Es geht um die Verbesserung verschiedener Infektions- und Transfektionsprotokolle zur in vitro Arbeit mit Cryptosporidium parvum in der Zellkultur. Des weiteren wurden Mausdarmorganoide ohne Hilfe eines Mikroinjektors mit C. parvum Sporozoiten infiziert. Außerdem wurde ein Protein (Trap-C1) von C. parvum welches ein putativer Virulenzfaktor ist, molekularbiologisch untersucht.

Introduction: Cryptosporidium parvum is one of the most common causes of diarrhea worldwide in neonatal calves. This pathogen is also life-threatening in malnourished children and immunodeficient patients. There is no vaccine and a single drug nitazoxanide (NTZ), of only the moderate efficacy has been approved by FDA for cryptosporidiosis treatment in human. Octaarginine is known to facilitate the transport of other molecules across cell membranes and has been use to transfect protozoan organisms. It is also proposed to increase the efficacy of drugs against intracellular pathogens. Aims of the study: The capacity of octaarginine to support transfection of C. parvum as an alternative to electroporation was evaluated. Furthermore, it was studied whether octaarginine covalently bound to NTZ (NTZ-R8) improves efficacy against the parasite. Animals, materials and methods: FAM-octaarginine was added to either intact oocysts, short-time excystation exposed (STE) oocysts, excysted sporozoites, intracellular stages of C. parvum to assess the permeability of the Cryptosporidium membrane to the peptide. The optimal conditions for condensation of plasmid for transfection experiments were evaluated by testing different N/P ratios applying by gel retardation assay. The transfection complex octaarginine/polyethyleneimine (PEI)/DNA was also incubated with intact oocysts, STE oocysts, and excysted sporozoites. Transfected parasites were transferred to HCT-8 cell cultures and further incubated for 24 h. Immunoflourescence assay (IFA) was performed to detect successfully transfected parasites. To evaluate the suitability of octaarginine as a vehicle supporting transport of NTZ across membranes, octaarginine was coupled to NTZ to produce NTZ-R8. Cryptosporidium oocysts were inoculated into HCT-8 cell monolayers in the presence of NTZ and NTZ-R8 at concentrations of 1, 5, 10, 50, 100 or 1000 ng/ml. Parasite growth was monitored by RT- qPCR after RNA extraction from C. parvum exposed HTC-8 cell cultures. RT-qPCR was performed on the target gene 18S rRNA of Cryptosporidium and normalized to the expression of the housekeeping gene 18S rRNA of host cells. To evaluate the efficacy of NTZ-R8 in vivo, IFN-γ knockout mice were orally inoculated with 1000 oocysts each, except for the non-infected controls. Infected mice were treated with NTZ (10 mg/kg BW) or NTZ-R8 (2 mg/kg BW) in 7 days. The efficacy of treatment was evaluated by oocyst excretion, survival rate, clinical symptoms, and histopathological changes in the ileum. Results: Octaarginine easily penetrated into Cryptosporidium sporozoites and STE oocysts, and intracellular stages while the membrane of intact oocysts remained impermeable. The optimal N/P ratio for the full DNA plasmid condensation starts from 10 when octaarginine was also added to the complex. Successful transfection of excysted sporozoites and STE oocysts was observed with only 1µg plasmid in the transfection complex. Transfection was not achieve when intact oocysts were used. The half-maximal inhibition concentration (IC50) of NTZ and NTZ-R8 was 60.54 ng/ml (197 nM) and 4.499 ng/ml (2.9 nM), respectively. Therefore, octaarginine significantly improved inhibition C. parvum growth by NTZ around 68 times (P < 0.05). During in vivo studies, it was observed that infected mice displayed symptoms of cryptosporidiosis such as anorexia, weight loss and ruffled fur. Mice treated with NTZ at 10 mg/kg BW displayed in 40% survival while mice treated with NTZ-R8 at 2 mg/ kg BW showed a distinctly higher survival rate of 80%, albeit non- significant (P > 0.05). Conclusion: DNA condensation by PEI and DNA delivery by octaarginine allows simple and rapid transfection that requires a small amount of plasmid DNA only and does not depend on sophisticated equipment. Best results were obtained using STE oocysts. Octaarginine also successfully transported the anticryptosporidial compound NTZ into extracellular and intracellular stages of C. parvum and is therefore a suitable vehicle for drug delivery, thus being a promising tool for improvement of treatment efficacy.

The apicomplexan Cryptosporidium parvum causes thousands of human deaths yearly. Since bovines represent the most important reservoir of C. parvum, the analysis of infected bovine small intestinal (BSI) explants cultured under physioxia offers a realistic model to study C. parvum–host cell–microbiome interactions. Here, C. parvum-infected BSI explants and primary bovine small intestinal epithelial cells were analysed for parasite development and metabolic reactions. Metabolic conversion rates in supernatants of BSI explants were measured after infection, documenting an immediate parasite-driven metabolic interference. Given that oxygen concentrations affect cellular metabolism, measurements were performed at both 5% O2 (physiological intestinal conditions) and 21% O2 (commonly used, hyperoxic lab conditions). Overall, analyses of C. parvum-infected BSI explants revealed a downregulation of conversion rates of key metabolites—such as glucose, lactate, pyruvate, alanine, and aspartate—at 3 hpi, followed by a rapid increase in the same conversion rates at 6 hpi. Moreover, PCA revealed physioxia as a driving factor of metabolic responses in C. parvum-infected BSI explants. Overall, the ex vivo model described here may allow scientists to address pending questions as to how host cell–microbiome alliances influence intestinal epithelial integrity and support the development of protective intestinal immune reactions against C. parvum infections in a realistic scenario under physioxic conditions.

Plasmodien spp. sind obligat intrazellulär lebende Parasiten, welche einen evolutionär konservierten aktinabhängigen molekularen Motor für die Fortbewegung und den Wirtszellein- und -austritt nutzen. In dieser Arbeit werden die Aktinregulatoren Adenylyl- Zyklase- assoziierte Protein (C-CAP), Profilin sowie die Aktin depolymerizierenden Faktoren 1 und 2 (ADF1, ADF2) in Plasmodium berghei charakterisiert. Die Geninaktivierung von C-CAP besitzt keinen Einfluss auf die Entwicklung von pathogenen Blutstadien. C-cap(-) Ookineten bewegen sich jedoch deutlich langsamer, sind aber in der Lage den invertebraten Wirt zu infizieren. Defekte treten während der extrazellulären Replikationsphase im Mosquito auf und führen zu Abbruch des Lebenszykluses. Die erfolgreiche Komplementierung der Defekte mit dem orthologen Gen aus Cryptosporidium parvum CpC-CAP bestätigt die funktionale Redundanz zwischen beiden Proteinen. Profilin, als ein weiteres G-Aktin bindendes Protein, ist hingegen nicht in der Lage die Defekte des c-cap(-) Parasiten auszugleichen. Mittels transgener Parasiten welche ein C-CAPmCherry Fusionsprotein exprimieren, wird das C-CAP Protein im Zytoplasma lokalisiert. Erstmals wird mit dieser Arbeit ein G-Aktin bindendes Protein, C-CAP beschrieben, welches eine essentielle Funktion während der Oozystenreifung in Plasmodium berghei besitzt. Die Transkription der Aktinregulatoren Profilin, ADF1 und ADF2 wird in Sporozoiten drastisch herunterreguliert und Profilin kann als Protein nicht mehr nachgewiesen werden. Um die Funktion von C-CAP und Profilin zu überprüfen, wurden beide Proteine spezifisch in Sporozoiten überexprimiert. Diese Parasiten sind nicht in der Lage die Speicheldrüsen des Wirtes zu besiedeln, was zum Abbruch des Lebenszykluses führt. Anhand dieser Ergebnisse entwickele ich ein „minimalistisches“ Model zur Beschreibung der Aktinregulation in Sporozoiten in welchem das ADF1 als regulatorisches Protein im Mittelpunkt steht. / Plasmodium spp. are obligate intracellular parasites, which employ an conserved actin-dependent molecular motor machinery that facilitates their motility, host cell invasion and egress. In this work I report implications of the actin-regulators adenylyl cyclase-associated protein (C-CAP), profilin and actin depolymerization factor 1 and 2 (ADF1, ADF2) in distinct and previously unanticipated cellular processes during the life cycle of in the rodent malarial parasite Plasmodium berghei. Fluorescent tagging of the endogenous C-CAP genetic locus with mCherry revealed cytosolic distribution of the protein. Gene deletion demonstrates that the G-actin binding protein C-CAP is entirely dispensable for the pathogenic blood stages. Ookinetes show reduced motility, but are competent infecting the mosquito host. Defects emerging in the extracellular replication phase, leading to attenuation of oocyst maturation. Successful trans-species complementation with the C. parvum C-CAP ortholog, rescues the c-cap(-) phenotype and proves functional redundancy. The actin regulator profilin fails to rescue the defects of c-cap(-) parasites, despite sharing its actin sequestering activity with C-CAP. Taken together, C-CAP is the first G-actin sequestering protein of Plasmodium species that is not required for motility but performs essential functions during oocyst maturation. Characterization of the actin regulators profilin, ADF1 and ADF2 revealed dramatic transcriptional down-regulation and the absence of the profilin protein in sporozoites. To test whether G-actin binding proteins interfere with sporozoite functions, I ectopically overexpressed of profilin and C-CAP stage-specifically in sporozoites. This conducted to abolishment of salivary gland invasion and lifecycle arrest. Based on these unexpected findings and the available literature data, I developed a “minimalistic model” for actin regulation in sporozoites that predicts ADF1 as the main actin-turnover regulating factor.

MSc (Microbiology) / Department of Microbiology / Background: Diarrheal diseases constitute an important problem among children but also among HIV positive patients particularly in developing countries such as South Africa. Cryptosporidium infect humans and has been shown to be an important cause of infection among different types of animals. Because of its small size, Cryptosporidium can easily go through the water purification system and can easily become a cause of an epidemic. Previous studies have shown that Cryptosporidium is an important cause of diarrhea in Limpopo Province. However, very few studies have been conducted on the genetic diversity of these organisms in the region. Therefore, the aim of this study was to detect and identify the genetic diversity of Cryptosporidium species from humans and animals in Giyani situated in the northern part of South Africa and Pretoria situated in the central part of the country. Methodology: A total of 560 samples were collected from human and animals and were all screened by microscopy using modified Ziehl-Neelsen staining technique. All the samples were tested by Enzyme-Linked Immunosorbent Assay (ELISA) using the Cryptosporidium II kits from Techlab, Virginia, USA. Positive samples from microscopy and ELISA were examined by different PCR protocols including conventional PCR for amplification of Cryptosporidium oocyst wall protein (COWP) region; Real-time PCR employing SYBR Green detection format for amplification of 18S rRNA region; Real-time PCR employing Hydrolysis probes detection format for amplification of SSU rRNA region; Real-time PCR specific for amplification of C. hominis region and C. parvum region. Positive samples from real-time PCR that gave clear bands on gel electrophoresis were sent for sequencing. The sequences were analysed using Staden package software to edit the nucleotides, Bioedit and MEGA6 software were used to align sequences and draw phylogenetic trees. The SPSS software was used for statistical analysis. xiii Results: The overall prevalence of Cryptosporidium as detected by ELISA method from the samples collected from humans was 41.2% (239/580). The prevalence was higher from the rural area 73.0% (159/218) compared to the urban area 22.1% (80/362) and the difference was statistically significant (χ2 = 145.1; p = 0.0001). Due to the limited amount of samples, only 134 ELISA-positive samples were tested using real-time PCR. Of these samples, 35.8% (48/134) tested positive. Of 48 real-time positive samples 25 were successfully sequenced and two different species (C. hominis and C. muris) were identified. Of all the sequences obtained, one (4.0%) was C. muris and 20 (80%) were C. hominis isolated from rural area, whereas 16.0% (4/25) were also C. hominis isolated from samples obtained from urban area. Cryptosporidium was not associated with diarrhea in the present study. A total of 85 samples were collected from animals (52 from cattle and 33 from goats) and of these 4 (4.7%) were positive by microscopy and ELISA. All these samples were non diarrheal. Conventional PCR also detected a similar number. Of these 4 positive samples, 1 was from a male goat, while the 3 others were obtained from female adult goats. Real-time PCR detected 56.5% (48/85) positive samples. Only 12 of the 85 animal samples were diarrheal and of these 4 were positive for Cryptosporidium. The prevalence of Cryptosporidium infection was higher 68.4% (13/19) in male animals compared to female animals 53.0% (35/66). The prevalence rates in cattle and goats were 55.8% (29/52) and 60.6% (20/33) respectively. Of 48 real-time positive samples from animals, 12 (25.0%) were successfully sequenced and two species (C. parvum and C. andersoni) were identified. Of these 6 were from cattle and the other 6 were from goats. Out of the 12 samples 10 (83%) were C. parvum while 2 (17%) were C. andersoni. Of the two C. andersoni, one was from a goat and one was from a cow. Of the 10 C. parvum, 5 were from goats and 5 were from cattle. xiv In conclusion, microscopy remains the low sensitive tool for the detection of Cryptosporidium while real time PCR appeared to be far much more sensitive by detecting more samples than all the three other methods combined. Closer to the real time PCR was ELISA that detected also more samples compared to conventional PCR and microscopy. The present study identified C. muris from humans’ samples in our area for the first time. However, C. hominis remains the dominant species that infects humans in our area. Cryptosporidium species was mostly found in samples from asymptomatic individuals. In animals, C. parvum was the most commonly isolated organism while C. andersoni was identified in our region for the first time as well and occurred in both goats and cattle. Populations in the affected areas need to be made aware of the infections so that care should be taken to avoid the spread of infection in water sources or in immunocompromised individuals.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below