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.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-01-29T18:42:14Z No. of bitstreams: 2 Tese - Luiz Augusto Pereira - 2008.pdf: 11628724 bytes, checksum: 97ca17282a858a05078e31d8a06bfefe (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-01-29T18:42:52Z (GMT) No. of bitstreams: 2 Tese - Luiz Augusto Pereira - 2008.pdf: 11628724 bytes, checksum: 97ca17282a858a05078e31d8a06bfefe (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-01-29T18:42:52Z (GMT). No. of bitstreams: 2 Tese - Luiz Augusto Pereira - 2008.pdf: 11628724 bytes, checksum: 97ca17282a858a05078e31d8a06bfefe (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2008-09-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioides brasiliensis, an important human pathogen causative of paracoccidioidomycosis (PCM), a systemic mycosis with broad distribution in Latin America. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. An adhesin of P. brasiliensiswas isolated from two dimensional electrophoresis and characterized. Peptides obtained by partial sequencing of the isolated protein, which presenteda molecular mass of 29 kDa and pI 5.8, were subjected to sequence analysis of their amino acids, that revealed strong homology to triose phosphate isomerase (TPI) from several sources. The complete cDNA and gene encoding TPI of P. brasiliensis (PbTPI) were characterized and both contained an open reading frame predicted to encode a 249 amino acid protein that presented all the peptides characterized in the native PbTPI. The complete coding PbTPI cDNA was cloned and over expressed in Escherichia coli host. The purified recombinant TPI was used to produce polyclonal antibody in rabbit. By immunoelectron microscopy and Western blot analysis, TPI was detected in the cell wall and the cytoplasm of the yeast phase of P. brasiliensis. The expression of PbTPI was analyzed in transition from mycelia to yeast phase. The native PbTPI is preferentially expressed in the yeast parasitic phase of P. brasiliensis. The recombinant PbTPI was found to bind to laminin and fibronectin in ligand far-Western blot assays. TPI binds preferentially to laminin, as determined by peptide inhibition assays. Of special note, the treatment of P. brasiliensisyeast cells with anti-PbTPI polyclonal antibody and the incubation of pneumocytes and VERO cells with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensisto those in vitrocultured cells. These observations indicate that TPI could be contribute to the adhesion of the microorganism to host tissues and to the dissemination of infection. / Paracoccidioides brasiliensis é um importante patógeno humano que causa a paracoccidioidomicose (PCM), uma micose sistêmica com ampla distribuição na América Latina. A adesão e a invasão de células são eventos essenciais envolvidos na infecção e disseminação do patógeno. Para isso, patógenos utilizam suas moléculas de superfície para se ligar a componentes da matriz extracelular e estabelecer a infecção. Uma proteína antigênica de P. brasiliensisfoi isolada a partir do gel de eletroforese bidimensional de proteínas totais do fungo e caracterizada. Peptídeos obtidos por sequenciamento parcial da proteína de 29 kDa e pI 5.8 mostraram homologia com triose fosfato isomerase (TPI) de diversos organismos. O cDNA e o gene completos que codificam para TPI de P. brasiliensis (PbTPI) foram caracterizados, e ambos contém uma ORF que codifica para uma proteína com 249 aminoácidos que apresenta todos os peptídeos caracterizados na PbTPI nativa. O cDNA completo que codifica para PbTPI foi expresso em Escherichia coli. A proteína recombinante TPI foi utilizada para produção de anticorpo policlonal em coelho. Através de imunomicroscopia de transmissão eletrônica e análises por Western blotting, foi detectada a presença da TPI, na parede celular de leveduras de P. brasiliensis e no citoplasma. A expressão da PbTPI foi analisada na transição das fases de micélio para levedura. A PbTPI nativa está preferencialmente expressa na fase parasitária de P. brasiliensis. A PbTPI recombinante foi capaz de se ligar a laminina e fibronectina em ensaios de Western blotting de afinidade. PbTPI se liga preferencialmente a laminina, como foi determinado por ensaio de inibição com peptídeos sintéticos. Uma observação importante, é que tanto o tratamento de P. brasiliensiscom anticorpo anti-PbTPI, quanto de pneumócitos e células VERO tratados com a TPI recombinante, promoveram considerável inibição da aderência e internalização de P. brasiliensisàs células cultivadas in vitro. Essas observações indicam que a TPI possivelmente contribui para a adesão do microrganismo aos tecidos do hospedeiro e para a disseminação da infecção.

Les réactions de phosphorylation / déphosphorylation chez les bactéries régulent plusieurs fonctions cellulaires telles que croissance, différenciation, pathogénie, résistance aux antibiotiques, réponse au stress, formation des biofilms ainsi que plusieurs processus impliqués dans le métabolisme secondaire. Cependant, les signaux qui déclenchent la cascade de signalisation par phosphorylation/déphosphorylation intracellulaire restent encore peu connus. Staphylococcus aureus est une bactérie à Gram-positif pathogène pour l‟homme. Elle est l‟une des principales causes des infections nosocomiales et ce pathogène opportuniste est capable de provoquer de multiples infections allant du furoncle à la septicémie. Nos études se sont basées sur la caractérisation aux niveaux structural et fonctionnel de deux protéines de cette bactérie : une sérine/thréonine kinase nommée Stk1 ainsi que l‟un de ses substrats, la triose phosphate isomérase. Stk1 a déjà été identifiée comme responsable de la phosphorylation de plusieurs enzymes impliquées dans le métabolisme central de la bactérie ainsi que dans les phénomènes de virulence et de résistance à l‟antibiotique phosphomycine. Cependant, à ce jour, aucune caractérisation structurale n‟a été conduite sur cette kinase. Nous avons ainsi mené une étude cristallographique de plusieurs domaines de cette protéine et nous présentons, plus particulièrement, la structure de trois domaines extracellulaires dits « PASTA », ainsi qu‟un modèle tridimensionnel de la protéine entière. Les domaines PASTA sont spécifiques des Ser/Thr kinases et des Penicillin-Binding Proteins et sont impliqués dans la synthèse du peptidoglycane. Par conséquent, la connaissance de la structure de ces domaines chez Stk1 pourrait servir de base à la conception rationnelle de nouveaux inhibiteurs à visée thérapeutique. Enfin, nous avons démontré que l‟activité de l‟un des substrats de Stk1, la triose phosphate isomérase, était régulée par phosphorylation / déphosphorylation, et nous avons décrit le mécanisme qui contrôle son activation/inactivation réversible. / The phosphorylation /dephosphorylation reactions in bacteria regulate various cellular functions such as growth, differentiation, pathogenicity, antibiotic resistance, stress response, biofilm formation as well as several processes involved in secondary metabolism. However, detailed understanding of their complete signaling pathways induced by phosphorylation/dephosphorylation is still unclear. Staphylococcus aureus is a Gram-positive bacterium and a human pathogen. It is one of the primary causes of nosocomial infections and this opportunist pathogen is able to cause multiple infections ranging from furuncle to septicemia. This study is focused on the structural and functional characterizations of two proteins from this bacterium: the serine/threonine kinase Stk1 and one of its substrates, the triose phosphate isomerase. Stk1 has been previously identified as responsible for the phosphorylation of several enzymes involved in the central metabolism of this bacterium as well as virulence and resistance to the antibiotic phosphomycin. However, no structural characterization has been done to date. We have performed a crystallographic study of several domains of this protein. We now present the structure of three extracellular domains designated “PASTA” in addition to the 3D molecular model of the entire protein. PASTA domains are specific to bacterial Ser/Thr kinases and to Penicillin-Binding Proteins which are involved in the peptidoglycan synthesis. Thus, the structural knowledge of PASTA domains from Stk1 could be of particular interest in the rational drug-design of new inhibitors with therapeutic aims. Finally, we have demonstrated that triose phosphate isomerase activity is regulated by phosphorylation/dephosphorylation and we have described the reversible activation/inactivation mechanism.