Orientador: Ricardo Luiz Dantas Machado / Banca: Paula Rahal / Banca: Carlos Eugênio Cavasini / Resumo: O presente estudo avaliou a relação entre a resposta de anticorpos contra AMA-1 das variantes de P. vivax (VK210, VK247 e P. vivax-like) e os polimorfismos do gene HLA-DRB1 em populações endêmicas da Amazônia brasileira, para melhor entendimento dos mecanismos que modulam a resposta imune contra a malária. A resposta sorológica foi analisada em indivíduos maláricos e não-maláricos por testes de ELISA para AMA-1. Um subgrupo de amostras foi utilizado para genotipagem do gene HLA-DRB1 por PCR-SSP. Foram detectados 13 alelos diferentes do gene HLA- DRB1, sendo o alelo HLA-DRB1*04 o prevalente na população estudada. Foi detectada uma alta freqüência de respondedores para o antígeno AMA-1, com níveis crescentes de acordo com exposição prévia a malária. Nenhuma associação significativa foi observada entre as variantes da CSP do P.vivax e a resposta a AMA- 1, bem como aos polimorfismos do HLA-DRB1. O HLA-DRB1 apresenta uma distribuição heterogênea na população estudada, evidenciando uma contribuição característica de descendência ameríndia. A resposta de anticorpos contra o antígeno AMA-1 parece não influenciar na epidemiologia das variantes da CSP de P. vivax. Os polimorfismos do gene HLA-DRB1 não influenciam no desenvolvimento de reposta de anticorpos contra o AMA-1 na malária vivax na Amazônia brasileira / Abstract: To better understand the mechanisms of the immune response modulation against malaria, this study evaluated the relationship among the antibody response to AMA-1 and variants of the circumsporozoite protein (CSP) of the P. vivax (VK210, VK247 and P. vivax-like) and the polymorphisms of HLA-DRB1 gene in populations endemic from the Brazilian Amazon. The antibody response was analyzed in malarial and non-malarial individuals by AMA-1ELISA test. A subset of samples was genotyping of HLA-DRB1 by PCR-SSP. We detected 13 different alleles of HLA-DRB gene, where the HLA-DRB1*04 was the commonest allele. A high frequency of responders to the antigen AMA-1 was detected, with increasing levels according to previous malaria experience. No significant association was observed among the response to P. vivax AMA-1 and, the variants of the CSP and the polymorphisms of HLA-DRB gene. The HLA-DRB1 has a heterogeneous distribution in the population studied, showing an effective contribution of Amerindian groups. The antibody response against the antigen AMA-1 does not influence the epidemiology of variants of the CSP of P. vivax. The polymorphisms of HLA-DRB1 gene do not influence the development of antibody response against AMA-1 in vivax malaria around the Brazilian Amazon region / Mestre

abstract: Malaria is a vector-borne parasitic disease affecting tropical and subtropical regions. Regardless control efforts, malaria incidence is still incredible high with 219 million clinical cases and an estimated 660,000 related deaths (WHO, 2012). In this project, different population genetic approaches were explored to characterize parasite populations. The goal was to create a framework that considered temporal and spatial changes of Plasmodium populations in malaria surveillance. This is critical in a vector borne disease in areas of low transmission where there is not accurate information of when and where a patient was infected. In this study, fragment analysis data and single nucleotide polymorphism (SNPs) from South American samples were used to characterize Plasmodium population structure, patterns of migration and gene flow, and discuss approaches to differentiate reinfection vs. recrudescence cases in clinical trials. A Bayesian approach was also applied to analyze the Plasmodium population history by inferring genealogies using microsatellites data. Specifically, fluctuations in the parasite population and the age of different parasite lineages were evaluated through time in order to relate them with the malaria control plan in force. These studies are important to understand the turnover or persistence of "clones" circulating in a specific area through time and consider them in drug efficacy studies. Moreover, this methodology is useful for assessing changes in malaria transmission and for more efficiently manage resources to deploy control measures in locations that act as parasite "sources" for other regions. Overall, these results stress the importance of monitoring malaria demographic changes when assessing the success of elimination programs in areas of low transmission. / Dissertation/Thesis / Ph.D. Microbiology 2014

Made available in DSpace on 2014-06-12T15:05:39Z (GMT). No. of bitstreams: 2 arquivo1800_1.pdf: 966444 bytes, checksum: ab2d1d73e5179a8e16442fa325514e9b (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / A malária representa grave problema mundial de saúde pública. Estima-se sua incidência em 400 milhões de casos anuais principalmente nas regiões tropicais e subtropicais. Este projeto avaliou aspectos eco-epidemiológicos da malária em Roraima, onde há grande diversidade de ecossistemas naturais. Inicialmente é descrita a carta anofélica do estado, com 12 espécies identificadas e dos ecossistemas ocupados por elas. Em dois ecossistemas, floresta e savana, foram realizados testes imunológicos para determinar a prevalência de infecção em mosquitos adultos para identificar vetores locais. Uma área de estudo situa-se num projeto de assentamento na floresta tropical de alta transmissão de malária. A segunda encontra-se na beira da mata ciliar característica dos cursos hídricos da savana. Durante um ano, coletamos larvas e adultos de anofelinos, determinando e caracterizando criadouros potenciais; analisamos variações na densidade dos mosquitos, e estudamos a variação sazonal da idade das populações de mosquitos, duração do ciclo gonotrófico, capacidade de dispersão e horário de picada. Também calculamos curvas de sobrevivência e analisamos técnicas de determinação etária das espécies. Foi analisada a variação da distribuição das larvas ao longo do ano e sua resposta ao desmatamento. A prevalência de infecção em mosquitos adultos implicou An. albitarsis s.l., além de An. darlingi, como transmissor. As taxas de sobrevivência se correlacionaram bem com os registros da literatura, mesmo sendo estes discrepantes. Houve aumento da longevidade durante a estação seca na floresta, correlacionado com aumento da malária, e queda durante a época de chuvas. Houve diferenças na duração do ciclo gonotrófico de An. darlingi encontrado na floresta em comparação com o da savana. Formas imaturas de An. darlingi se agregaram próximo a residências, mas o desmatamento exerceu papel negativo sobre seus criadouros. Construções de reservas hídricas proporcionaram redutos para An. darlingi, permitindo sua sobrevivência durante a seca e transmissão de malária durante todo o ano, mas apenas nos seus arredores, devido à limitada dispersão da espécie em condições naturais. Observaram-se picos de densidade de An. darlingi e An. albitarsis s.l. no fim da estação seca, na floresta, e durante as chuvas, na savana. An. albitarsis s.l. na savana também apresentou aumento de densidade correlacionado com as chuvas. Esses dados foram correlacionados com os de densidade larvária e de paridade para criar um modelo de transmissão de malária distinto para dois locais com diferentes tipos de drenagem hídrica. Finalmente, descrevemos a ocorrência de um parasito de mosquitos sem relato prévio na América Latina, presumivelmente com efeitos deletérios sobre o desenvolvimento ovariano de fêmeas de An. darlingi

O Antígeno-1 de Membrana Apical (AMA-1) de merozoítas de Plasmodium é um dos principais candidatos a compor uma vacina contra a malária. A função biológica de AMA-1 não é totalmente conhecida, entretanto, existem evidências que sugerem a participação dessa proteína na ligação a eritrócitos de diferentes espécies de Plasmodium. O objetivo deste estudo foi investigar a participação de AMA-1 de P. vivax (PvAMA-1) na ligação a eritrócitos humanos utilizando células COS-7 transfectadas com plasmídios recombinantes codificando diferentes regiões do ectodomínio de PvAMA-1. Para isso, os genes que codificam os domínios I-II ou III de PvAMA-1 foram inseridos no vetor pDisplay-EGFP, que permite a expressão das proteínas recombinantes em fusão com a porção N-terminal da Proteína Fluorescente Verde (GFP). Em paralelo, utilizamos construções contendo os genes que codificam a região C-terminal de 19 kDa da Proteína 1 de Superfície do Merozoíta (PvMSP119) e a região II da Duffy Binding Protein (PvDBP-RII). As quatro construções foram utilizadas para transfectar células COS-7 na presença de lipofectamina. A eficiência das transfecções transientes foi confirmada por imunofluorescência utilizando anticorpos específicos. Em seguida, estudamos a participação dos diferentes domínios de PvAMA-1 na ligação aos eritrócitos. Nossos resultados mostraram que os domínios contíguos I-II, ao contrário do domínio III, foram capazes de se ligar a eritrócitos in vitro. Essa ligação foi específica, pois soros de indivíduos infectados por P. vivax e soros policlonais de camundongos contendo anticorpos anti-PvAMA-1 foram capazes de inibir essa ligação em 82,0% e 79,8%, respectivamente. Além disso, anticorpos monoclonais dirigidos contra o domínio II dessa proteína foram capazes de inibir parcialmente essa ligação. Após o tratamento de eritrócitos com tripsina ou quimiotripsina, estas células perderam grande parte de sua capacidade de ligação, sugerindo que o receptor para PvAMA-1 tenha constituição predominantemente protéica. Em conjunto, nossos resultados podem servir de base para futuros estudos visando um melhor entendimento da função de anticorpos gerados durante a infecção natural ou induzidos após vacinação com PvAMA-1. / The Apical Membrane Antigen (AMA-1) of Plasmodium merozoites is one of the main candidates to be part of a vaccine against malaria. The biological function of AMA-1 is unknown. However, there are evidences that suggest the participation of this protein in the interaction with erythrocytes (RBC) of different Plasmodium species. Using transfected COS-7 cells with recombinant plasmids encoding different portions of the PvAMA-1 ectodomain, our aim was to identify possible domains of PvAMA-1 able to interact with human RBC. The genes that encoded domains I and II in combination or domain III of PvAMA-1 were cloned into the pDisplay-EGFP vector. This vector allows expression of the protein fused to the N-terminus of enhanced green fluorescent protein (GFP). In parallel, we also used constructions containing the genes that encoded the 19 kDa C-terminal region of Merozoite Surface Protein 1 (PvMSP119) and region II of the Duffy Binding Protein (PvDBP-RII). Constructions were used to transiently transfect COS-7 cells. The efficiency of expression of all constructs was confirmed by immunofluorescence assay using specific antibodies. After that, we studied the participation of the different domains of PvAMA-1 in the binding to human RBC. We found that COS-7 cells expressing domains I-II, but not domain III, bound to human RBC in vitro. This binding was specific, because sera from malaria-infected patients and mouse polyclonal sera containing antibodies to PvAMA-1 were able to block the adhesion by 82.0% and 79.8%, respectively. Moreover, monoclonal antibodies directed against domain II were partially inhibitory in the cytoadherence assays. The receptor recognized on the surface of COS-7 cells expressing the domains I and II was partially removed after human RBC were treated with trypsin or chymotrypsin, suggesting that its composition is predominantly protein. In conclusion, our results can be used as basis for future studies aimed at better understating the function of the antibodies generated during the natural infection or after vaccination with PVAMA-1.

Notre étude s’est proposée d’analyser l’incidence du paludisme et son évolution dans le temps et dans l’espace, ainsi que de rechercher les facteurs de risque d’accès palustres chez les enfants d’un village du Moyen-Oyapock (Camopi), peuplé d’Amérindiens wayampi et émerillon, d’une part, et d’un village du Haut-Maroni (Antecume Pata), peuplé d’Amérindiens wayana, d’autre part. L’approche a été multiple avec, pour chacun des deux sites d’étude :- Une analyse de survie (modèle de Cox) à partir des accès palustres confirmés biologiquement dans une cohorte d’enfants de moins de sept ans régulièrement suivis, ainsi qu’un questionnaire de type Connaissances, Attitudes et Pratiques (CAP), puis les caractéristiques des habitats et la description de leur environnement immédiat.- Une analyse spatiale avec une classification de l’occupation du sol à partir d’images satellites SPOT 5, l’extraction de variables environnementales d’intérêt, l’étude de leur effet sur la transmission du paludisme et la mise au point d’une méthode objective de sélection d’un rayon d’observation autour des habitations pour la caractérisation de l’environnement.- Une étude de séries temporelles (ARIMA) afin de déterminer l’effet des évènements climatiques et hydrologiques sur le paludisme, aux niveaux local et plus global (El Niño).Les taux d’incidence d’accès palustres sur la période 2001-2009 se sont révélés particulièrement élevés chez les jeunes enfants, notamment à Camopi avec une moyenne de 773‰ par année. Une diminution brutale de l’incidence a eue lieu en 2007 sur le Haut-Maroni et ce phénomène est observé à Camopi depuis 2010. Une prémunition se développe assez rapidement au cours de la vie (2-3 ans), surtout contre les reviviscences à Plasmodium vivax. Les facteurs environnementaux se sont avérés être les plus nombreux et les plus fortement liés à l’incidence palustre. En effet, le dégagement des alentours du carbet de toute végétation et une certaine distance de celui-ci à la forêt sont des facteurs protecteurs. La composante géographique est également apparue essentielle à Camopi avec une incidence qui variait selon le fleuve d’habitation et en fonction de la distance au hameau principal. Les facteurs météorologiques locaux (température et niveau du fleuve) se sont également révélés être liés à l’incidence du paludisme à court terme (0-3 mois). Par ailleurs, nos résultats ont permis d’émettre un certain nombre d’hypothèses quant à la transmission et au(x) vecteur(s) local(ux), et notamment de suggérer la participation d’un vecteur autre qu’An. darlingi dans la transmission du paludisme à Camopi. Nous avons également prouvé par ce travail que la télédétection et les systèmes d’information géographique sont très prometteurs pour la prise en compte de la dimension spatiale et environnementale dans l’étude des maladies transmissibles, notamment dans les zones d’accès difficile de Guyane. / The aim of our study was to analyze the incidence of malaria in children and its evolution through time and space, as well as to search for risk factors in a village in Mid-Oyapock (Camopi), populated by Amerindians Wayampi and Emerillon, on the one hand, and a village in Upper-Maroni (Antecume Pata), populated by Amerindians Wayana, on the other hand. The approach was multiple with, for both study sites:- A survival analysis (Cox modelling) completed out of biologically confirmed malaria attacks in a cohort of children under seven, as well as a Knowledge, Attitudes, Practices and Behavior (KAPB) questionnaire, and also the characteristics of the houses and the description of their immediate environment.- A spatial analysis with a land cover classification from SPOT 5 satellite images, the extraction of environmental variables, the study of their effect on malaria transmission and the development of an objective method for picking the proper observation horizon around houses in order to characterize the environment.- A time series study (ARIMA) to determine the effect of climatic and hydrological events on malaria at local and global (El Niño) scales.The incidence rates of malaria attacks during the period 2001-2009 were particularly high among young children, especially in Camopi with an average of 773‰ by year. A sharp decline in incidence occurred in 2007 on the Upper Maroni and this phenomenon has been observed in Camopi since 2010. An acquired immunity develops quite rapidly during the life (2-3 years old), especially against P. vivax relapses. Environmental factors were found to be the most strongly associated with malaria incidence. Indeed, living in a hut cleared from the surrounding vegetation and at a larger distance from the forest are protective factors. The geographic component also appeared essential in Camopi with an incidence which varied with the river of living and with the distance from the main hamlet. The local meteorological factors (temperature and river level) also proved to be linked to malaria incidence in the short term (0-3 months). Moreover, our results have allowed issuing a number of assumptions about the transmission and the local vector(s), in particular to suggest the involvement of another vector than An. darlingi in the malaria transmission in Camopi. We also proved by this work that remote sensing and geographic information systems hold great promise for the inclusion of the spatial and environmental dimensions in the study of transmitted diseases, especially in areas of difficult access in French Guiana.

Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.

Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

Antimalarial drug discovery remains a challenging endeavour as malaria parasites continue to develop resistance to drugs, including those which are currently the last line of defence against the disease. Plasmodium falciparum is the most virulent of the malaria parasites and it delivers its deadliest impact during the erythrocytic stages of the parasite’s life cycle; a stage characterised by elevated catabolism of haemoglobin and anabolism of parasite proteins. The present study investigates the use of nanotechnology in the form of metallic silver nanoparticles (AgNPs) against P. falciparum leucine aminopeptidase (PfLAP), a validated biomedical target involved in haemoglobin metabolism. AgNPs were also tested against the human homolog cytosolic Homo sapiens leucine aminopeptidase (HsLAP) to ascertain their selective abilities. PfLAP and HsLAP were successfully expressed in Escherichia coli BL21(DE3) cells. PfLAP showed optimal thermal stability at 25 °C and optimal pH stability at pH 8.0 with a Km of 42.7 mM towards leucine-p-nitroanilide (LpNA) and a Vmax of 59.9 μmol.ml⁻¹.min⁻¹. HsLAP was optimally stable at 37 °C and at pH 7.0 with a Km of 16.7 mM and a Vmax of 17.2 μmol.ml⁻¹.min⁻¹. Both enzymes exhibited optimal activity in the presence of 2 mM Mn²⁺. On interaction with polyvinylpyrrolidone (PVP) stabilised AgNPs, both enzymes were inhibited to differing extents with PfLAP losing three fold of its catalytic efficiency relative to HsLAP. These results show the ability of AgNPs to selectively inhibit PfLAP whilst having much lesser effects on its human homolog. With the use of available targeting techniques, the present study shows the potential use of nanotechnology based approaches as “silver bullets” that can target PfLAP without adversely affecting the host. However further research needs to be conducted to better understand the mechanisms of AgNP action, drug targeting and the health and safety issues associated with nanotechnology use.

The advent of advanced modern nanotechnology techniques offers new and exciting opportunities to develop novel nanotech-derived antimalarial nanodrugs with enhanced selective and targeting abilities that allow for lower effective drug dosages, longer drug persistence and reduced drug degradation within the body. Using a nanodrug approach also has the advantage of avoiding drug resistance problems that plague reconfigured versions of already-existing antimalarial drugs. In this study recombinant triosephosphate isomerase enzymes from Plasmodium falciparum (PfTIM) and Humans (hTIM) were recombinantly expressed, purified and characterised. PfTIM was shown to have optimal pH stability at pH 5.0-5.5 and thermal stability at 25°C with Km 4.34 mM and Vmax 0.876 μmol.ml⁻ₑmin⁻ₑ. For hTIM, these parameters were as follows: pH optima of 6.5-7.0; temperature optima of 30°C, with Km 2.27 mM and Vmax 0.714 μmol.ml⁻ₑmin⁻ₑ. Recombinant TIM enzymes were subjected to inhibition studies using polyvinylpyrrolidone (PVP) stabilised silver nanoparticles (AgNPs) of 4-12 nm in diameter. These studies showed that the AgNPs were able to selectively inhibit PfTIM over hTIM with an 8-fold greater decrease in enzymatic efficiency (Kcat/Km) observed for PfTIM, as compared to hTIM, for kinetics tests done using 0.06 μM of AgNPs. Complete inhibition of PfTIM under optimal conditions was achieved using 0.25 μM AgNPs after 45 minutes while hTIM maintained approximately 31% of its activity at this AgNP concentration. The above results indicate that selective enzymatic targeting of the important, key metabolic enzyme TIM, can be achieved using nanotechnology-derived nanodrugs. It was demonstrated that the key structural differences, between the two enzyme variants, were significant enough to create unique characteristics for each TIM variant, thereby allowing for selective enzyme targeting using AgNPs. If these AgNPs could be coupled with a nanotechnology-derived, targeted localization mechanism – possibly using apoferritin to deliver the AgNPs to infected erythrocytes (Burns and Pollock, 2008) – then such an approach could offer new opportunities for the development of viable antimalarial nanodrugs. For this to be achieved further research into several key areas will be required, including nanoparticle toxicity, drug localization and testing the lethality of the system on live parasite cultures.

Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.