Identifying and Characterising Developmental Regulators of Human Infectious Leishmania

Species of Leishmania threaten 350 million people worldwide on four continents. The World Health Organisation estimates 12 million are currently infected and over 1 million new cases occur annually. No vaccine currently exists and available Leishmaniasis treatments are often overwhelmed by acute epidemics that are increasing in occurrence and severity. New treatments and vaccines are desperately needed and the UK government has 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 parasite's appearance, metabolism and virulence proteins occur during these transitions that enable them to survive. Leishmania gene expression 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 changes these parasites go through enable them to adapt, survive in and infect humans. Such proteins are essential for the virulence and spread of the Leishmania parasite infection. By characterising such regulatory proteins and their downstream targets, we can find out what mechanisms the Leishmania parasites use to control their lifecycle changes. If we can isolate and stop control panel "Regulator" proteins, we can block the parasite from establishing an infection in humans. Significant findings would provide insight to Leishmaniasis research as well as diseases caused by related parasites, Chagas Disease and African Trypanosomiasis.

Leishmania proteins are different from human proteins; therefore we can exploit these differences to target Leishmania-specific developmental regulators, block their function and block the parasites' ability to invade. Unfortunately, no Leishmania regulator proteins have yet been identified.

I propose to find regulators of Leishmania spp. lifecycle changes, identify how they function, and test whether any are essential for parasite survival. Essential Regulators could serve as potential drug targets to block Leishmania parasite infection.

My aim is to use Leishmania mexicana as a cultured, genetically manipulable model to isolate and characterise post-transcriptional regulators essential to Leishmania spp. differentiation and virulence. Host immune cells provide the environmental cues, and signal the parasites to differentiate to virulent amastigote stage human infective forms. Factors that regulate and promote this differentiation enable the cells to adapt to the new host environment, survive and spread. Essential regulators may serve as potential drug targets as Leishmania spp. proteomes are very divergent from human.
I will initiate the investigation by uv-crosslinking Leishmania parasites of different stages and performing polysomal gradients to isolate putative mRNPs that associate with the translational machinery and are unique to human-infective lifecycle stages. Non-basal polysomal RNA-binding proteins will present potential trans-regulators while stage-specific polysomal mRNAs present putative targets and virulence factors to investigate. In addition, I will tag UTRs of known virulence factors to trap associating RNA binding proteins and potential regulators. Candidate regulatory proteins will be epitope-tagged to immunoprecipitate associating proteins (IP) and RNAs (RNA IP); validating isolated protein:transcript interactions and novel mRNPs.
The effect of candidate regulators on target transcripts and differentiation will be genetically tested by ectopic overexpression and protein depletion methods. The regulation of associating transcripts will be examined for impact on the protein level using reporter:UTR constructs and antibodies to promising trans-regulators and transcript targets. To determine the location and requirements of protein:transcript associations I will use uv-crosslinking IP (CLiP) techniques.
I will build upon significant findings of this work and investigate homologous factors in Leishmania donovani parasites, causative agent of deadly visceral Leishmaniasis.

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 my proposed experiments is to yield trans-regulators and transcript targets that are essential to differentiation 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 post-transcriptional regulators of Leishmania differentiation during mammalian invasion will drive an investigation into novel factors that enable Leishmania infection. I 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 this transition. 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 all other strains. 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.

POLITICAL POLICY: Initiatives to specifically address the neglected tropical diseases have been proposed by WHO and undertaken by the international community, in particular the UK and USA. Many basic biomedical researchers in the Leishmaniasis community are immunologists. Those that have investigated Leishmania spp. development have done so primarily in the sandfly vector or in an axenic cultured environment, likely missing essential signaling factors present in vivo.

PHARMACEUTICAL INDUSTRY: Would benefit significantly from findings that not only impact human disease, but canine Leishmaniasis as well. Regulators of Leishmania differentiation have yet to be identified and present a tremendous opportunity, as they are likely to be parasite-specific and fundamental to 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).