Development of the oocyst wall in Eimeria maxima and biochemical analysis of gametocyte wall forming bodies

University of Technology, Sydney. Faculty of Science.Eimeria is a cyst-forming intracellular parasite that causes the economically important disease, coccidiosis, in intensely reared broiler chickens worldwide. The ability of the Eimeria parasite to replicate very rapidly and to synthesize an impenetrable, highly resistant oocyst wall, allows it to build up to very large numbers in the litter of broiler flocks. The molecular machinery involved in the assembly of the oocyst wall is housed in the two types of wall forming bodies (WFB1 and WFB2) of the sexual stage parasites (macrogametocytes). The current project aimed to expand our understanding of the fundamental mechanisms involved in oocyst wall formation by: (1) characterising the morphological changes involved in oocyst wall assembly during parasite development; (2) developing a method to isolate gametocyte WFBs in order to characterise their molecular composition; and (3) studying the nature and characterizing the mechanisms of nutrient acquisition in developing E. maxima gametocytes in vitro. Extracted macrogametocytes were stained using cytochemical and immune-labelling methods, and morphological changes of the developing zygote characterised by bright-field, scanning electron and 3D confocal microscopy. Additionally, the WFBs of macrogametocytes were enriched by subcellular fractionation and fractions containing these organelles were analysed by microscopy, western blot and label-free quantitative shotgun proteomics. Data from these studies has shown that gametocytes and early stage oocysts contain surface pores and are capable of actively taking up and internalizing nano beads via endocytosis. In addition, microscopic analyses shows that E. maxima is selective in compartmentalizing neutral lipids to the type 1, and glycoproteins to the type 2 wall forming bodies during gametocytogenesis. Furthermore, it became possible to visualise both neutral lipids and glycoproteins during outer and inner oocyst wall formation. Thus, a model of outer oocyst wall formation was proposed and suggests that neutral lipids found in the WFB1s are translocated to the parasite’s surface where they deposit their cargo via exocytosis. The released molecules fuse with the parasite’s limiting membrane for incorporation into the neutral lipid rich outer oocyst wall. Finally, biochemical and proteomic methods were employed to identify and analyse vesicular trafficking proteins and other putative regulators of endocytosis and transport. The results reported here reveal valuable insights into the mechanisms by which the parasite is able to acquire nutrients essential for development, transport organelles and at the same time synthesise the impervious oocyst wall

Comparison of Protective Immune Responses to Apicomplexan Parasites

Members of the phylum Apicomplexa, which includes the species Plasmodium, Eimeria, Toxoplasma, and Babesia amongst others, are the most successful intracellular pathogens known to humankind. The widespread acquisition of antimicrobial resistance to most drugs used to date has sparked a great deal of research and commercial interest in the development of vaccines as alternative control strategies. A few antigens from the asexual and sexual stages of apicomplexan development have been identified and their genes characterised; however, the fine cellular and molecular details of the effector mechanisms crucial for parasite inhibition and stimulation of protective immunity are still not entirely understood. This paper provides an overview of what is currently known about the protective immune response against the various types of apicomplexan parasites and focuses mainly on the similarities of these pathogens and their host interaction. Finally, the evolutionary relationships of these parasites and their hosts, as well as the modulation of immune functions that are critical in determining the outcome of the infection by these pathogenic organisms, are discussed

Phytochemical and pharmacological investigations of traditional plant material used against malaria as a basis for developing new lead structures for Plasmodium falciparum

Titelblatt und Inhaltsverzeichnis Einleitung Ziel der Arbeit Experimenteller Teil Ergebnisse Diskussion Zusammenfassung Summary Abkürzungsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Literaturverzeichnis AnhangAus traditionell gegen Malaria verwendeten Heilpflanzen aus Latein-Amerika und Ost-Afrika konnten durch phytochemische Untersuchungen Substanzen unterschiedlicher Stoffklassen isoliert werden. Aus dem Blattmaterial von Exostema mexicanum A.Gray, Rubiaceae, wurden erstmalig 4-Phenylcumaringlykoside und zwei neue acylierte Flavonoltetraglykoside isoliert. Aus der bisher noch nicht phytochemisch untersuchten Verbenaceae Stachytarpheta guatemalensis Moldenke konnten fünf Phenylethanoid-Glykoside gewonnen werden, von denen drei zum ersten Mal in der Gattung Stachytarpheta nachgewiesen werden konnten. Aus dem Blattmaterial der zu den Kürbisgewächsen zählenden Momordica foetida Schum. ließen sich erstmalig klassische Flavonoidglykoside isolieren. Im pharmakologischen Teil dieser Arbeit wurden Untersuchungen zu möglichen Wirkmechanismen von Extrakten und isolierten Naturstoffen durchgeführt, und zwar wurde die Beeinflussung des glutathionabhängigen Hämabbaus und eine mögliche Cystein-Protease-Hemmung überprüft. Es wurden die Rohextrakte von M. foetida und deren Reinsubstanzen sowie antiplasmodial aktive Chalkone aus Humulus lupulus L., Cannabaceae, auf heminbindende Eigenschaften untersucht. Dabei zeigte das Flavanonglykosid Eriodicytol-7-O-β-D-glucopyranosid eine mit dem Anti-Malaria-Wirkstoff Chloroquindiphosphat vergleichbare Aktivität. Unter den aus Humulus lupulus isolierten Inhaltsstoffen wiesen die Substanzen Xanthohumol, 2 ,3 -Dihydroxanthohumol und das 3 ,4 -Pyranoxanthohumol Hemmungen des glutathionabhängigen Hemin-Abbaus von über 60 % im Vergleich zum Chloroquindiphosphat mit über 82 % auf, was mit den vorab bestimmten antiplasmodialen Aktivitäten an einem chloroquin-sensitiven (PoW) und einem chloroquin-resistenten (Dd2) P. falciparum Stamm nur partiell übereinstimmte. Eine mögliche Cystein-Protease-Hemmung wurde an der pflanzlichen Cystein- Protease Papain analysiert, da die rekombinate Herstellung der parasitären Falcipaine nicht nur schwierig und aufwendig, sondern auch sehr kostspielig ist. An diesem Testmodell wurden ebenfalls Chalkone aus H. lupulus überprüft. Von den Hopfenchalkonen wurden 5 Substanzen zusätzlich an den beiden parasitären Cystein-Proteasen Rhodesain (aus Trypanosoma brucei rhodesiense) und Cruzain (aus Trypanosoma cruzi) am Sandler Center der Universität von Kalifornien in San Francisco, USA getestet. Hier zeigte das Desmethylxanthohumol eine viel versprechende Aktivität.Plant remedies from Latin-America and East-Africa traditionally used against malaria were phytochemically investigated leading to the isolation of natural compounds of a variety of classes. From the leaves of Exostema mexicanum A.Gray, a member of the Rubiaceae, 4-phenylcoumarin glycosides and two new acylated flavonol tetraglycosides were isolated for the first time. Five phenylethanoid glycosides could be identified from the whole-plant of the previously non examined Verbenaceae, Stachytarpheta guatemalensis Moldenke. Three of these phenylethanoid glycosides were detected for the first time in the genus Stachytarpheta. Out of the leaves of Momordica foetida Schum. (belonging to the Cucurbitaceae) classical flavonoid glycosides were afforded for the first time. Additionally, investigations concerning possible mode of actions of extracts and isolated substances were performed, e.g. the influence on the glutathione dependent heme degradation and a possible inhibition of cysteine proteases. Raw extracts and pure substances of M. foetida, as well as antiplasmodially active chalcones derivatives, isolated from Humulus lupulus L., Cannabaceae, were examined for hemin binding properties. The flavanone glycoside eriodicytol-7-O-β-D-glucopyranoside showed an activity similar to the well- known antimalarial drug chloroquine. Furthermore xanthohumol, 2 ,3 -dihydroxanthohumol and 3 ,4 -pyranoxanthohumol displayed more than 60 % inhibition of hemin degradation, compared with 82 % for chloroquine. Nevertheless, these results are not totally in agreement with the antiplasmodial activity of these compounds. Another test model was used, in which the process of hemoglobin hydrolysis by cysteine proteases inside the food vacuole was simulated. Instead of falcipains, which are originally found in Plasmodium parasites and whose recombinant production is difficult, elaborate and expensive, the herbal cysteine protease papain was chosen. Furthermore, the chalcone derivatives from H. lupulus were tested regarding their inhibiting effects on the parasitic cysteine proteases rhodesain (from Trypanosoma brucei rhodesiense) and Cruzain (from Trypanosoma cruzi) at the Sandler Center, UCSF, USA, revealing a promising activity of desmethylxanthohumol

Cell biological analysis reveals an essential role for Pfcerli2 in erythrocyte invasion by malaria parasites

Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade and was associated with elongation of the rhoptry organelle during merozoite development and inhibition of rhoptry antigen processing. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion

DataSheet_1_Targeting malaria parasites with novel derivatives of azithromycin.pdf

IntroductionThe spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: ‘delayed death’ by inhibiting the bacterium-like ribosomes of the apicoplast, and ‘quick-killing’ that kills rapidly across the entire blood stage development.MethodsHere, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans).ResultsSeventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment.DiscussionThe azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.</p

DataSheet_2_Targeting malaria parasites with novel derivatives of azithromycin.pdf

IntroductionThe spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: ‘delayed death’ by inhibiting the bacterium-like ribosomes of the apicoplast, and ‘quick-killing’ that kills rapidly across the entire blood stage development.MethodsHere, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans).ResultsSeventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment.DiscussionThe azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.</p