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

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.

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.

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.