Use of polyomics approaches to understanding drug resistance in kinetoplatid protozoa

QH345 Biochemistry

Mwenechanya, Roy ; Barrett, Michael

University:University of Glasgow

Department:School of Life Sciences

关键词: Resistance mechanisms, Amphoteriscin B, Isometamidium, Leishmania mexicana, Trypanosoma brucei, polyomics, Lanosterol 14-alpha demethylase, ergosterol, sphingolipids, cross resistance, choline phosphorylceramide synthase.;   

Others  :  http://theses.gla.ac.uk/5416/1/2014mwenechanyaphd.pdf

来源: University of Glasgow

【 摘 要 】

The lack of new drugs coming through to the market, to add to those few available, for use against diseases caused by trypanosomatids, calls for ways of safe-guarding the use of the current drugs to prolong their usefulness. This can be achieved by studying mechanisms of resistance to the drugs currently in use. In this thesis, the complementary use of untargeted metabolomic and whole genome sequence analyses was applied to elucidate the mechanisms of amphotericin B (AmB) resistance in L. mexicana promastigotes and isometamidium (ISMM) resistance in bloodstream forms of T. brucei.Resistance to AmB and ISMM was induced by step-wise increase of the drug concentration in the growth medium of L. mexicana promastigotes and bloodstream forms of T. brucei, respectively. Untargeted metabolomics results were used to link a single SNP in the lanosterol 14α-demethylase (CYP51) gene of the sterol (ergosterol) biosynthetic pathway from a multitude of SNPs that were found in generated AmB resistant cells as compared to the parental wild-type cells. The identified SNP was found to be a non-synonymous mutation, causing the change N176I, outside the active sites of CYP51 and was accompanied by accumulation of the enzyme’s product, 4, 4-dimethylcholesta-8, 14, 24-trien-3β-ol. These results suggested a break down in a possible protein-protein interaction of CYP51 with the next enzyme in the pathway, sterol C14-reductase, for efficient flow of the metabolite. Although the main subcellular localisation is different for these enzymes, they had a common localisation in the ER. AmB resistance was accompanied by depleted levels of ergosterol in AmB resistant cells. Expression of the wild-type CYP51 in AmB resistant cells restored ergosterol levels to those of the parental wild-type. Associated with restoration of ergosterol synthesis was reversal of resistance to AmB and susceptibility to pentamidine observed in AmB resistant cells. Thus N176I mutation in CYP51 underlies resistance to AmB in L. mexicana promastigotes. T. brucei resistance to isometamidium was accompanied by loss of the kinetoplast and maintenance of a much reduced mitochondrial membrane potential as compared to the parental wild-type cells. Reduction of the mitochondrial membrane potential was not connected to any apparent alteration of the energy metabolism of related metabolites. However, a perturbation in sphingolipid metabolism was observed to lead to depletion of sphingomyelin and accumulation of its precursor ceramide in the resistant cells. The responsible reaction enzyme, choline phosphorylceramide synthase (SLS4), was found to have 4 non-synonymous mutations which were outside the active site. The ISMM resistant cells exhibited a slow growth phenotype which has also been associated with perturbation of the sphingolipid synthesis pathway. ISMM resistant cells also showed cross resistance to diminazene aceturate and ethidium bromide used for control of African animal trypanosomiasis.