithelial cells and THP-1 macrophages, expressing fluorescently tagged protein markers for phosphoinositide phosphates (PI4P and PI4,5P) or lysosomes (Lamp-2), which facilitate the visualisation of the trafficking of intracellular pathogens in host cells by fluorescence microscopy. 
Type Of Material Cell line 
Year Produced 2018 
Provided To Others? No  
Impact These cell lines enable my group now to follow Legionella-pathogen interaction by live-cell imaging, significantly enhancing our ability to resolve the time course of the infection process. We anticipate that they will be of use for many researchers working on host-pathogen interactions and basic cell biology in the future. 
 
Description Madrid Yeast Team 
Organisation Complutense University of Madrid
Country Spain 
Sector Academic/University 
PI Contribution We contacted our collaborator, who is a specialist in yeast biology, to support our effort to run yeast cytotoxicity assays to identifiy host cell targets of Legionella effectors. We shared preliminary data of yeast cytotoxicity assays with him, demonstrating that expression of our effector of interest has a phenotype in yeast, and provided yeast expression plasmids for further experiments in his laboratory. We will further perform Legionella infection assays and ectopic expression experiments in mammalian cells based on the information, e.g. potential host targets, delivered by our collaborator.
Collaborator Contribution Our collaborator in Madrid is a specialist in yeast biology. He has been validating our findings in yeast and is now performing a yeast cytotoxic suppressor screen with our proteins of interest to identify potential host cell targets, which will inform our future experiments.
Impact First of all, our collaborator reproduced our findings in his laboratory using additional yeast strains, validating our findings. No publications or other tangible outputs have arisen from this collaboration yet.
Start Year 2018
 
Description Proteomics QUB 
Organisation Queen's University Belfast
Department School of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution My team opimised sample preparation and processing for interactome analysis
Collaborator Contribution The collaborator is providing advice for experimental design, performs the mass spectrometry and supports the data analysis.
Impact Microbiology and Proteomics
Start Year 2020
 
Description Structural Biology Hamburg 
Organisation University Medical Center Hamburg-Eppendorf
Department Institute of Biochemistry and Signal Transduction
Country Germany 
Sector Academic/University 
PI Contribution We have carried out preliminary expression tests for the Legionella effector proteins, which are in the focus of this grant. With this information we have approached the Prof. Aymelt Itzen to establish this collaboration.
Collaborator Contribution Professor Dr. Aymelt Itzen is director of Institute of Biochemistry and Signal Transduction, UKE, and academic lead for the protein purification facility at the Centre for Structural Systems Biology (CSSB), Hamburg, Germany. With the help of his team and the facility we will attempt to determine the crystal structure of the effectors.
Impact Cellular Microbiology & Structural biology
Start Year 2020
 
Description Contributor to the Lab View 360 Event NI Science Festival 2020 
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 Public/other audiences
Results and Impact In a new pilot event, 75 people from the general public over the age of 12 were offerend the opportunity to visit the laboratories in the Wellcome-Wolfson Instistute for Experimental Medicine. They had the opportunity to get hands-on experience at different stations illustrating the reserach carried out in the centre and discuss science with researchers. My team and I contributed one station.
Year(s) Of Engagement Activity 2020
 
Description Northern Ireland Science Festival 2019 
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 Public/other audiences
Results and Impact As part of the NI Science Festival we contributed to the session: "Know Your Enemy: Disease-Focussed Research at Queen's University", which involved a day of the open door in the Wellcome-Wolfson Institute for Experimental Medicine. I would estimate that the event was attended by 100-150 visitors. We contributed a stand at which kids and teenagers could learn about bacteria and antibiotic resistance in a playfull way. Moreover, we ansered questions about our ongoing research in the centre, and I guided a tour with app. 25 visitors through the centre, explaining the research facilities and daily routines of researchers. The group consisted of interested adults and a few kinds at the end of school, who consider studying natural sciences. The group was very enganged and ask questions regarding the centre, but also about more general topics such as the future of personalised medicine.
Year(s) Of Engagement Activity 2019