Thesis (PhD)--Stellenbosch University, 2013. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/79843 |
| Date | 03 1900 |
| Creators | Penkler, Gerald Patrick |
| Contributors | Snoep, J. L., Westerhoff, H. V., Rautenbach, M. R., Stellenbosch University. Faculty of Science. Dept. of Biochemistry., Vrije Universiteit Amsterdam. Faculteit der Aard- en Levenswetenschappen. Dept. of Molecular Cell Physiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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