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Transmission of the malaria parasite from man to the mosquito requires the formation of sexual parasite stages, the gametocytes. The gametocytes are the only parasite stage that is able to survive in the mosquito midgut and to undergo further development – gamete
formation and fertilization. Numerous sexual stage-specific proteins have been discovered, some of which play crucial roles for parasite transmission. However, the functions of many sexual stage proteins remain elusive. Amongst the sexual stage-specific proteins
are the proteins of the PfCCp proteins family, which exhibit numerous adhesion domains in their protein structures. For four members of the protein family, PfCCp1 to PfCCp4 gene-disruptant parasite lines had been already studied. Amongst these, PfCCp2 and PfCCp3 showed an important role for development of the parasites in the
mosquito. In the present work the study of gene-disrupted parasites of the PfCCp Protein family was completed. PfCCp5-KO and PfFNPA-KO parasite lines were characterized
to a great extent and many properties were similar to those of other PfCCp proteins. The co-dependent expression previously reported to be a phenomenon of PfCCp
proteins was also observed in these two mutants, although to lesser extent. When either PfCCp5 or PfFNPA were absent, all other proteins were detected in reduced abundance only. Co-dependent expression manifests exclusively on the protein level. Transcript
levels were not altered as RT-PCR showed. Amongst PfCCp proteins numerous proteinproteins interactions are taking place. The previously described multimeric protein
complexes also include further sexual stage-specific proteins like Pfs230, Pfs48/45 and Pfs25. Recently, a new component of PfCCp-based multimeric protein complexes had
been identified. The protein was named PfWLP1 (WD repeat protein-like protein 1) due to its possession of several WD40 repeats. In the present study expression of this uncharacterized protein was investigated via indirect IFA. It was expressed in asexual blood stages and gametocytes. Upon gamete formation and fertilization its expression ceased. Another sexual stage protein studied in this work was PfactinII. It was shown to be exclusively
expressed in sexual stages. In gametocytes it co-localizes with Pfs230 and correct localization of PfactinII depends on presence of Pfs230. Transcript analysis by means of RT-PCR revealed the expression of several components of the IMC in gametocytes. Furthermore, five or six myosin genes encoded in the
P. falciparum genome were detected in gametocytes. Gametocyte egress was studied on the ultrastructural level via transmission electron microscopy and an inside-out type of egress was observed. Firstly, the membrane of the parasitophorous vacuole (PVM) was lysed and only thereafter the membrane of the red blood cell (RBCM) ruptured. Furthermore, a new inductor of gametogenesis was identified: The K+/H+ ionophore nigericin induced gametocytes activation in the absence of xanthurenic acid (XA), which is responsible for gamtetocyte activation in the mosquito midgut. Selective permeabilization of RBCM and PVM by the mild detergent saponin, showed that in the absence of these membranes male gametocytes were still able to perceive both XA and the drop in temperature. Thus, the receptors for both factors signaling the parasite transmission to the mosquito, seem to be of parasitic origin. LC/MS/MS analysis confirmed the ability
of RBCs to take up XA. With malaria eradication on the agenda of malaria research targeting the sexual stages
becomes a crucial part of intervention strategies. The sexual stages are especially attractive target as they represent a population bottleneck. The here reported findings on P. falciparum gametocytes provide several potential candidate proteins for developing tools to interrupt transmission from man to mosquito. Such tools might include Transmission blocking vaccines and drugs.
Malaria is a vector-borne disease caused by the protozoan parasite of the genus Plasmodium and it is transmitted from human to human by female Anopheles mosquitoes during a blood meal. For malaria transmission to occur, the malaria parasite must undergo a crucial developmental sexual phase inside the mosquito midgut. In this study, we sought to investigate the interplay of the malaria parasite in the mosquito midgut with regard to the identification of novel types of transmission blocking intervention strategies. These strategies are aimed at reducing the spread of malaria by blocking the development of the mosquito midgut-specific stages of Plasmodium. We focused on three aspects. The first aspect was to investigate the interplay between mosquito midgut bacteria and malaria parasites in order to determine the potential influence of malaria parasites on the composition of the mosquito gut microbiota and also determine midgut bacteria which could be exploited as vehicles for the generation of paratransgenic Anopheles mosquitoes. We analyzed the microbial diversity of gut bacteria of the Asian malaria vector Anopheles stephensi during development and under different feeding regimes, including feeds on malaria parasite-infected blood, using the human pathogenic P. falciparum as well as the rodent malaria model P. berghei. 16S rRNA and DGGE analyses demonstrated a reduction in the microbial diversity during mosquito development from egg to adult and identified the gram-negative bacterium Elizabethkingia meningoseptica as the dominant species in the midgut of laboratory-reared male and female mosquitoes. E. meningoseptica is transmitted between generations and its predominance in the mosquito midgut was not altered by diet, when the gut microbiota was compared between sugar-fed and blood-fed female mosquitoes. Furthermore, feeds on blood infected with malaria parasites did not impact the presence of E. men-ingoseptica in the gut. Interestingly, extracts from E. meningoseptica exhibited antibacterial, antifungal and antiplasmodial activities, which may account for its dominance in the midgut of the malaria vector. Isolates of E. meningoseptica were cultivable, making the bacterium a potential candidate vehicle for the generation of paratransgenic Anopheles mosquitoes. The second aspect of this thesis was to determine transcriptome changes that occur during the first half hour following transmission of P. falciparum to the mosquito vector in order to better understand gene regulation mechanisms important for the change of hosts and determine novel proteins which could be exploited in malaria transmission blocking interventions. We initially used suppression subtractive hybridization (SSH) to compare mRNA levels of P. falciparum gametocytes before and 30 min fol-lowing activation. We identified a total of 126 genes for which transcript expression changed during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or motor complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were genes encoding for cell surface associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. We further selected a subset of 34 genes from all the above ontology groups and analyzed the transcript changes during gametogenesis in detail by quantitative realtime RT-PCR. Of these, 29 genes were expressed in gametocytes, and for 20 genes transcript expres-sion in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of eight genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito which could be exploited in malaria transmission blocking strategies. The last aspect of this thesis was to determine the transmission blocking effect of a range of antimicrobial molecules as transmission blocking agents. The molecules were either isolated from insect hemolymph or recombinantly expressed in tobacco and designed to act either directly on the mosquito midgut stages or cover receptors on mosquito tissues like the midgut epithelium which the parasite would need for transit. We were able to show an antiplasmodial and transmission blocking effect of the anti-microbial molecule harmonine, a defense compound isolated from the hemolymph of the Asian ladybug Harmonia axyridis. Harmonine thus represents a potential lead structure for the development of novel antimalarials.
Die vorliegende Arbeit, die im Rahmen des SFB 630 „Erkennung, Gewinnung und funktiona-le Analyse von Wirkstoffen gegen Infektionskrankheiten“ erstellt worden ist, beschäftigt sich mit der Entwicklung und Synthese der bisquartären Bisnaphthalimide und deren antimikro-biellen Eigenschaften, speziell gegen Erreger tropischer Infektionskrankheiten, wie Plasmo-dien und Trypanosomen aber auch Bakterien wie Staphylococcus aureus. Erste Testungen einer kleinen Bibliothek verschiedener mono- und bisquartärer Phthal- und Naphthalimide im SFB 630 offenbarten deren antimikrobielles Potenzial. Daher war es das Hauptziel dieser Arbeit, durch systematische Variation der verschiedenen Strukturbestandteile diese Bibliothek zu erweitern. Dazu mussten zuerst die entsprechenden Naphthalin-1,8-dicarbonsäure-Anhydride hergestellt werden. Im nächsten Schritt wurden diese mit einem N,N-Dimethylaminopropylamin-Derivat zum Imid kondensiert und abschließend mit einem Alkyl-Linker zur bisquartären Verbindung alkyliert. So konnte die Bibliothek um 25 Verbin-dungen erweitert werden. Dabei umfassten die Variationen die Alkylkettenlänge zwischen den quartären Stickstoffen mit 3–14 Methylen-Einheiten, das aromatische Substitutionsmuster mit Amino- bzw. Nitrogruppen und symmetrische, wie asymmetrische Bisnaphthalimide. Durch anschließende antimikrobielle Testung und qualitativen Vergleich der ermittelten IC50-Werte konnten verschiedene strukturelle Merkmale der Bisnaphthalimide identifiziert werden, die einen positiven Einfluss auf die Aktivität gegen den untersuchten Mikroorganismus haben.
Die paläotropischen Pflanzenfamilien der Ancistrocladaceae und Dioncophyllaceae sind die bisher einzig bekannten Produzenten von Naphthylisochinolin-Alkaloiden. Diese spezielle Klasse acetogeniner Sekundärmetabolite weist durch die verschiedenen Kupplungspositionen der beiden namensgebenden Molekülbausteine eine breite strukturelle Diversität auf und zeichnet sich durch vielfältige pharmakologische Wirksamkeiten, z.B. antiplasmodiale, antileishmaniale oder antitrypanosomale Aktivitäten, aus. Zur Synthese dieser Naturstoffe wurde im Arbeitskreis Bringmann eigens eine Methodik entwickelt, das Lacton -Konzept. Diese Methode erlaubt durch eine Vorfixierung der beiden Molekülhälften durch eine Esterbrücke, anschließender intramolekularer Kupplungsreaktion und der stereoselektiven Öffnung des erhaltenen Lactons den atropselektiven Aufbau der Naphthylisochinoline. Als Ziele dieser Arbeit ergaben sich somit die Synthese pharmakologisch und strukturell interessanter Naphthylisochinolin-Alkaloide mittels des Lacton-Konzepts sowie die Isolierung und Strukturaufklärung weiterer Sekundärmetabolite aus Triphyophyllum peltatum (Dioncophyllaceae), welche anschließend auf ihre Bioaktivität hin untersucht werden sollten, um potenziell neue Leitstrukturen für neue Wirkstoffe zu finden.