Analysen zum Metabolismus Cryptosporidium parvum-infizierter Wirtszellen sowie Feldstudien zu C. parvum-Infektionen bei Kälbern und Seekühen
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Beschreibung
Cryptosporidium parvum is an obligate intracellular protozoan that infects epithelial cells of the small intestine and can induce severe diarrhoeal diseases in humans and animals. In its development, C. parvum is largely dependent on the metabolic performance of its host cell, since the parasite itself has only minimal metabolic capacities of its own. In the present study, metabolic reactions of C. parvum-infected host cells were therefore investigated. Laboratory conditions (21% O2 = hyperoxia) and physiological oxygen conditions prevailing in the intestine (5% O2 = physioxia) were taken into account, since it is known that the metabolism of a cell is significantly influenced by the oxygen partial pressure. To detect metabolic signatures of infected cells, metabolic conversion rates of the key metabolites glucose, lactate, pyruvate, glutamine, glutamate, alanine, serine and aspartate were analysed and metabolic pathways derived from the obtained metabolic data were selectively inhibited to test their relevance for parasite development. In summary, C. parvum-infected HCT-8 cells showed a marked increase in glucose and glutamine consumption with concomitant increased lactate release, indicating both glycolysis and glutaminolysis as important energy-providing pathways for parasite replication. Accordingly, the effect in terms of glucose and lactate was enhanced when cells were cultured under glutamine-minimal conditions. Based on these results, inhibition experiments were carried out using inhibitors of the glucose-lactate axis [lonidamine = inhibitor of hexokinase, mitochondrial carrier protein and monocarboxylate transporters (MCT) 1, 2, 4; galloflavin = lactate dehydrogenase inhibitor; syrosingopine = MCT1/4 inhibitor] as well as glutaminolysis (compound 968 = glutaminase inhibitor). In accordance with the metabolic data obtained, all inhibitors led to a significant inhibition of the parasite under both physioxia and hyperoxia. These data confirmed the relevance of glycoyse and glutaminolysis, but also of lactate export for C. parvum infections.
In order to generate even more realistic data, a C. parvum infection model was additionally established using bovine small intestine explants and metabolic signatures of infected samples were analysed under hyperoxia and physioxia. Here, interestingly, initially decreased turnover rates of the important key metabolites glucose, lactate, pyruvate, glutamine, glutamates, alanine, serines and aspartates were shown early after infection (3 hours p. i.) with a subsequent reversal of the reactions three hours later (6 hours p. i.). Studies on C. parvum infections in equally newly established primary bovine inflammatory epithelial cells (BSIEC) confirmed the upregulation of glycolytic responses in the early phase of merogony. Interestingly, a principal component analysis confirmed an oxygen-dependent clustering of the metabolic data and thus the important influence of the prevailing oxygen partial pressure.
Since it is known that the composition of the enteric microbiome of a host can influence the outcome of cryptosporidiosis on the one hand and that cryptosporidial infections modulate microbiome diversity on the other hand, a field study was additionally conducted in this work to test the efficacy of orally administered yeast fermentation products compared to halofuginone administration in natural C. parvum infections of calves. However, it was found that neither treatment had a significant positive effect on oocyst excretion, clinical symptoms or daily gain.
Since the zoonotically relevant parasite C. parvum can infect a variety of hosts and can be transmitted in particular via water bodies, different populations of Colombian manatees were examined for their parasite spectrum in a final work package of the present dissertation. These animals could not be identified as excretors of cryptosporidial oocysts or coproantigen, but a differentiated spectrum of specific parasite infections was found in the geographically differently located manatee populations. Among them were several first descriptions of parasites in this mammalian species threatened with extinction.
Autor*in
Juan Diego Vélez Muñoz