Bridging epigenetics, metabolism and cell cycle in pathogenic trypanosomatids

We propose to bring together a group of researchers across the UK and in Sao Paulo to share expertise that will allow us to understand the biology of parasites that cause neglected tropical diseases and improve our ability to intervene against them. Disease-causing microbes remain a major problem for health the world over. This is seen disproportionately in tropical countries, were a combination of environmental factors (e.g. climatic conditions supporting the propagation of insects that can transmit disease) and relatively scarce economic resource ensure the maintenance of infectious disease. Parts of rural Brazil, like other areas in Latin America, suffer from diseases caused by parasitic protozoa, which are single-celled organisms. One parasite, Trypanosoma cruzi, causes Chagas disease, affecting several million people across Latin America. Closely-related parasites, belonging to the genus Leishmania, cause a range of diseases called the leishmaniases in many parts of the world. Chagas disease is transmitted by triatomine bugs that suck the blood of mammals including humans and in the process defecate upon their victims. Parasites in the faeces then enter the body and over a long period of time, sometimes twenty years or more, the insidious Chagas disease develops. Quite often patients do not know they have the disease. In around 30% of cases though, a chronic inflammatory response to the heart or digestive system can lead to death. The leishmaniases are transmitted by sandflies through the saliva whilst they feed on their victims. Depending on which species of the Leishmania parasite is transmitted, the disease can either occur in the skin (cutaneous leishmaniasis) or else migrate to the liver and spleen (visceral leishmaniasis). Some species, including Leishmania braziliensis, causes the horrible disease known in Brazil as Espundia, where human cells called macrophages (in which the parasite resides) migrate to the mucosal membranes of our lips and nostrils and cause them to disintegrate. Sometimes, following treatment, after a lapse that can last a few years, the visceral form of the disease relapses into a form we call post Kalar-Azar dermal leishmaniasis (PKDL).

Although drugs exist to treat both Chagas disease and the leishmaniases, they are far from ideal. Some are toxic, others have to be given over a protracted period, and several are available only by needle injections. Resistance has emerged to existing drugs, and it has been known for a long time that some parasites do not respond to treatment even if resistance has not been selected.

This collaborative project aims to bring together a team of researchers across the UK and Sao Paulo to look specifically at the processes that enable parasites to thrive in the insect vectors that carry them and then within the very different environment of their mammalian host. Since the parasites keep exactly the same genome (which is the blueprint that encodes every aspect of their ultimate makeup) they need to choose which parts of the genome to express in different environments. This involves subtle changes to the structure of the protein architecture that holds their genes together, in a structure we call chromatin. In other organisms it has, in recent years, become clear that the addition of small molecules to those chromatin-associated proteins can change their function. Removing those adaptations causes them to change back again. These alterations, which do not involve modifying the sequence of the DNA blueprint itself, and yet do influence how that DNA is expressed, are described as "epigenetic" alterations. We will investigate how epigenetic alterations impact on gene expression in T. cruzi and leishmanias, and work out how we can manipulate this epigenetic process by altering the availability of the small molecule metabolites responsible for those changes. We hope to find new therapeutic drugs treatment interventions to act on these epigenetic targets.

This programme of research will create a network of researchers studying the regulation of gene expression in the kinetoplastid protozoa, allowing us to dissect the processes that link metabolism, epigenetic change to chromatin, gene expression and cell differentiation. This will give insights into the processes that enable these organisms to enter a quiescent state that protects them from both immunological and chemotherapeutic intervention, and also provide novel targets against which to develop new drugs.

Specifically, we aim to characterise the range and extent of post-translational modifications to proteins (specifically histones) involved in chromatin formation in Trypanosoma cruzi and Leishmania infantum and L. mexicana cells undergoing passage through the life cycle and in response to metabolic pertubations. This will involve the isolation of chromatin from different parasite lifecycle stages, and isolation of the individual proteins of the complex (using immunopreciptation methodologies with antibodies specific for the trypanosomatid histones, and BioID approaches to identify neighbouring members of the complex). Proteomic approaches will determine the repertoire of proteins involved. Post-translational modifications (PTMs) on individual proteins will be assessed using specific antibodies and also mass spectrometry of digested proteins to seek mass changes consistent with different PTMs. The abundance of individual metabolites in cells at different cell/lifecycle stages will be determined using LC-MS based untargeted metabolomics. CRISPR/cas9 approaches will allow rapid knockout of non-essential enzymes producing metabolites implicated in PTM, and use of different metabolic substrates will also be used to modulate their abundance to asses impact on epigenetic change and gene transcription by RNAseq. Cell culture techniques will assess life cycle progression in mutants

The programme we propose will bring together a group of researchers from four UK institutions and the University of Sao Paulo in Brazil to investigate a crucial and yet hitherto relatively poorly studied aspect of the biology of protozoan parasites belonging to the order Kinetoplastida. Trypanosoma cruzi afflicts millions of people in Latin American causing Chagas disease, while the leishmaniases have a distribution spanning the world's tropical and sub-tropical regions. The groups that we are bringing together have expertise in many aspects of the biology of the causative parasites and covers parasite biochemistry, molecular and cell biology, metabolomics, proteomics and nucleotide sequencing. The group collectively brings together all of the skills required to learn about the epigenetic processes that allow the parasites to remodel their metabolic pathways to survive within the different environmental niches in which they find themselves. These can include the bloodstream and different intracellular environments in their mammalian hosts as well as different anatomical locations within the insect vectors that transmit them.

Epigenetic processes are those which enable profound changes in the physiology of an organism without requiring changes in the nucleotide sequence of their genomes. Generally, epigenetics involves remodelling of the structure of chromatin, which is the combined protein and nucleic acid structure that contains the genetic information. Chromatin remodelling can involve the post-translational modification of its protein components, for example histones. The modifications to chromatin-associated proteins are generally those of addition of small molecules, derived from cellular metabolism. As such, metabolism, and the accumulation or loss of particular metabolites, have a profound impact on how cells behave.

We will identify and characterise the post-translational modification in trypanosomes and leishmanias. We will then determine how these modifications influence gene expression as parasites move from one environment to another. As metabolism is ultimately responsible for providing the molecules that modulate chromatin, we will also probe the metabolism of these cells progressing through their life cycle.

Critically, it has recently become clear that T. cruzi and Leishmania parasites can enter a quiescent, non-proliferating state where they become tolerant of both immunological and chemotherapeutic assault. It is likely this process evolved (as in other microbes) to enable protection of the population against environmental fluctuations of many types (manifest in humans as immunological or drug intervention). As changes to genetic sequence are not associated with quiescence, epigenetics must be at play.

A key outcome of discovering the processes underpinning epigenetic influence on gene expression and phenotypic variation in these parasites will be to identify the process that drive quiescence. Since this process is responsible for long-term parasite persistence and drug treatment, this information will open the way to new strategies to prevent, or reverse, the formation of quiescent parasites, and thus create new avenues to assure successful treatment that will ultimately lead to control and elimination of Chagas disease and the leishmaniases, diseases which afflict tens of millions of people across the world today.