The sexual phase of Plasmodium falciparum begins with the differentiation of intraerythrocytic sexual stages, termed gametocytes, in the human host. Mature gametocytes circulate in the peripheral blood and are taken up by the mosquito during the blood meal. These stages are essential for the spread of the malaria disease and form gametes in the mosquito midgut within minutes. A highly conserved family of six secreted proteins has been identified in Plasmodium falciparum. They comprise multiple adhesive domains and are termed PfCCp1 through PfCCp5, and PfFNPA. It was revealed in this work that PfCCp multi-domain adhesion proteins form protein complexes in gametocytes and on the surface of newly emerged macrogametes by adhesion domain-mediated binding. Co-Immunoprecipitation assays with activated gametocyte lysates show interactions between PfCCp proteins and indicate surface association via Pfs230 and Pfs25. Pfs230 is connected with the plasma membrane of the parasite by its interaction partner Pfs48/45. This protein is linked to the plasma membrane by a GPI anchor and presumably retains the multi-protein complex on the surface of newly emerged macrogametes in the mosquito midgut. A WD40 domain containing protein was identified to be part of this protein complex. It might serve as platform for the assembly of the multi protein complex or mediate the interplay among proteins, as suggested from known functions of the WD40 domain repeats. During egress from the host erythrocyte, the emerging gametes become vulnerable to factors of the human complement, which is taken up with the blood meal. In this thesis it was found that the complement system is active for about one hour post feeding. Macrogametes defend against complement-mediated lysis by co-opting the human complement regulators Factor H and FHL-1 from the blood-meal. These serum proteins bind via its SCR domains 5-7 to the surface of macrogametes. Once bound, they trigger complement inactivation of the alternative pathway, which prevents induction of complement lysis on the surface of the malaria parasite. Antibodies against Factor H are able to impair the sexual development in vitro and are able to block transmission to the mosquito. Interaction studies on endogenous proteins and immobilized recombinant proteins revealed the PfGAP50 protein as binding partner of Factor H and FHL-1. This protein was hitherto described as a glideosome-associated protein in invasive parasite stages, but has not yet been characterized in gametes. First localization studies indicate a relocation of PfGAP50 from the inner membrane complex to the surface of macrogametes. Malaria still persists as one of the deadliest infectious diseases worldwide. Investigations on the essential transmissive stages, gametocytes and gametes of Plasmodium falciparum, stood in the background of research for a long time. This work deciphered details on protein interactions on the surface of the malaria parasite and provides first information about coactions between the parasite and the human complement in the mosquito midgut. / Die Sexualphase von Plasmodium falciparum beginnt mit der Ausbildung von intraerythrozytären Sexualstadien, sogenannten Gametozyten, im menschlichen Wirt. Reife Gametozyten zirkulieren im peripheren Blut und werden während der Blutmahlzeit von der Mücke aufgenommen. Dieses Parasitenstadium ist ausschlaggebend für die Verbreitung von Malaria und bildet im Mückendarm innerhalb von Minuten Gameten. In Plasmodium falciparum wurde eine hochkonservierte Familie bestehend aus sechs sekretierten Proteinen entdeckt. Diese bestehen aus verschiedenen Adhäsionsdomänen und werden PfCCp1 bis PfCCp5 und PfFNPA genannt. In dieser Arbeit wurde gezeigt, dass PfCCp Multiadhäsionsproteine Komplexe in Gametozyten und auf der Oberfläche von jungen Makrogameten mittels domänenvermittelter Bindungen bilden. Ko-Immunpräzipitationen mit Lysat aus aktivierten Gametozyten zeigten oberflächenvermittelte Interaktionen der PfCCp Proteine durch Pfs230 und Pfs25. Pfs230 ist mit seinen Interaktionspartner Pfs48/45 durch einen GPI-Anker mit der Plasmamembran des Parasiten verbunden. Der Multi-Proteinkomplex wird somit auf der Oberfläche von jungen weiblichen Gameten festgehalten. Zudem wurde in dem neu identifizierten Proteinkomplex ein Protein entschlüsselt welches WD40-Domänen aufweist. Bereits bekannte Funktionen von sich wiederholenden WD40-Domänen lassen vermuten, dass dieses Protein möglicher-weise als Plattform für den Zusammenbau des Proteinkomplexes dient oder das Wechselspiel zwischen Proteinen vermittelt. Während des Ausbruchs aus der Wirtszelle, dem Erythrozyten, werden Gameten angreifbar für Faktoren des humanen Komplements, welches mit der Blutmahlzeit in den Mückendarm aufgenommen wird. In dieser Arbeit wurde ermittelt, dass das Komplementsystem nach der Blutmahlzeit etwa eine Stunde lang im Mückendarm aktiv ist. Durch die Bindung der Regulatoren Faktor H und FHL-1 des menschlichen Komplementsystems aus der Blutmahlzeit, schützen sich Makrogameten gegen eine komplementvermittelte Lyse. Diese Serumproteine binden mittels ihrer SCR-Domänen 5-7 an die Oberfläche von Makrogameten und vermitteln damit die Inaktivierung des alternativen Komplementweges. Dadurch schützen sie sich vor der komplementinduzierten Lyse auf der Oberfläche des Parasiten. Antikörper gegen Faktor H vermindern die sexuelle Entwicklung in vitro und können die Weiterentwicklung des Erregers in der Mücke blockieren. Interaktionsstudien mit endogenen Proteinen und immoblilisierten rekombinanten Proteinen offenbarten PfGAP50 als Bindungspartner von Faktor H und FHL-1. PfGAP50 wurde bislang einem Motorkomplex zugeschrieben, welcher für die Parasitenbewegung von invasiven Stadien zuständig ist. Es wurde jedoch bis heute nicht in Gameten charakterisiert. Erste Lokalisationsstudien weisen auf eine Relokalisierung von PfGAP50 vom inneren Membrankomplex zur Oberfläche von Makrogameten hin. Malaria ist weiterhin eine der tödlichsten Infektionskrankheiten weltweit. Die Erforschung dieser für die Übertragung essentiellen Stadien, den Gametozyten und Gameten von Plasmodium falciparum, stand lange im Hintergrund der Forschung. Diese Arbeit entschlüsselt Details über Proteininteraktionen auf der Oberfläche des Malariaparasiten und beschreibt das Zusammenwirken des Parasiten mit dem menschlichen Komplementsystem im Darm der Mücke.

Le paludisme reste une des principales causes de mortalité infantile dans le monde tropical. L'émergence continue des résistances du parasite aux anti-paludiques constitue un sérieux problème de santé publique. La recherche de nouvelles cibles thérapeutiques, basée sur une connaissance plus approfondie des mécanismes moléculaires de la vie du parasite, est une nécessité permanente dans un paradigme de « reine rouge » qui s'applique parfaitement à la capacité d'adaptation du parasite. La découverte récente d'une métacaspase de Plasmodium falciparum (PfMCA1) et la mise en évidence de son rôle potentiel dans l'apoptose du parasite, fait qu'elle est une cible thérapeutique contre le paludisme. Dans le but de mieux approfondir les connaissances sur cette protéine cible, nous avons voulu, dans un premier temps, déterminer la structure tridimensionnelle de PfMCA1, afin de confirmer les différentes structures prédites in silico, et chercher de nouvelles molécules candidates par le docking moléculaire. Cependant cet objectif n'a pas pu être atteint, à cause d'un phénomène d'autoclivage de la protéine suite à son expression, ce qui fait que nous n'avons pas réussi à récupérer la protéine. Dans un second temps, nous avons étudié la métacaspase de Plasmodium vivax (PvMCA1) en comparaison avec PfMCA1, et nous avons montré que les résidus histidine et cystéine dans la dyade catalytique sont bien conservés. Nous avons identifié un deuxième site potentiel dans le domaine catalytique de PvMCA1. A partir d'échantillons collectés en Mauritanie, au Soudan et à Oman, nous avons montré que les résidus histidine et cystéine, ainsi, que les résidus du second site du domaine catalytique de PvMCA1 sont très variables. Les mutations de ces résidus doivent faire l'objet d'étude approfondie de leurs effets sur la fonction de la protéine PvMCA1. Ce polymorphisme trouvé dans les résidus catalytiques de PvMCA1, doit-être évalué comme marqueurs moléculaires de résistance / Malaria remains one of the main causes of infant mortality in the tropical world.The continuous emergence of parasite resistant to drug treatment is a serious threat to public health. Exploring new therapeutics targets based on depth knowledge on molecular mechanism of the parasite’s life is utmost needed in a paradigm of « red queen», which applies perfectly on the ability of the parasitic adaptation. The recent discovery of metacaspase of Plasmodium falciparum (PfMCA1) and the demonstration of its potential role in apoptosis, make it a therapeutic target against malaria. In order to increase knowledge about this protein, we planned, to determine the three-dimensional structure of PfMCA1, to confirm the different structures predicted in silico, and to look for new drug using molecular docking. However, this goal was not reached, since autoprocessing occurred during expression, and we failed to obtain the full-length protein. Then we studied the metacaspase of Plasmodium vivax (PvMCA1) in comparison with PfMCA1 and, we shown that histidine and cysteine residues in the dyad catalytic are well conserved. We have identified a second potential site in the catalytic domain of PvMCA1. We shown that residues in both putative sites are highly polymorphic in samples from Mauritania, Sudan and Oman. Mutations on these residues need to be deeply studied for their effects on the PvMCA1 function. This polymorphism found in catalytic residues of PvMCA1should be evaluated as new molecular marker of resistance

The malaria pre-erythrocytic stages have been identified as an ideal therapeutic target, but complex in vitro models for Plasmodium vivax and Plasmodium falciparum lack the efficiency needed for rapid screening and evaluation of new vaccines and drugs, especially targeting the P. vivax hypnozoite. To address this challenge, we employed a multi-parameter approach using “omics’” to identify pre-erythrocytic targets and biomarkers, guide phenotypic therapeutic screening, and study parasite functionality with innovative bioassays using highcontent screening. Herein, we discuss three novel bioassays formatted in 384-well plate systems with utilization of commercially-available materials and application of high-content imaging for rapid bio-image analysis. To refine functional assessment of pre-erythrocytic targets in early infection phases, we developed a real-time, ‘live’ sporozoite motility assay and a live sporozoite hepatocyte cell traversal assay to examine chemotherapeutic and immunoprophylactic interventions in biologically relevant environments. Furthermore, our 384-well primary hepatocyte culture system and methodology maintains stable hepatocyte physiology of cryopreserved primary human hepatocytes in addition to primary non-human primate hepatocytes for greater than 30 days, thus ideal for robust liver parasite development following infection with P. vivax, P. falciparum or P. cynomolgi sporozoites. We report antimalarial drug and vaccine studies performed in all bioassays with identification of novel anti-LS inhibition mechanisms. Additionally, this research discusses the discovery of potential sporozoite and liver stage targets identified through transcriptomic profiling of freshly isolated P. vivax and P. cynomolgi sporozoites using a candid approach of recapitulating the pivotal transition period from mosquito to human through microenvironment reconstruction and exposure to biological stimuli. We further characterize sporozoite invasive phenotypes through the application of the bioassays. Together, these novel functional assays enable us to rapidly evaluate potential preerythrocytic therapeutic candidates and analyze complex Plasmodium sporozoite phenotypes.

Mediante trabajos en campo y laboratorio, se investigó la respuesta de Anopheles pseudopunctipennis (Theobald, 1901) a la exposición de diferentes concentraciónes de insecticidas tipo piretroides 0.1%, 0.075%, 0.050%, 0.025%, 0.0125%. Las muestras biológicas fueron colectadas dentro y fuera de las viviendas, el método utilizado para la captura fue el método del cebo humano y trampa luz, el trabajo de campo se realizó en el Anexo Pampa Baja y San Isidro. La investigación realizada con los insecticidas piretroides según la metodología de la Organización Mundial de la salud (OPS) y en el Perú por el Instituto Nacional de Salud (INS) de concentración 0,1% por 24 horas se determinó una mortalidad de 81% para insecticida Deltametrina categorizado como especie en vigilancia por la Organización Mundial; para insecticidas Lamdaciaholotrina, Ciflutrina Permetrina, Cipermetrinala mortalidad fue superior al 98 % catalogados como especie sensible a este grupo de insecticidas. En el estudio en campo de Anopheles pseudopunctipennis, se ha evaluado el índice de Picadura por Hombre/Noche (IPHN) y el Índice de Picadura Hombre/hora (IPHH), en donde se ha observado el nivel máximo en el mes de enero llegando a 23 mosquitos por hora y 272 mosquitos por noche. Los resultados observados han permitido dar la información de la situación en que se encuentra la especie en estudio de nuestra región que es un vector principal de la malaria y si no se toma medidas de un control integrado, incrementará el riesgo de transmisión por los factores ambientales favorables y las inadecuadas prácticas para su control.

Plasmodium falciparum is the causative agent of malaria, one of the world‟s most prevalent infectious diseases. The emergence of strains resistant to current therapeutics creates the urgent need to identify new classes of antimalarials. Here we present and analyse a constraints-based model (iMPMP427) of P. falciparum metabolism. Consisting of 427 genes, 513 reactions, 457 metabolites, and 5 intracellular compartments, iMPMP427 is relatively streamlined and contains an abundance of transport reactions consistent with P. falciparum’s observed reliance on host nutrients. Flux Balance Analysis simulations reveal the model to be predictive in regards to nutrient transport requirements, amino acid efflux characteristics, and glycolytic flux calculation, which are validated by a wealth of experimental data. Furthermore, enzymes deemed to be essential for parasitic growth by iMPMP427 lend support to several previously computationally hypothesized metabolic drug targets, while discrepancies between essential enzymes and experimentally annotated drug targets highlight areas of malarial metabolism that could benefit from further research.

Plasmodium falciparum is the causative agent of malaria, one of the world‟s most prevalent infectious diseases. The emergence of strains resistant to current therapeutics creates the urgent need to identify new classes of antimalarials. Here we present and analyse a constraints-based model (iMPMP427) of P. falciparum metabolism. Consisting of 427 genes, 513 reactions, 457 metabolites, and 5 intracellular compartments, iMPMP427 is relatively streamlined and contains an abundance of transport reactions consistent with P. falciparum’s observed reliance on host nutrients. Flux Balance Analysis simulations reveal the model to be predictive in regards to nutrient transport requirements, amino acid efflux characteristics, and glycolytic flux calculation, which are validated by a wealth of experimental data. Furthermore, enzymes deemed to be essential for parasitic growth by iMPMP427 lend support to several previously computationally hypothesized metabolic drug targets, while discrepancies between essential enzymes and experimentally annotated drug targets highlight areas of malarial metabolism that could benefit from further research.

“Plasmodium vivax” és el paràsit causant de malària humana amb més àmplia distribució i és responsable cada any de 100-300 milions de casos clinics, incloent algunes manifestacions severes i mort. P. vivax envaeix principalment reticulòcits I és àmpliament acceptat que els reticulòcits infectats no citoadhereixen en els capil•lars d'òrgans interns, tenint un pas obligat per la melsa. Es desconeix com P. vivax escapa del pas per la melsa, el nostre major òrgan limfoide, i evita així ser fagocitat. La nostra hipòtesi postula que aquest paràsit indueix la formació de cèl•lules de barrera a la melsa, on reticulòcits infectats citoadhereixen de manera específica per protegir-se de l’atac dels macròfags. Per avançar en aquesta hipòtesi, hem utilitzat el model murí de malària en ratolins Balb/c infectats amb una soca noletal que infecta principalment reticulòcits, P. yoelii 17X, semblant a P. vivax, i una soca letal amb tropisme per normòcits, P. yoelii 17XL, semblant a P. falciparum. Per investigar la funció i l’estructura de la melsa en la malària, així com les característiques dinàmiques dels processos impulsats pel paràsit, hem implementat la microscòpia intravital i la ressonància magnètica de la melsa del ratolí en infeccions experimentals. Notablement, hem observat major acumulació de paràsits, motilitat reduïda, pèrdua de direccionalitat, augment en el temps de residència i ressonància magnètica alterada només a la melsa dels ratolins infectats amb 17X. D'altra banda, aquestes diferències s’han associat amb la formació d'una barrera d'origen fibroblàstic induïda en la polpa vermella de les melses de ratolins infectats amb la soca no-letal, acompanyada d'evasió dels macròfags de la polpa vermella i adherència dels glòbuls vermells infectats a la barrera. A més, hem confirmat l'adhesió de reticulòcits infectats per P. vivax de pacients humans a crioseccions de melsa humana i explorat el paper de les proteïnes Vir en aquesta adhesió. Les nostres dades suggereixen que en la malària no-letal el pas per la melsa és diferent del que es coneix en altres espècies de Plasmodium i obren noves vies per a estudis funcionals i estructurals d'aquest òrgan limfoide en la malària. / Plasmodium vivax is the most widely distributed human malaria parasite and responsible each year for 100-300 million clinical cases including severe disease and death. P. vivax invades predominantly, if not exclusively, reticulocytes and it is amply accepted that it does not sequester in the deep capillaries of inner organs having an obligate passage through the spleen. Questions thus remain as how P. vivax escapes spleen-clearance and establishes chronic infections. We have advanced a hypothesis postulating that this parasite induces the formation of spleen barrier cells where infected reticulocytes specifically cytoadhere protecting themselves from spleen macrophage-clearance. To advance this hypothesis, we have used the rodent malaria model of Balb/c mice infected with the reticulocyteprone non-lethal P. yoelii 17X strain, resembling P. vivax, and the normocyte-prone lethal P. yoelii 17XL strain, resembling P. falciparum. To investigate the function and structure of the spleen in malaria, as well as the dynamic characteristics of parasitedriven processes, we implemented the intravital microscopy and magnetic resonance imaging of the mouse spleen in experimental infections. Noticeably, there was higher parasite accumulation, reduced motility, loss of directionality, increased residence time and altered magnetic resonance only in the spleens of mice infected with 17X. Moreover, these differences were associated with the formation of a strain-specific induced spleen tissue barrier of fibroblastic origin, with red pulp macrophage-clearance evasion and adherence of infected red blood cells to this barrier. We further reported adhesion of P. vivax-infected reticulocytes from human patients to cryosections of human spleens and explored the role of Vir proteins in such an adhesion. Our data suggest that in reticulocyte-prone non-lethal malaria, passage through the spleen is different from what is known in other Plasmodium species and open new avenues for functional/structural studies of this lymphoid organ in malaria.

Thèse de doctorat : Sciences du vivant. Aspects moléculaires et cellulaires de la biologie : Strasbourg 1 : 2008. Thèse de doctorat : Sciences du vivant. Aspects moléculaires et cellulaires de la biologie : Université d'Abomey-calavi : 2008. / Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Bibliogr. p. 227-252.

Thesis (M.S.)--Florida State University, 2004. / Advisor: Dr. Dean Falk, Florida State University, College of Arts and Sciences, Dept. of Anthropology. Title and description from dissertation home page (viewed June 21, 2004). Includes bibliographical references.

Malaria is a global health concern accounting for approximately 219 million cases and an estimated 660 000 deaths in 2010. The most fatal strain of malarial parasite, Plasmodium falciparum is found to contain 3 Adenylate Kinases (PfAK1, PfAK2 and PfGAK). Adenylate Kinases are important enzymes that essentially catalyze and regulate energy metabolism processes. PfAK1 and PfAK2 catalyze the reversible MG2+ reaction ATP + AMP ←→ 2ADP whereas, the PfGAK catalyzes the Mg2+ dependent reaction GTP+AMP ←→ ADP+GDP. Of all malarial strains, only the Plasmodium falciparum Adenylate Kinase 2 (PfAK2) was found to contain a N-myristoylation sequence and subsequently formed a stable heterodimer with Plasmodium falciparum N-myristoyl transferase (PfNMT). The myristoylation of PfAK2 by PfNMT is believed to help transport PfAK2 to the parasitophorous vacuole membrane (PVM) so that the enzyme can perform its essential functions. With these enzymes being key components in the parasite’s survival, the structural study of these enzymes would provide a lot of insight into targeting these proteins for drug design that would effectively kill the parasite without affecting the human host. In this study, PfAK1 was able to be expressed, purified and crystallized with a dataset collected at 4.3Å. PfGAK was expressed and purified. A GTP analogue called GP5A was used to soak the purified PfGAKand the PfGAK bound to GP5A was crystallized and diffracted. Moreover, PfAK2 and PfNMT was successfully expressed and co-purified. The purified PfAK2-PfNMT heterodimer are undergoing crystal screening for possible crystallization conditions. / published_or_final_version / Physiology / Master / Master of Philosophy