Regulating the trans-regulators: Investigating the PRMT7 molecular pathway as an epigenetic regulator of Leishmania spp. virulence

Species of Leishmania threaten 350 million people worldwide on four continents. The Leishmaniases are the second deadliest parasitic disease and all pathologies of this diverse disease are present in Brazil. The World Health Organisation estimates 12 million people globally are currently infected and over 1 million new cases occur annually. No vaccine currently exists and available Leishmaniasis treatments are threatened by growing resistances and often overwhelmed by acute epidemics that are increasing in occurrence and severity. New treatments and vaccines are desperately needed and the UK and Brazilian governments are committed to the World Health Organisation's recent call to further support Neglected Tropical Disease research.

The single-cell Leishmania parasite transforms into many different forms during its lifecycle to adapt to very different hosts; moving from mammals to sandflies and back to mammals by sandfly bites. Only Leishmania parasites of certain lifecycle stage forms can infect and survive in humans. Major changes to the Leishmania parasite's appearance, metabolism and virulence occur during these transitions that enable them to survive. Gene expression in Leishmania relies almost exclusively upon mRNA regulation. In response to changes in the environment, specific parasite proteins bind mRNAs and target them for protein production to guide and promote adaptation. Proteins that control the adaptation of these parasites enable them to survive in and infect humans. Such proteins are essential for the virulence and spread of the Leishmania parasite infection.

We have recently isolated a major control panel "Regulator" protein, PRMT7, which controls Leishmania parasite virulence in mammalian infections. Very few Leishmania regulator proteins have yet been identified and this finding represents a major leap forward to isolate and examine this regulatory pathway and block parasite virulence. To study the PRMT7 regulation pathway and identify the way this protein functions, we have assembled a team of experts in Leishmania parasite PRMT proteins, RNA regulators and protein interactions. We believe insight into this pathway will enable us to block the parasite from establishing human infections.

We have identified some downstream target proteins of PRMT7 and now seek to determine if they are regulated by PRMT7 and whether they participate in Leishmania parasite virulence. Leishmania proteins are different from human proteins; therefore we can use these differences to target Leishmania- specific virulence factors, block their function and block Leishmaniasis from developing. Significant findings will provide insight to Leishmaniasis research as well as diseases caused by related parasites, Chagas Disease and African Trypanosomiasis.

We propose to find more regulators of Leishmania spp. virulence using the PRMT7 virulence pathway. We will identify how these regulators function, and test whether any are essential for parasite survival. Essential Regulators could serve as potential drug targets to block Leishmania parasite infection.

The novelty and importance of our project is four fold:

1. PRMT7 is the only Protein aRginine Methyl Transferase enzyme that has been characterised in Leishmania spp. parasites thus far and it appears to block virulence (Ferreira et al., 2014).
2. Methylation as a protein modification is uncharacterised in Leishmania spp. parasites.
3. Regulatory RNA binding proteins (RBPs) that are important in parasite lifecycle differentiation, human infectivity and virulence are largely unknown in Leishmania spp.
4. The 3-dimensional molecular structures of RBPs and mRNA:protein complexes are largely unknown in all parasites and are absent in Leishmania spp.

Our aim is to use Leishmania major parasites to isolate and characterise the PRMT7-driven molecular pathway which regulates Leishmania spp. virulence in vivo. Previously, we demonstrated that PRMT7 levels are inversely proportional to Leishmania parasite virulence (Ferreira et al., 2014).

We will first dissect exactly how PRMT7 levels impact parasite virulence in mammalian cells. We will do this by genetically increasing and decreasing levels of PRMT7 enzyme and tracking parasite differentiation capacity to metacyclic (mammalian transmissive) versus amastigote (mammalian-infective) lifecycle stage parasites using stage-specific markers including RNA and protein expression, human serum sensitivity and proliferation. We will further examine whether levels of PRMT7 impact the disease-causing amastigotes' ability to infect and proliferate within human immune cells to discriminate differentiation capacity from virulence.

We will verify candidate trans-regulatory RNA binding protein (RBP) targets of PRMT7 by in vitro biochemical methylation analyses and genetically manipulate target RBP levels to examine their potential role in parasite virulence. Preliminary in vitro data supports a direct interaction in which E.coli-derived PRMT7 modifies target RBPs in an arginine glycine (RGG-motif) dependent manner. If a candidate trans-regulator is confirmed to be both a target of PRMT7 and implicit in parasite differentiation and/or virulence, we will isolate associating transcripts and examine whether PRMT7 and RBP levels regulate these RNAs.

Lastly, we will examine the structure/function relationship of target RBP trans-regulators in the presence/absence of PRMT7, methylation, protein binding partners, and associating RNAs using a variety of complementary biochemical, biophysical and structural techniques. We have already established protocols for production of recombinant RBPs which we can methylate in vitro using recombinant PRMT7.

TRANSLATIONAL MEDICINE POTENTIAL:
The Leishmania-focused laboratories already in place in the Centre for Immunology and Infection (http://www.york.ac.uk/res/cii/) provide the perfect platform for immediate investigations. Significant findings from this research could be directly translated into potential treatments using in-house expertise of the CII. As a whole, this Centre provides an inspiring opportunity to impact drug target testing and witness application of research achievements.
The purpose of our proposed experiments is to yield targets trans-regulators and transcript that impact virulence and/or lifecycle progression in Leishmania parasites and enable the parasite to infect, adapt and thrive within the human host immune cells. Since the majority of RNA binding proteins (potential trans-regulators) are not well conserved between these ancient eukaryotes and their human hosts, there is great potential to target Leishmania-specific virulence factors without dangerous side effects.

RESEARCH DIRECTIONS: The proposed isolation and characterization of the PRMT7-pathway will drive an investigation into novel factors that regulate Leishmania parasite virulence. We will build upon the foundation of this initial research and explore the function of additional cofactors isolated by the study; including potential non-RNA binding regulators of infectivity. It is highly likely that new virulence factors will be discovered and verified during this project and I will actively pursue the regulation of these. The mechanisms responsible for quorum and pH sensing in the parasite that trigger metacyclogenesis and differentiation have yet to be isolated, as well as many factors responsible for parasite-dependent manipulation of mammalian phagolysosomes. The directed aim of this research will be to isolate weaknesses in the parasite lifecycle and highlight potential drug targets to block transmission or efficiency of parasite infection. Regulators that coordinate the adaptation of these parasites to different host cells and promote parasite infection are critical to further understanding of this disease.

LOCAL POPULATIONS AT THREAT OF LEISHMANIASIS:
Leishmaniasis is the ninth largest disease burden amongst infectious diseases with WHO estimates of over a million new cases annually, killing 10-20% of them. 70-75% Leishmaniasis cutaneous infections occur in only 10 countries, and over 90% of visceral Leishmaniasis cases occur in only 6 countries. Over 70% of those infected are under 15 years of age. Despite over 100 years of effort, no vaccine is yet available and treatments are very limited. A vaccine should be possible as a single patient can only get Leishmaniasis once, as one infection conveys resistance against subsequent infections yet injection of killed Leishmania parasites does not convey immunity. There is an urgent need for a vaccine as epidemics often overwhelm available resources and treatments are limited and costly.

PHARMACEUTICAL INDUSTRY: Would benefit significantly from findings that not only impact human disease, but canine Leishmaniasis as well. This regulatory pathway of Leishmania virulence is completely novel and uncharacterised, thus presents a tremendous opportunity for new drug targeting as factors are likely to be parasite-specific and fundamental to parasite survival during transmission. Over 80% of drug discoveries in the past 40 years from the US originated in government-funded academic research (Stevens et al., NEJM, Feb. 2011).