A kinetic model of glucose catabolism in Plasmodium falciparum

Penkler, Gerald Patrick (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Malaria infects over 200 million individuals and leads to the death of over 600 000 people annually. Currently artemisinin combination therapy treatments are effective in treating the disease, but resistance has started to emerge in Cambodia and it is suspected in parts of Vietnam. To maintain the drive to eradicate malaria globally, a great deal of research is aimed at identifying novel prevention strategies, vaccines and antimalarial compounds. Plasmodium falciparum, the most deadly of the malaria parasites, is entirely dependent on glycolysis for ATP. Several of the enzymes within this pathway have been proposed as drug targets and studied in isolation, but the pathway as a whole has not been considered. In this study we employ a bottom up approach for drug target identification in P. falciparum glycolysis. In this thesis we present the biochemical characterisation each of the glycolytic enzymes in P. falciparum trophozoites. The kinetic rate equations, which described the kinetic behaviour of the individual enzymes, were incorporated into a kinetic model. The unfitted model was validated in its ability to predict experimentally measured steady state metabolite concentrations and fluxes as well as the experimental inhibition of the glucose transporter. The validated model provided a tool for drug target identification in P. falciparum glycolysis. Metabolic control analysis and differential control analysis identified the glucose transporter, PfHT1, as a drug target based on its high control of glycolytic flux in the parasite, but low control of flux in the host erythrocyte. This differential control makes the transporter an attractive drug target, as even if both the erythrocyte and parasite glucose transporters are inhibited to the same degree, it is expected that the parasite glycolytic flux would be inhibited to a much greater degree. To demonstrate the differential control of the glucose transporter on the flux and provide further evidence that PfHT1 is an attractive drug target, we investigated the inhibition of the glucose transporter in isolated trophozoites by cytochalasin B. We also measured the inhibition of lactate production flux by cytochalasin B in both isolated P. falciparum trophozoites as well as in erythrocytes. Our findings demonstrated that differential control analysis can be used as a tool for drug target identification and that PfHT1 is an attractive drug target. In this study the fields of biochemistry and systems biology were merged to create a detailed kinetic model of asexual P. falciparum glycolysis and identify several drug targets in the pathway. The model prediction and experimental evidence of differential flux control of the glucose transporter in the host and parasite, has highlighted PfHT1 as a drug target and also demonstrates the strength of differential control analysis in identifying drug targets within a system. The kinetic model is a valuable tool for furthering our understanding of P. falciparum glycolysis and it provides a good foundation for expansion to identify drug targets in the entire central carbon metabolism of P. falciparum.

AFRIKAANSE OPSOMMING: Malaria infekteer meer as 200 miljoen mense en veroorsaak jaarliks tot 600 000 sterftes. Tans is die artemisinien-kombinasieterapie effektief in die bestryding van die siekte, maar weerstandbiedendheid van die parasiet teen die middel blyk reeds ’n merkbare effek in Kambodja en vermoedelik ook in dele van Viëtnam te hê. Om ’n wêreldwye bestryding van malaria moontlik te maak, is ’n groot deel van die huidige navorsing gemik op die identifisering van nuwe voorkomingsstrategieë, entstowwe en malariateenmiddels. Plasmodium falciparum, die dodelikste van die malaria-parasiete, is geheel en al afhanklik van glikolise vir ATP vorming. Verskeie van die ensieme in hierdie metaboliese pad is as teenmiddelteikens voorgestel, en in isolasie bestudeer, maar die pad as ’n geheel is nie bestudeer nie. In hierdie studie het ons ’n ’bottom-up’ benadering vir teenmiddel teikenidentifisering in P. falciparum glikolise gebruik. In hierdie tesis bied ons die biochemiese karakterisering van elk van die glikolitiese ensieme in P. falciparum trofozoïete aan. Die kinetiese vergelykings wat die kinetiese gedrag van die individuele ensieme beskryf, is geintegreer in ’n enkele kinetiese model. Die model waarop geen datapassing toegepas is nie, is gevalideer om eksperimenteel bepaalde bestendige-toestand metabolietkonsentrasies en fluksiewaardes, asook die eksperimentele inhibisie van die glukose transporter, te voorspel. Die gevalideerde model verskaf ’n bykomende hulpmiddel om teenmiddelteikens te identifiseer in P. falciparum glikolise. Metaboliese kontrole-analise en differensiële kontrole-analise het die glukose transporter, PfHT1, as ’n teenmiddelteiken geïdentifiseer, gebaseer op sy hoë kontrole van glikolitiese fluksie in die parasiet, tesame met ’n lae beheer van die glukose transporter op die fluksie in die gasheer eritrosiet. Dié differensiële kontrole maak die glukose transporter ’n aantreklike teenmiddelteiken, want selfs as beide die eritrosiet en die parasiet glukose transporters tot dieselfde mate geïnhibeer word, sal dit steeds ’n hoër glikolietiese fluksieinhibisie van die parasiet tot gevolg hê. Om die differensiële kontrole van die glukose transporter op die fluks te demonstreer en verdere bewyse te lewer dat PfHT1 ’n teenmiddelteiken kan wees, het ons die inhibisie van die glukosetransporter in geïsoleerde trofozoïete deur sitokalasien B ondersoek. Ons het ook die inhibisie van die laktaatproduksiefluksie deur sitokalasien B in beide geïsoleerde P. falciparum trofozoïete sowel as in eritrosiete ondersoek. Ons bevindings bewys dat differensiële kontroleanalise as ’n hulpmiddel vir teenmiddelteikenidentifikasie gebruik kan word en dat PfHT1 ’n aantreklike teenmiddelteiken is. In hierdie studie is die velde van biochemie en sisteembiologie gekombineer om ’n gedetaileerde kinetiese model van ongeslagtelike P. falciparum glikolise te konstueer en verskeie teenmiddelteikens in die metaboliese pad te identifiseer. Die modelvoorspelling sowel as eksperimentele bewyse van die differensiële flukskontrole van die glukose transporter in die gasheer en parasiet het PfHT1 uitgelig as ’n teenmiddelteiken en demonstreer ook die krag van differensiële kontrole analise in die identifisering van teenmiddelteikens binne ’n biologiese stelsel. Die kinetiese model is ’n waardevolle hulpmiddel vir die bevordering van ons begrip van P. falciparum glikolise en dit bied ’n goeie basis vir uitbreiding om teenmiddelteikens in die hele sentrale koolstofmetabolisme van P. falciparum te identifiseer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79843
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