Investigating post Transcriptional Essential Gene Regulation In Leishmania (InTEGRL)

Leishmania are the cause of leishmaniasis, the 9th greatest infectious disease burden. Nearly 50% of all patients are children. No vaccine currently exists and epidemics are increasing in occurrence and severity. Treatments have toxic and painful side effects, are inappropriate for children and have growing resistances developing.

The single-cell Leishmania 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 cells 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 Leishmania infection. By characterising such regulatory proteins and their downstream targets, we can find out what mechanisms Leishmania use to control their lifecycle changes. If we can isolate and stop control panel "Regulator" proteins, we can block Leishmania from establishing an infection in humans. Significant findings would provide insight to leishmaniasis research and related diseases.

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. Recently, the Walrad lab has not only identified the full sets of proteins that bind genes in Leishmania, but from these have found 12 key Leishmania regulator proteins that are essential for cellular survival and infection. The majority of these regulators are pathogen-specific and not present in animals, making these potential druggable targets. We want to find out now how these regulators function; what other proteins and RNAs they bind to enable Leishmania to survive and infect. It is possible these essential Regulators or their cofactors could inform new drug discovery to block Leishmania infection.

My aim is to use Leishmania mexicana as a cultured, genetically manipulable model to characterise key posttranscriptional regulators we have identified that are 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 proteomes are very divergent from human.

I will initiate the investigation by endogenously epitope-tagging the 12 essential RBPs that have been shown to drive differentiation and virulence in human-infective Leishmania parasites. These transgenic cells will then be uv-crosslinked in vivo to immunoprecipitate associating proteins (IP), RNAs (RNA IP) of regulators. To determine the location and requirements of protein:transcript associations I will use uv-crosslinking RIP CLIP techniques. Quantitative RNASeq and LC mass spectroscopy will be used to identify isolated mRNP factors which will be validated by additional RIPs and qRTPCR (RNAs) and associating proteins will be endogenous tagged with complementary IPs confirming novel mRNP interactions.

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 epitope tags, reporter:UTR constructs and antibodies to promising trans-regulators and transcript targets. The mRNP interactions will be mapped and fuctional contributions toward parasite survival and infectivity will be determined and compared. Whether these mRNPs function in the same, divergent or independent pathways in differentiation and virulence will be investigated.