Development of an inducible system for Leishmania gene deletion: application to the cell cycle protein kinase CRK3

Duncan, Samuel Martin (2015) Development of an inducible system for Leishmania gene deletion: application to the cell cycle protein kinase CRK3. PhD thesis, University of Glasgow.

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Abstract

Leishmania spp. are protozoan parasites that infect humans and other vertebrates to cause a spectrum of disease, ranging from cutaneous ulceration to visceral dissemination dependent on the species. Leishmaniasis is prevalent across the developing world and is a major global health issue, yet difficulties in the efficacy and administration route of current anti-leishmanial treatments means the existing drug repertoire is inadequate. To address this, further research and development measures are necessary to identify Leishmania proteins representing useful targets for drug inhibition. Essential genes encode proteins that are necessary for parasite survival and therefore represent suitable drug targets, but the study of such genes is limited by the absence of a conditional deletion system. A family of proteins which has previously been shown to regulate crucial aspects of Leishmania biology are the protein kinases. Protein kinases have been validated in mammalian systems as drug targets in cancer therapy, therefore they represent a promising avenue for research into anti-leishmanial drugs. The cdc-related kinases CRK3 has been studied in particular depth in Leishmania, and current reverse genetic techniques have implicated expression of CRK3 as essential to promastigote survival. CRK3 regulates the cell cycle as demonstrated by treatment of cdc2 inhibitors, but a lack of a system to regulate expression prevents more specific phenotypic dissection of the role of CRK3. In addition the validation of CRK3 as a drug target has been limited by an absence of a conditional genetic system to ablate the gene in mammalian infective amastigotes. To regulate CRK3 expression in a conditional manner to assess its function in the cell cycle of promastigotes and validate it as essential for amastigotes, we have implemented an inducible gene deletion system based on a dimerised Cre recombinase (diCre) for use in L. mexicana. Cre recombinase mediates the excision of DNA sequences flanked by 34bp loxP sites (‘floxed’). diCre is encoded as two separate subunits each linked to rapamycin binding domains (FRB and FKBP12); therefore recombinase activity is induced by rapamycin treatment which causes dimerisation of the subunits. Our method involves replacing both CRK3 alleles with a ‘floxed’ CRK3 open reading frame and the diCre coding sequence through promastigote transfection and homologous recombination. Induction of diCre through rapamycin treatment of promastigotes results in highly efficient deletion of CRK3 and a distinct growth arrest phenotype corresponding to a block in G2/M. Induced loss of CRK3 can be complemented by expression of a CRK3 transgene but not by expression of an inactive site (T178E) CRK3 mutant, showing that protein kinase activity is crucial for CRK3 function. Significantly, inducible deletion of CRK3 in stationary phase promastigotes prevents the establishment of murine infection, thereby demonstrating an essential role in the amastigote cell cycle to further validate CRK3 as a drug target. Promisingly, inducible deletion is functional in lesion-derived amastigotes and will enable direct phenotypic assessment following essential gene loss in this life cycle stage. To establish a basis for future in vivo application of diCre in Leishmania, a murine infection model was developed with which to track bioluminescent parasite burden by in vivo imaging and assess innate immune cell recruitment to the site of infection by flow cytometry analysis. The combination of functional gene regulation in amastigotes and measures of parasite burden and immune response will yield a powerful tool for the further study of Leishmania genes encoding suitable drug targets. The application of the diCre technique to Leishmania would be greatly benefitted by targeting genes where there is evidence of a regulatory role of orthologous genes in model organisms. The utilisation of genome or protein family-wide RNAi screens in Trypanosoma brucei has identified a number of protein kinases which regulate the differentiation of the parasite between life cycle stages. The repressor of differentiation (RDK1) protein regulates bloodstream form to procyclic form differentiation in T. brucei, and the identification of a protein in L. mexicana with high sequence identity suggested a potentially analogous role in preventing Leishmania from undergoing amastigote to promastigote differentiation in vivo. To assess this, a cell line was generated deficient in RDK1 but no effect on differentiation was identified, as parasites were able to maintain murine infection and differentiate between life cycle stages. This study represents an important addition to the reverse genetic toolkit to study aspects of cell cycle regulation in vitro, and further assess essential genes as drug targets by deletion in amastigotes. The application of the diCre conditional deletion method will enhance the discovery and evaluation of suitable drug targets in Leishmania by phenotypic analysis.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Leishmania, genome engineering, diCre recombinase, cyclin-dependent kinase
Subjects: Q Science > QH Natural history > QH426 Genetics
Q Science > QR Microbiology
R Medicine > RB Pathology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Parasitology
Funder's Name: UNSPECIFIED
Supervisor's Name: Mottram, Professor Jeremy and Garside, Professor Paul and Brewer, Professor James
Date of Award: 2015
Depositing User: Mr Samuel Duncan
Unique ID: glathesis:2015-6813
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 09 Nov 2015 10:47
Last Modified: 11 Nov 2015 15:07
URI: http://theses.gla.ac.uk/id/eprint/6813

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