Dissecting the role of the Leishmania flagellum in pathogenicity

Lead Research Organisation: University of Oxford
Department Name: Sir William Dunn Sch of Pathology

Abstract

Leishmania are parasites that are transmitted by the bite of the sand fly. A person infected with Leishmania can develop mild symptoms, with localized skin lesions, or a life-threatening infection of the whole body. About 12 million people across the world are currently infected; about 30,000 people die every year from the disease and those that survive are often left with disfiguring scars. Leishmania parasites are small single cells and in the human body they live inside blood cells called macrophages. One of the most important jobs of the macrophage is the discovery and destruction of microbes that cause disease. Simply put, macrophages swallow and digest some of the invading microbes and then inform other cells of the immune system to mount a coordinated attack and destroy the microbes. When a macrophage swallows a Leishmania parasite, the parasite does not die. Instead it changes its shape, multiplies and then spreads to infect other cells. We want to find out how the parasites communicate with the macrophage to block its killing mechanisms and promote their own survival. We think a thin projection on the parasite, called the flagellum, could function as a 'cellular antennae' or signalling device. We now want to discover what the building blocks (proteins) of this flagellum are and why it appears to be required for parasite survival in the host cell. We have generated a list of flagellar proteins and will now use a new technology called gene editing to produce a set of several hundred mutant parasites, where different parts of the flagellum are disrupted. We will then mix these mutant parasites with macrophages in the laboratory and look a few days later to see which mutants were successful in entering the macrophages and which ones didn't make it. Those that fail to infect will be studied in detail to discover which parts of the flagellum are required for infection and how the flagellum and the connections it makes help the parasite to survive the destruction mechanisms of the macrophage.

Technical Summary

The protist Leishmania is an obligate intracellular parasite that infects macrophages and causes disease in humans. Leishmania have a single flagellum required for motility and attachment of promastigote forms in the insect vector. Surprisingly, promastigotes are viable in culture even if they build no flagellum, yet intracellular amastigote forms, whose short flagellum was long considered non-functional, cannot survive in macrophages if protein trafficking to the flagellar membrane is disrupted. This points to a role for the flagellum in infection. We previously showed that the short amastigote flagellum structurally resembles sensory cilia and observed that the flagellum tip forms a junction with the parastiophorous vacuole membrane. Cilia and flagella are organelles found on diverse cell types, from protists to cells in the human body, acting as motile appendages or antenna-like signal transducers and we hypothesise that the flagellum of intracellular Leishmania is a sensory organelle. The flagellum is a complex organelle composed of hundreds of proteins. We have developed a new method for rapid generation of gene knockouts (KO) using CRISPR-Cas9 tools which enable us for the first time to conduct a systematic KO screen to test which types of flagellar mutants (cytoskeletal, membrane, tip defects) lose the ability to infect macrophages. We will also use biochemical methods to discover new proteins localized to the flagellum for inclusion in the KO screen. Mutants that fail to survive in macrophages will be subjected to detailed phenotype analysis. This research will uncover new information about the role of the Leishmania flagellum in infection and test the hypothesis that a physical connection between flagellum tip and host cell contributes to virulence. We will use pluripotent stem cell-derived human macrophages as in vitro infection models which allow us to dissect molecular mechanisms of host-pathogen interactions with relevance to human disease.

Planned Impact

The primary objective of this research is to understand the mechanisms through which the Leishmania parasite interacts with the blood cells it infects and how it escapes the killing mechanisms of the immune system. The beneficiaries of this research outside the academic research community are the 350 million people worldwide estimated to be at risk of infection with the Leishmania parasite. Leishmaniasis belongs to a group of neglected tropical diseases (NTD) that poses a huge health burden on some of the poorest communities in the world with a detrimental impact on economic development in endemic areas.
This fundamental research employs advanced cell biology methods and to dissect the interactions between the parasite and its host cell and links dissection of molecular mechanisms with structural studies of flagellum/cilium biogenesis and function.
This work will link important areas of molecular parasitology, infection and immunity, and cell biology and generate new hypotheses with relevance to studies on drug and vaccine development, which in time could be translated into tangible health benefits for the affected communities. This project will train researcher and PhD students in powerful new gene editing methods for discovery of mutant phenotypes and gene function. Tools and protocols for genetic modification of Leishmania and forward genetic screens resulting from this work are likely to find applications in other areas of Leishmaniasis research. The project will generate new knowledge about a parasite's strategy for hijacking cells the human body and pinpoint which parasite genes are important for virulence. These outputs will be publicised to a wider audience through articles and science exhibitions so that the public can engage with basic discovery science and understand the impact of scientific research on health, and to inspire the next generation of school children to become researchers themselves.

Publications

10 25 50
 
Description Lord Kelvin / Adam Smith (LKAS) Fellowship
Amount £100,000 (GBP)
Organisation University of Glasgow 
Sector Academic/University
Country United Kingdom
Start 03/2020 
End 03/2021
 
Title LeishGEdit bar-seq 
Description To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we previously developed a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit (Beneke et al., 2017. R Soc Open Sci. 4(5):170095). To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations ("bar-seq" screens; Beneke et al., 2019. PLoS Pathogens 15:e1007828). To facilitate the application of this method in a wide range of kinetoplastid parasites, we developed tools for the design and analysis of bar-seq screens, including a standalone pipeline for CRISPR primer design and a list of 14,995 barcodes. Barcode and primer sequences are accessible through our website www.leishgedit.net. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact This tool allows for phenotype analysis of mixed mutant pools. This accelerates studies of gene function in kinetoplastid parasites and it greatly reduces the number of animals needed for in vivo fitness screens. 
URL http://www.leishgedit.net
 
Description In vivo phenotyping - mice 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution My research team shares with the collaborator innovations and refinements of the method used for pooled phentoyping of Leishmania in mice.
Collaborator Contribution The collaborator's laboratory performs the in vivo experiments and advises on experimental design for follow-up studies to build on our preliminary data.
Impact This collaboration has produced preliminary data, showing that our method for pooled phentoyping of Leishmania in mice works.
Start Year 2017
 
Description In vivo phenotyping - sand flies 
Organisation Charles University
Country Czech Republic 
Sector Academic/University 
PI Contribution My research team has trained a visiting student from the collaborator's laboratory and helped to generate mutant Leishmania strains. Preliminary data from experiments with these strains is now being used as the basis for a collaborative research grant application to the Czech science foundation. To enable travel between the laboratories and complete an initial set of experiments I secured a Royal Society International Exchanges grant in 2017 (GBP 4610.00).
Collaborator Contribution The collaborator's laboratory performs sand fly infections with Leishmania cell lines generated in my lab to determine the fitness phenotypes of mutant parasites and their ability to complete their life cycle in the insect vector. This allows us to use the mutant library, which we are generating as part of my MRC grant, to investigate more wide-ranging questions about the parasite's life cycle. The collaborator paid for my travel expenses when I visited their laboratory in 2018 to discuss the collaboration and future grant applications.
Impact We reported the results of the firs set of experiments in this collaboration in this preprint: Beneke T, Demay F, Hookway E, Ashman N, Jeffery H, Smith J, Valli J, Becvar T, Myskova J, Lestinova T, Sadlova J, Volf P, Wheeler RJ and Gluenz E (2018). Genetic dissection of the Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections. biorXiv doi: https://doi.org/10.1101/476994
Start Year 2017
 
Description Translation factors 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution My group generated 16 cell lines to enable a new project to study how the loss of transcription factors affects survival and pathogenicity of the Leishmania parasites.
Collaborator Contribution The collaborator is PI on a new BBSRC project grant application on which I am a collaborator.
Impact None as yet, we are awaiting the outcome of the grant application. The project is interdisciplinary, involving structural biologists as well as molecular and cell biology labs.
Start Year 2019
 
Description CRISPR workshops 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Organised practical workshop teaching gene editing methods to researchers in Brazil and India so that they can be applied in research on leishmaniasis in countries where these neglected diseases are endemic. Following a successful run in Brazil in 2018 we repeated the workshop in India in 2019. The workshop is funded by a GCRF-funded Global Network for Neglected Tropical Diseases.
Year(s) Of Engagement Activity 2018,2019
URL https://ntd-network.org/event/3rd-advanced-school-in-genetic-manipulation-of-parasitic-protozoa-rece...