A latent ability to persist: differentiation in Toxoplasma gondii

A critical factor in the transmission and pathogenesis of Toxoplasma gondii is the ability to convert from an acute disease-causing, proliferative stage (tachyzoite), to a chronic, dormant stage (bradyzoite). The conversion of the tachyzoite-containing parasitophorous vacuole membrane into the less permeable bradyzoite cyst wall allows the parasite to persist for years within the host to maximize transmissibility to both primary (felids) and secondary (virtually all other warm-blooded vertebrates) hosts. This review presents our current understanding of the latent stage, including the factors that are important in bradyzoite induction and maintenance. Also discussed are the recent studies that have begun to unravel the mechanisms behind stage switching

Metabolic Signatures of Cryptosporidium parvum-Infected HCT-8 Cells and Impact of Selected Metabolic Inhibitors on C. parvum Infection under Physioxia and Hyperoxia

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C. parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals

First Metabolic Insights into Ex Vivo Cryptosporidium parvum-Infected Bovine Small Intestinal Explants Studied under Physioxic Conditions

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

Cryptosporidium in water reuse. Evaluation of new disinfection technologies

Water reclamation is considered as one of the most important priorities worldwide. However, the reuse of treated wastewater involves health risk related with the transmission of certain pathogens. This Doctoral Thesis evaluates the efficacy of new technologies based on advanced oxidation processes against Cryptosporidium, which infectious forms have a robust nature, and they are often detected in wastewater treatment plant effluents, indicating that conventional sewage treatments do not remove this waterborne enteroparasite. The results obtained suggest that these methods are promising alternatives for improving the microbiological quality of reclaimed water, reducing the risk of transmission of infectious diseases

Solute uptake into B. divergens and P. falciparum infected erythrocytes: same theme but different mechanisms

Plasmodium falciparum and Babesia divergens are parasites of the same phylogeny apicomplexa and they invade and replicate in the human erythrocytes. Both the parasites differently modify the host cell membrane during and after infection, providing an excellent example of parallel but distinct adaptations of the parasites to survive in the RBC. During invasion of the host erythrocyte, the parasite targets specific entry sites on the RBCs membrane, which is also the initial step in the biogenesis of the parasitophorous vacuole (PV). The parasitophorous vacuolar membrane (PVM) surrounds the parasite and thus P. falciparum remains surrounded by the vacuolar membrane for most of the parasite’s development. On the other hand in B. divergens, vacuolar membrane fate is unclear, owing to the lack of suitable markers. The electron microscopic studies suggest the disintegration of the PVM in the later stages of parasite maturation. As the differentiated erythrocyte does not endocytose or phagocytose, events leading to the formation of the PVM, in particular the contribution of the erythrocyte membrane (RBCM) are unknown. In order to understand better the internalization of RBCM proteins by parasites, we wanted to determine whether the lipid rafts, which are concentrated patches of lipids and proteins on the erythrocyte membrane, play any such role as specific entry site´ in the biogenesis of PV and also determine whether both parasites respectively internalize the same type of lipids/proteins or not. Recent work has shown that there is no internalization of major RBCM proteins, but the proteins which were found internalized are comparatively present in low abundance in the RBCM and are associated with the lipid rafts. In this work I used B. divergens and also P. falciparum infected erythrocytes to do a comparative investigation of the host cell lipids/proteins, whether they are included or excluded in PV. As a result (i) we found that both parasites recruit and exclude the same set of proteins. (ii) we used cholera toxin B subunit which binds to the ganglioside GM1, to follow the fate of this lipid raft associated glycophingolipid during PVM formation and disintegration. (iii) we observed that GM1 remains in the vicinity of P. falciparum during maturation but disappears from the vicinity of B. divergens as the parasite matures, consistent with a disintegration of the PVM. In conclusion, it appears that there is similarity between the early events in the PVM formation in both the parasites. All the biochemical pathways are dependent on the balance of the transport mechanisms and counteractions like the import of extracellular metabolites, export of the intracellular metabolites and proper waste disposal of unused metabolites. Thus the second part of my work was focused on the increased permeability of the host cell membrane after infection and appearance of new permeability pathways (NPP) in the infected erythrocyte. The erythrocyte which is save heaven´ for the parasite have limited resources and metabolism, that the parasite can use. The intracellular parasite is far more active inside the erythrocyte than outside cell. After invasion the small metabolically active merozoite stage of each genus (Plasmodium and Babesia) grows rapidly into other developmental stages (ring form and trophozoite form) inside the erythrocyte and thereby synthesizing DNA, RNA, proteins and lipids in large amounts. In order to fulfil all the essential requirements, the growing parasite induces changes in the membrane permeability of the erythrocyte and takes up solutes from the extracellular media via NPP. One among the other important amino acids required by the parasite is L-glutamate. The main aim of my work was to observe and characterize the transport of L- glutamate into B. divergens infected erythrocytes, and compare it with P. falciparum infected erythrocytes. As it is known that P. falciparum infected erythrocytes have both low- and high-affinity (EAAT3) glutamate transporter activated. The transport system present in B. divergens infected erythrocytes is unclear, therefore it is interesting to know the details of transport mechanism and see if it is parasite specific mechanism. Like P. falciparum, after infection of erythrocytes, B. divergens also induces the uptake of several solutes which are not taken up by non-infected erythrocytes. We found that B. divergens infected erythrocytes show uptake of glutamate via a low-affinity transport system. The other important characteristics of the glutamate transport observed was Na+-independency, non-saturability and non-stereo selectivity. There was also enhanced uptake of glutamate in the presence of choline. In conclusion, it appears that both parasites induce different mechanisms or transport systems for glutamate uptake and also the activation of the transport system is parasite specific

Toxoplasma gondii and behavioral modification in hosts

Thesis (M.A.)--Boston UniversityToxoplasma gondii is a heteroxenous protozoan parasite that is found in nearly every species of mammal and billions of latently infected humans worldwide. The symptoms and morbidities associated with acute, congenital, and AIDS-associated toxoplasmosis are familiar to many, while those associated with latent toxoplasmosis are not nearly as well known. Behavioral manipulation is a common strategy of parasite and parasitoid species, and recent research into T. gondii has revealed that T. gondii infection alters the way rodents respond to the odor of the urine of its feline predators, which are also the definitive hosts of T. gondii. Humans have been found to be potentially affected by T. gondii as well: associations have been identified between latent T. gondii infection and psychiatric diseases (including schizophrenia), personality changes, and traffic accidents. This review investigates the state of current scientific knowledge related to Toxoplasma gondii, analyzes recent developments, and examines the implications on public health. We also provide critical analysis of the published literature and make suggestions for future research

Apicomplexan Parasite Interactions with Host Organelles: Recruitment, Lipid Scavenging, and Consequences

Apicomplexa parasites are harmful pathogens of humans and animals. They invade mammalian cells wherein they reside within a parasitophorous vacuole (PV) that protects them from cytosolic destructive pathways but forms a physical barrier from host-derived nutrients. Among Apicomplexa, Toxoplasma gondii has proficiently evolved to recruit host organelles to its PV to facilitate nutrient uptake. Almost nothing is known about the intracellular development of Neospora caninum, a closely relative of Toxoplasma. We show that N. caninum is also able to attract host organelles (ER, Golgi, endosomes) to its PV from which it retrieves cholesterol and ceramides, revealing conserved strategies among these parasites to exploit nutrient-filled host organelles. We next explore the role of host lipid droplets (LD) as sources of neutral lipids for Toxoplasma. We demonstrate that host LD cluster around the PV, and infection leads to an increase, then decrease in host LD numbers, suggesting a manipulation of these structures by Toxoplasma. Indeed, Toxoplasma is able to scavenge lipids from host LD, in part through the interception of Rab vesicles associated with LD and the translocation of host LD into its PV. Ectopic addition of oleic acid (OA) up to 1 mM (non toxic concentration for mammalian cells) stimulates LD biogenesis. When exposed to 0.2 mM OA, Toxoplasma scavenges this fatty acid in excess, channels it to LD that accumulate in the cytoplasm, as a result of increased transcription of its enzymes generating neutral lipids. However, this condition slows down both parasite replication and egress. By comparison, 0.2 mM palmitic acid does not affect the parasite’s intracellular development. Interestingly, ultrastructural analyses of OA-loaded Toxoplasma reveal for the first time, the presence of coated pits at the plasma membrane and structures potentially involved in endocytosis. More dramatically, addition of 0.4 mM OA to the medium results in massive accumulation of lipid deposits in the PV and the parasite, leading to replication defects and death. This highlights the high sensitivity of Toxoplasma towards deleterious effects of accumulating fatty acids. Deciphering the lipotoxic response of the parasite would reveal new vulnerabilities amenable to controlling Toxoplasma infections