Department of Biochemistry, Cell and Molecular Biology
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Item In Vitro Studies Of The Effect Of Anopheles Gambiae Midgut Bacteria On The Development Of Plasmodium Falciparum(University Of Ghana, 2021-12) Ametsi, Williams GodwinDuring blood feeding, female Anopheles mosquitoes may ingest Plasmodium gametocytes which undergo transformation in the gut and develop into sporozoites that are infectious to humans. Bacteria inhabit the mosquito gut, and the number and diversity of these bacteria change following blood feeding. The presence of some bacteria species results in the reduced intensity of developing Plasmodium parasites. Little attention has been given to understanding this direct mechanism of bacteria on Plasmodium parasites, and the effects of bacteria on malaria parasite developmental genes are not completely understood. This limits the scope of how gut bacteria, for example Enterobacter and Serratia, which have been found with anti-Plasmodium effects can be further explored for alternative disease control strategies. Therefore, this study investigated the lethal effect of cell-free secreted bio-products of E. cloacae and S. marcescens on a key Plasmodium parasite developmental gene (Gamete release gene, GAMER) for its potential as a target for malaria transmission-blocking. Plasmodium falciparum 3D7 and Dd2 cultures at 1% parasitaemia were independently exposed to spent Luria-Bertani (LB) medium from varying concentrations of Enterobacter cloacae and Serratia marcescens. The parasite killing effect of the bacteria were assessed with SYBR green fluorescent assay after 48 hours of co-culture. Spent media with final bacteria concentration between 10e+10-10e+20 reduced parasitaemia (P<0.001) compared to parasite culture without bacteria treatment. Using real-time (quantitative) PCR, it was found that the expression of GAMER was down regulated by 2 folds after 1 hour of screening P. falciparum 3D7 with cell-free spent medium of E. cloacae cultured for 8 hours in LB broth (Ec-8). However, the expression of GAMER was unaffected after 6 and 12 hours of screening P. falciparum 3D7 with Ec-8. These data provide information for further studies on gene and protein targets for transmission blocking interventions.Item Functional Characterization of Plasmodium Falciparum Claudin-Like Apicomplexan Microneme Protein (Pfclamp) (Pf3d7_1030200)(University of Ghana, 2019-07) Quansah, E.B.Malaria is still a public health burden. With the recent reports of artemisinin resistance in Plasmodium falciparum coupled with the low efficacy of the RTS, S vaccine currently under a WHO-recommended pilot malaria vaccine implementation programme (MVIP), there is the need to continue identifying new targets by functionally characterizing some of the ~60% of the parasite’s genes with unknown functions. This will foster the identification of viable vaccine and possible drug targets for the development of interventions against the parasite. This project focused on studying P. falciparum Claudin-Like Apicomplexan Microneme Protein (PfCLAMP) with gene ID (Pf3D7_1030200) and its role during invasion of the parasite. CLAMP has been shown to be highly conserved in apicomplexans, with its orthologue in P. falciparum being essential for parasite growth and invasion. Here, using specific antibodies raised against the extracellular domain of the PfCLAMP, we report that PfCLAMP localizes at the apical portion of merozoites. The generated antibodies inhibited parasite invasion in a dose dependent manner. We have also observed that PfCLAMP is differentially expressed across the different asexual stages of the parasite, with peak expression occurring at the late stages of the parasite. We also discovered that some clinical isolates habour multiple copies of the PfCLAMP gene. Additionally, we show that PfCLAMP conditional gene knockout reduced invasion by up to 30% in the first cycle of the parasite development. Altogether, our data demonstrates that PfCLAMP provides a potentially attractive target for further investigation for drug and vaccine development.Item Functional Characterization of Plasmodium Falciparum Pf10_0351 Protein(University Of Ghana, 2017-07) Appiah Essel , C.C.N.Inadequate understanding of the biology of Plasmodium falciparum and the aetiology of malaria is a hindrance to the development of effective drugs and vaccines against the backdrop of resistance to Anopheles insecticides and antimalarial drugs, including artemisinin-based combination therapies (ACTs). About 60% of proteins of the P. falciparum genome has not been characterized. Since clinical symptoms of malaria are manifested at the blood stage, parasite proteins involved in erythrocyte invasion are of major research interest in vaccine development. This study aimed to functionally characterize a probable P. falciparum PF10_0351 protein, and establish its role during invasion. To achieve this, bioinformatic and immunoinformatic analyses were used to map out three highly antigenic epitopes for synthesis and antibody production. ELISA was performed to test whether the peptides will react with natural human plasma from malaria-endemic areas in Ghana with varying transmission intensities (Kintampo>Navrongo>Accra). Invasion inhibition assays were also performed to assess the ability of the peptide-specific antibodies to block invasion, using flow cytometry. Immunolocalization experiments were also performed in schizont, merozoite and gametocyte stages by immunofluorescence assay (IFA) and fluorescent microscopy. The peptides, particularly PF10_0351-2 and -3, could be recognized by naturally-induced antibodies in human plasma. However, there was no significant association between plasma antibody levels and age or parasite density by Spearman’s correlation. Peptide-specific antibodies inhibited parasite invasion of erythrocytes in a dose-dependent manner with significant differences in their invasion inhibitory effects (Tukey’s range test, p<0.0001). PF10_0351 appears to co-localize with Pfs48/45 on the surface of gametocyte stages, and also partially co-localize with MSP-1 on the surface of schizonts. Taken together, these findings suggest that the PF10_0351 protein may be localized to the surface of schizont, merozoite and gametocyte, and that it may be considered for further studies to explore its role in invasion.Item Assessment of the Effects of Cocoa and Tea Kombucha on Asexual Stages of Plasmodium Falciparum(University of Ghana, 2017-07) Agbemafle, S.Tea and Cocoa Kombucha are fermented beverages produced by the action of a symbiotic culture of bacteria and yeasts. Their health benefits have been hailed and staunchly declared by Kombucha drinkers worldwide. However, there is minimal scientific evidence on their anti-malarial properties. This research aimed to further investigate the anti-plasmodial properties and possible mechanism of action of tea Kombucha and cocoa Kombucha by determining the inhibitory concentrations, evaluate the stage specificity, assess the inhibition of Plasmodium falciparum erythrocyte invasion and determine the antioxidant and cytotoxic activities of Kombucha extracts and fractions. Kombucha black and green tea and Kombucha cocoa were prepared, neutralized and fractionated using three different solvents (hexane, ethyl acetate and water) to obtain the respective extracts for in-vitro assays. The growth inhibition effects of the various extracts on in vitro P. falciparum were assessed using the SYBR Green I assay. Ring stage survival assays and schizont stage specific assays were evaluated after 72 hours using flow cytometry. Inhibition of P. falciparum invasion of human red blood cells (RBCs) treated with Kombucha extracts/fractions were also assessed using flow cytometry. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) method was used to assess antioxidant activity of Kombucha on human peripheral blood mononuclear cells (PBMCs). The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) method was used to assess cytotoxicity profiles of Kombucha extracts and their fractions. The neutralized ethyl acetate fraction of cocoa Kombucha exhibited over 20 times (20x) the inhibitory activity of the corresponding unneutralized ethyl acetate fraction on Dd2 P. falciparum, and on NF54, the activity of neutral cocoa was almost 6 times (5.8x) its unneutralized counterpart. The ethyl acetate fraction of the neutralized GTK was 6x less potent than the non-neutralized ethyl acetate fractionof green tea Kombucha in inhibiting the ring stage of P. falciparum. The ethyl acetate fraction of green tea extract demonstrated schizonticidal activity at concentrations above its IC50(2mg/ml). The aqueous CTK extract displayed more potency in inhibiting invasion (46.6%) of P. falciparum Dd2 at its IC50 (280 μg/ml). The neutral cocoa Kombucha (NCTK) was 66% more effective at radical scavenging than the fermented CTK extract. The ethyl acetate NCTK fraction displayed the highest selective index (4.21). Generally, ethyl acetate selectively extracted bioactive components of Kombucha that exhibited anti-plasmodial and antioxidant activities coupled with good selectivity index compared to all other extracts and fractions. Cocoa Kombucha and its fractions exhibited the most active anti-plasmodial and stage specific activity on P. falciparum and could be explored as a food supplement for malaria prophylaxis.Item Copy Number Variation of GTP Cyclohydrolase 1 (Gch1) Gene and Its Impact on Antifolate Drug Resistance of Plasmodium Falciparum in Ghana.(University Of Ghana, 2017-07) Osei, M.As part of the malaria control measures in Ghana, sulfadoxine-pyrimethamine (SP) is used as an Intermittent Preventive Treatment (IPTp) among pregnant women and now as a seasonal chemoprophylaxis (SMC) in children on pilot basis. However, parasite resistance to the drug has been reported in the country and this has been linked to point mutations in the dhps (dihydroptoreate synthase) and dhfr (dihydrofolate reductase) genes, the targets of SP. There is also an evidence of amplification of GTP cyclohydrolase 1 (gch1) gene, which codes for the first enzyme in the parasite denovo folate pathway, amongst parasites which harbor the highest SP resistance point mutation (164L) in South East Asia. These point mutations make the parasites less fit, but the acquisition of multiple copies of the gch1 gene may compensate for this fitness cost. This study sought to determine the prevalence and effects of Pfgch1 copy number variations (CNV) on SP resistance amongst clinical isolates in Ghana. Two hundred and two (202) blood samples collected from children aged 14 years and below, with uncomplicated malaria presenting at health centres in Accra, Kintampo, Cape coast, and Navrongo were used in this study. Quantitative real-time PCR (qPCR) and RT qPCR were used to estimate the copy numbers and expression levels respectively. The pfdhps and pfdhfr regions were PCR amplified and directly sequenced. Almost ninety-three percent (92.6%) and 7.4% of the parasite isolates harbored single and double copies of the gch1 gene respectively. Duplication of gch1 gene was independent of the different study sites (P=0.696). Point mutations at dhfr108N (P<0.001) and dhfr108T (P<0.001) were found to be associated with the study sites. Mutations at dhfr108 appear to be fixed in the parasite population and mutations at dhfrS108T and dhpsA581G were observed for the first time in Ghana. However, there were no point mutations observed at codons 164L, 50R and 163T as reported elsewhere. For correlation between the mutations and gch1 CNV, only mutations at dhps540E (P=0.001), and dhps581G (P=0.002) were found to be significant. The relative expression between parasites with gch1 CN of 1 and 2 was about 3-folds. The findings from this study revealed that mutations at dhps540E and dhps581G correlated with double gch1 gene, implying that gch1 may compensate for the fitness cost in parasites harboring these mutations. Continuous monitoring of gch1, dhfr & dhps genes and also further studies to discover component drugs that can inhibit the gch1 gene product are required.Item The Carotenoid Biosynthesis Pathway In The Asexual Intraerythrocytic Stages Of Plasmodium Falciparum: In Vitro Inhibition Assays, Hplc Profiling Of Carotenoids And Bioinformatics Analysis(University of Ghana, 2016-03) Nortey-Mensah, R.; Teye, M. A.; Ofori, M. F.; University of Ghana, College of Basic and Applied Sciences, School of Biological Sciences, Department of Biochemistry, Cell and Molecular BiologyPlasmodium falciparum, like other Apicomplexans, has retained a relict plastid known as the apicoplast. This organelle represents a new and exciting target for the chemotherapeutic management of malaria because it houses metabolic pathways that are unique to the parasite such as isoprenoid biosynthesis. The phytoene synthase (PSY) gene, has been demonstrated to be very important in carotenogenesis, however, little is known about the evolutionary relatedness of this gene in P. falciparum and other Apicomplexans. This study therefore aimed at profiling the carotenoids synthesized in the asexual intraerythrocytic stages of P. falciparum and to determine the evolutionary history and relatedness of the PSY gene in Apicomplexans and other organisms. In vitro inhibition assays were performed on the asexual intraerythrocytic stages of P. falciparum using fluridone to determine its IC50 and effect on parasite population. HPLC was used to profile carotenoids synthesized at the asexual stages and to determine the evolutionary history and relatedness of the PSY gene in Apicomplexans and other organisms, an unrooted phylogenetic tree was generated using MEGA 6. A dose-dependent inhibition of parasite population was observed with fluridone treatment on all the asexual stages, with the ring stages being the most susceptible. The carotenoid profiles showed that synthesis of carotenoids in P. falciparum is cumulative through the asexual intraerythrocytic stages with carotenoids such lycopene, α-, β-carotene among others being synthesized. An exciting novel finding of this study was the discovery of relatively high amounts of abscisic acid (ABA) in the schizont stages and not in the other stages. This is the first time ABA has been demonstrated to be synthesized by P. falciparum and it would be pioneering to further investigate the specific role of ABA in P. falciparum schizont stages. The phylogenetic analysis showed that the P. falciparum PSY was most related to P. University of Ghana http://ugspace.ug.edu.gh xii reichenowi, the chimpanzee strain of the malaria causing parasites further lending support to the proposed origin of malaria species in humans.