Dissecting the role of the Leishmania flagellum in pathogenicity

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.

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.

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.