The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. ...However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions.
Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable.
A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4%. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident.
With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broad-diversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.
The life cycle of the malaria parasite Plasmodium falciparum is tightly regulated, oscillating between stages of intense proliferation and quiescence. Cyclic 48-hour asexual replication of Plasmodium ...is markedly different from cell division in higher eukaryotes, and mechanistically poorly understood. Here, we report tight synchronisation of malaria parasites during the early phases of the cell cycle by exposure to DL-α-difluoromethylornithine (DFMO), which results in the depletion of polyamines. This induces an inescapable cell cycle arrest in G
(~15 hours post-invasion) by blocking G
/S transition. Cell cycle-arrested parasites enter a quiescent G
-like state but, upon addition of exogenous polyamines, re-initiate their cell cycle. This ability to halt malaria parasites at a specific point in their cell cycle, and to subsequently trigger re-entry into the cell cycle, provides a valuable framework to investigate cell cycle regulation in these parasites. We subsequently used gene expression analyses to show that re-entry into the cell cycle involves expression of Ca
-sensitive (cdpk4 and pk2) and mitotic kinases (nima and ark2), with deregulation of the pre-replicative complex associated with expression of pk2. Changes in gene expression could be driven through transcription factors MYB1 and two ApiAP2 family members. This new approach to parasite synchronisation therefore expands our currently limited toolkit to investigate cell cycle regulation in malaria parasites.
Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic ...disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites, information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries.
Phenolic acids, which are generally esterified with tartaric acid, are natural constituents of grape must and wine and can be released as free acids (principally p-coumaric, caffeic, and ferulic ...acids) by certain cinnamoyl esterase activities during the wine-making process. Some of the microorganisms present in grape can metabolize the free phenolic acids into 4-vinyl and 4-ethyl derivatives. These volatile phenols contribute to the aroma of wine. The Saccharomyces cerevisiae phenyl acrylic acid decarboxylase gene (PAD1) is steadily transcribed, but its encoded product, Pad1p, shows low activity. In contrast, the phenolic acid decarboxylase (PADC) from Bacillus subtilis and the p-coumaric acid decarboxylase (PDC) from Lactobacillus plantarum display substrate-inducible decarboxylating activity in the presence of phenolic acids. In an attempt to develop wine yeasts with optimized decarboxylation activity on phenolic acids, the padc, pdc, and PAD1 genes were cloned under the control of S. cerevisiae's constitutive phosphoglyceratekinase I gene promoter (PGK1P ) and terminator (PGK1T ) sequences. These gene constructs were integrated into the URA3 locus of a laboratory strain of S. cerevisiae, Σ1278b. The overexpression of the two bacterial genes, padc and pdc, in S. cerevisiae showed high enzyme activity. However, this was not the case for PAD1. The padc and pdc genes were also integrated into an industrial wine yeast strain, S. cerevisiae VIN13. As an additional control, both alleles of PAD1 were disrupted in the VIN13 strain. In microvinification trials, all of the laboratory and industrial yeast transformants carrying the padc and pdc gene constructs showed an increase in volatile phenol formation as compared to the untransformed host strains (Σ1278b and VIN13). This study offers prospects for the development of wine yeast starter strains with optimized decarboxylation activity on phenolic acids and the improvement of wine aroma in the future. Keywords: Phenolic acid decarboxylation; volatile phenols; wine yeast; wine aroma
Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic ...disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites, information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries.
Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Phenolic acids (principally p-coumaric and ferulic acids), which are generally esterified with;
tartaric acid, are natural ...constituents of grape must and wine, and can be released as free;
acids during the winemaking process by certain cinnamoyl esterase activities. Free phenolic;
acids can be metabolised into 4-vinyl and 4-ethyl derivatives by several microorganisms;
present in wine. These volatile phenols contribute to the aroma of the wine.;
The Bretfanomyces yeasts are well known for their ability to form volatile phenols in;
wine. However, these species are associated with the more unpleasant and odorous;
formation of the ethylphenols and the formation of high concentrations of volatile phenols.;
Other organisms, including some bacterial species, are responsible for the formation of;
volatile phenols at low concentrations, especially the 4-vinylphenols, and this enhances the;
organoleptic properties of the wine.;
The enzymes responsible for the decarboxylation of phenolic acids are called phenolic;
acid decarboxylases; and several bacteria and fungi have been found to contain the genes;
encoding these enzymes. The following genes have been characterised: PAD1 from;
Saccharomyces cerevisiae, fdc from Bacillus pumilus, pdc from Lactobacillus plantarum and;
padc from Bacillus subtilis. PadA from Pediococcus pentosaceus was also identified.;
S. cerevisiae contains the PAD1 (phenyl acrylic acid decarboxylase) gene, which is;
steadily transcribed in yeast. The activity of the PAD1-encoded enzyme is low. Phenolic;
acid decarboxylase from B. subtilis, as well as p-coumaric acid decarboxylase from;
L. plantarum displays substrate inducible decarboxylating activity with phenolic acids. Both;
the p-coumaric acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) genes;
were cloned into PGK1 PT expression cassette. The PGK1 PT expression cassette consisted;
of the promoter (PGK1 p) and terminator (PGK1 T) sequence of the yeast;
phosphoglyceratekinase I gene (PGK1). Episomal and yeast integration plasmids were;
constructed for the PAD1 gene under the control of the PGK1 PT for overexpresion in yeast.;
Industrial strains with the PAD1 gene disrupted were also made. Overexpression of pcoumaric;
acid decarboxylase (pdc) and phenolic acid decarboxylase (padc) in S. cerevisiae;
showed high enzyme activity in laboratory strains. The overexpressed PAD1 gene did not;
show any higher enzyme activity than the control strain. Both bacterial genes, under the;
control of the PGK1 PT cassette, were also cloned into a yeast-integrating plasmid, with the;
SMR1 gene as selective marker. The cloning and transformation of pdc and padc into;
industrial wine yeast strains can therefore be used to detect the effect of phenolic acid;
decarboxylase genes in the winemaking process for the possible improvement of wine;
aroma. Wine was made with all three strains (the bacterial genes overexpressed and PAD1;
disrupted). The effect of these genes in wine was determined through GC analysis. The results showed that the bacterial genes could effectively produce higher levels of volatile;
phenols in the wine. The manipulated strains also produced enzymes capable of producing;
large amounts of favourable monoterpenes in the wine.;
This study paves the way for the development of wine yeast starter culture strains for;
the production of optimal levels of volatile phenols, thereby improving the sensorial quality of;
wine.
AFRIKAANSE OPSOMMING: Die fenoliese sure (p-kumaarsuur en ferolsuur), wat as natuurlike komponente in mos en wyn;
voorkom, word gewoonlik as esterverbindings in wynsteensuur gevind. Seker;
esterase-aktiwiteite kan die fenoliese sure as vrye sure vrystel gedurende die;
wynmaakproses. Hierdie vrye fenoliese sure kan dan weer deur verskillende mikroorganismes;
na 4-viniel en 4-etiel derivate omgesit word. Hierdie derivate staan as vlugtige;
fenole bekend en kan tot die aroma van wyn bydra.;
Die Brettanomyces giste is baie bekend vir hulle vermoeë om vlugtige fenole in wyn te;
vorm, maar dit is gewoonlik die formasie van hoë konsentrasies van vlugtige fenole, veral die;
4-etiel derivate, wat met af geure geassosieer word. Ander organismes besit egter die;
vermoeë om vlugtige fenole teen lae konsentrasies te vorm, veral die 4-viniel derivate, wat 'n;
aanvullende effek op die wyn aroma kan hê. .;
Die ensieme wat verantwoordelik is vir die dekarboksilasie van fenoliese sure staan as;
fenolsuurdekarboksilases bekend. Verskeie bakterieë en fungi bevat gene wat vir hiedie;
ensieme kodeer. Die volgende gene is reeds gekarakteriseer: PAD1 van;
Saccharomyces cerevisiae, fdc van Bacillus pumilus, pdc van Lactobacillus plantarum en;
padc van Bacillus subtilis. PadA van Pediococcus pentosaceus is ook reeds geïdentifiseer.;
S. cerevisiae bevat die PAD1- (fenielakrielsuurdekarboksilase) geen, wat teen 'n vaste;
tempo in gis getranskribeer word. Die aktiwiteit van hierdie ensiem is egter laag.;
Fenolsuurdekarboksilase van B. subtilis, sowel as p-kumaarsuurdekarboksilase van;
L. plantarum, vertoon "n substraat-induseerbare dekarboksilerende aktiwiteit met fenoliese;
sure. Beide die p-kumaarsuur dekarboksilase en die fenolsuurdekarboksilase gene is in die;
PGK1PT ekspressie kasset gekloneer. Episomale en gisintegreringsplasmiede is vir die;
PAD1-geen onder beheer van die PGK1 PT ekspressiekasset gekonstrueer vir die;
ooruitdrukking van hierdie geen in gis. Die PGK1 PT ekspressiekasset het bestaan uit die;
promotor- (PGK1 p) en termineerdersekwense (PGK1 T) van die gisfosfogliseraatkinasegeen;
(PGK1). Industriële gisrasse is ontwikkel waarin die PAD1-geen onderbreek is.;
Ooruitdrukking van p-kumaarsuurdekarboksilase (Pdc) en fenolsuurdekarboksilase (pade) in;
S. cerevisiae toon hoë ensiemaktiwiteit in laboratoriumgisrasse. Die ooruitdrukking van die;
PAD1-geen het nie hoër aktiwiteit as die kontroleras gewys nie. Albei die bakteriële gene,;
onder die beheer van die PGK1 PT ekspressiekasset, is ook in "n gisintegreringsplasmied met;
die SMR1-geen as selektiewe merker geplaas. Die klonering en transformasie van pdc en;
padc in industriële wyngiste kan dus gebruik word vir die bepaling van die effek van fenolsuur;
dekarboksilases in die wynmaakproses en die moontlike verbetering van wynaroma. Wyn is;
met al drie die industriële rasse (die ooruitgedrukte bakteriële gene en die ontwrigte PAD1- geen) gemaak. Die effek van die teenwoordigheid van hierdie gene in die wynmaakproses is;
deur gaschromatografie bepaal. Die resultate het aangedui dat die bakteriële gene op In;
effektiewe wyse vlugtige fenole in die wyn kan produseer. Sekere monoterpene is ook in In;
verhoogde mate gedurende hierdie proses gevorm.;
Hierdie studie baan die weg vir die ontwikkeling van reingisinentingskulture vir die;
produksie van optimale vlakke van vlugtige fenole om sodoende die sensoriese gehalte van;
die wyn te verbeter.
Dissertation (PhD)--University of Stellenbosch, 2007.
ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been;
intertwined with human culture and long as ...civilized man has existed. This fermentation;
process has been dominated by the micro-organism Saccharomyces cerevisiae and from;
providing ancient seafaring explorers of a non perishable beverage to equipping bakers;
with a raising agent to turn flour into bread; this organism with its fermentative potential,;
has formed an essential part of most societies.;
In more recent times, many industries still rely on this basic principle. The;
complexities and efficiencies of the conversion of sugar into its various fermentative byproducts;
have been studied and optimised extensively to meet the specific demands of;
industries. Depending on the raw material used as starting point, the major beneficiaries of;
the useful characteristics have been alcoholic beverage producers (wine, beer, and;
whiskey amongst others), bakers (bread leavening) and biofuel producers.;
One of the obstacles in fermentation optimisation is the sugar consumption;
preferences displayed by the organism used. S. cerevisiae can consume a wide variety of;
sugars. Depending on the complexities of its structures, it shows a preference for the;
simpler saccharides. The fermentation of certain more complex sugars is delayed and runs;
the risk of being left residually after fermentation. Many of the crops utilised in;
fermentation-based products contain large amounts of starch. During the starch;
degradation process many different forms of sugars are made available for fermentation.;
Improved fermentation of starch and its dextrin products would benefit the brewing,;
whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and;
maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin;
products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to;
transport the aforementioned mono-, di- and trisaccharides into the cytosol. The;
inefficiency of maltotriose transport has been identified as the main cause for residual;
maltotriose. The maltotriose transporting efficiency also varies between different;
Saccharomyces strains.;
By advancing the understanding of maltotriose transport in yeast, efforts can be;
made to minimise incomplete fermentation. This aim can be reached by investigating the;
existing transporters in the yeast cell membrane that show affinity for maltotriose. This;
study focuses on optimising maltotriose transport through the comparison of the alpha;
glucoside transporter obtained from different strains of Saccharomyces. Through specific;
genetic manipulations the areas important for maltotriose transport could be identified and;
characterised.;
This study offers prospects for the development of yeast strains with improved maltose;
and maltotriose uptake capabilities that, in turn, could increase the overall fermentation;
efficiencies in the beer, whiskey, and biofuel industries.
AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde;
maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die;
verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.
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