Phenotypic characterisation of glucose transporter knockout Leishmania mexicana

QR Microbiology

Lamasudin, Dhilia Ude ; Burchmore, Richard

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: QR Microbiology;   

Others  :  http://theses.gla.ac.uk/4161/1/2012lamasudinphd.pdf

来源: University of Glasgow

【 摘 要 】

The aim of this project was to investigate nutrient acquisition by glucosetransporter null mutant Leishmania mexicana, to understand how geneticablation of glucose transporter genes in these protozoan parasites affects theirphenotype. It has previously been demonstrated that glucose transporter nullmutant Leishmania (ΔLmGT) promastigotes have lost the capacity to transportglucose as well other hexoses. Glucose is a potential carbon source forpromastigotes, which are typically maintained in glucose-rich media and whichencounter high levels of glucose in the sandfly midgut. Despite inability totransport glucose, the null mutant parasite is able to survive and grow, althoughat a reduced rate in media that contains non-carbohydrate potential carbonsources. This finding suggesting that the null mutant may utilise other carbonsources. In this project, a variety of approaches have been applied to investigatethe nature of these alternative carbon source and the mechanisms by which theyare acquired and metabolised.Amino acid uptake assays revealed that glucose transporter null mutantpromastigotes take up several key amino acids at a significantly enhanced rate,compared with wild type promastigotes. A comparative metabolomic analysiswas applied for more comprehensive comparison between the wild type andglucose transporter null mutant. Though the uptake assay and metabolomicanalysis showed distinct changes in nutrient uptake and metabolisms betweenthe two cell lines, these results does not tell whether the changes are due tochanges in transporter activity or expression, nor does it inform on the enzymesinvolved in these processess. Thus, a proteomic approach was applied. Sincemembrane transporter were a focus of interest, but are known to be highlyhydrophobic and of relatively low abundance, it was necessary to developprotocols for membrane protein enrichment and fractionation in Leishmaniamexicana.A previous comparative proteomic analysis, which did not representmembrane proteins, had highlighted changes in the abundance of a majorcomponent of the paraflagellar rod. This unexpected result was furtherinvestigated, by Western blotting, electron microscopy and taxis assay. Theresults point to changes in flagellar structure of function in glucose transporternull mutant Leishmania.It is no doubt the experiments carried out in this project have showninteresting changes in glucose transporter null mutant Leishmania, in terms ofnutrient acquisition and phenotypic characteristics. The data provide a new setof ideas as to how Leishmania thrive in a condition where glucose is scarce.These data may have relevance to leishmaniasis in mammals, as it is likely thatglucose is constitutively scarce in the intracellular environment inhabited by theamastigote. However, due to time constraints, it was not possible to extendthese investigations to the amastigote stage of Leishmania.