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The pellicle of the apicomplexan parasite Toxoplasma gondii is a shared structure among the supergroup Alveolata, which consists of a patchwork of flattened vesicles underneath the plasma membrane. The apical complex, from which the phylum obtains its name, is the defining feature of apicomplexans. The apical complex is a dedicated invasion-related structure that is integrated into the parasite pellicle architecture, and these two structures are both essential for the pathogenesis of T. gondii. The aim of this study is to discover and characterize new components of the apical complex and the pellicle in T. gondii by implementing a three-pronged strategy that involves the study of the peripheral apical annuli, the exocytic organelle microneme and the membrane occupation and recognition nexus (MORN) domain-containing proteins. The peripheral apical annuli of T. gondii, which consist of 5-7 ring-like structures at the lower edge of the apical cap, are intriguing compartments but their function and significance remain unknown. We have identified a novel apical annulus protein AAP6, and I have then demonstrated that it is an integral plasma membrane protein, which makes AAP6 the only membrane component found exclusively at the annuli so far. Depletion of AAP6 revealed that this protein is essential for the parasite lytic cycle in vitro, as the loss of AAP6 caused defects in parasite gliding motility, invasion and intracellular replication. A possible explanation for these defects is that the annuli might participate in the regulation of secretion processes as constitutive microneme exocytosis was impaired after AAP6 knockdown. Furthermore, I found that AAP6 is required to maintain the peripheral distribution of a transmembrane transporter HP03 which is sorting from the post-Golgi endosome-like compartment (ECL) to the parasite plasma membrane. Next, to uncover novel components and protein interactions of the T. gondii apical annuli, proximity-dependent biotin identification (BioID) was applied using AAP6 as bait. Two novel annuli proteins, which may be related to vesicle trafficking, named TgSnare and TgSyntaxin were discovered from the BioID study. Collectively, these studies tell us for the first time that the annuli span the plasma membrane to the extracellular environment, that they are important structures for parasite growth and invasion, and that the annuli are likely to function as vesicle delivery structures which maintain homeostasis of PM proteins. Preliminary investigations have been made into two other components of the pellicle and apical complex by two further approaches. Micronemes are one of the secretory organelles of the apical complex, and their exocytosis promotes parasite motility and invasion. The MORN domain is a common motif that is considered to have a role in lipid- binding and membrane-targeting. I have sought to identify new microneme proteins based on the new proteomic method LOPIT and to identify if MORN domain-containing proteins will comprise further elements of the pellicle beyond those already known. The locations of six novel microneme proteins (MC1-MC6) and five MORN domain- containing proteins (MORN2-MORN6) were revealed by protein endogenous tagging and microscopy. Preliminary functional investigations of some of these proteins were performed via inducible protein depletion studies.

Toxoplasma gondii is an intracellular protozoan parasite that infects nearly 1 in 3 individuals in the world. In immunocompromised individuals T. gondii infection can disseminate and cause serious disease and even death. Manifestations of the disease caused by T. gondii, toxoplasmosis, can include ocular lesions, vision loss, miscarriage, hydrocephaly, mental retardation, and even death. T. gondii is closely related to the malaria-causing parasite, Plasmodium sp., and both of which are without effective protective vaccines. For the development of an effective treatment or vaccine against parasitic infections, more about the battle between host and pathogen must be understood. What allows for some mouse strains to survive infections, while others succumb? What antigens on T. gondii elicit the strongest antibody response? What receptors and pathways must be triggered to provide protection? The glycosylphosphatidylinositol (GPI)-anchor is a highly conserved glycolipid that anchors proteins to the external membrane of T. gondii and is also found on all protozoan pathogens. Most of the major surface antigens (SAGs) of T. gondii are GPI-anchored proteins, and express GPI without attached proteins on their surface called GIPLs. The GPI is known to be recognized by innate pattern recognition receptors Toll-like Receptors (TLR) -2 and -4 and is robustly targeted by IgM antibodies after infection. While the glycan portion of the GPI consists of a conserved set of 3 mannose sugars, a GlcNAc, and an inositol, species differ in additional sugars or sidechains added to the mannose backbone. The functional significance of GPI sidechains has remained largely unknown because only a few GPI sidechain modifying enzymes have been identified. Since the GPI is such a potent antigen and in high abundance, we sought out to address the role of the GPI sidechain in parasite pathogenesis.We have successfully identified and knocked out both of the enzymes responsible for GPI sidechain modification in T. gondii. Named here PIGJ and PIGE, the former adds the first GalNAc sugar to the core mannose backbone of the GPI, while the latter adds a terminal Glc to the GalNAc as confirmed by mass spectrometry. Parasites lacking the entire GPI sidechain exhibit increased virulence during primary and secondary infections, demonstrating the GPI sidechain as an important pathogenesis factor. To elucidate the mechanism for increased virulence we characterized innate immune responses, parasite burden in various organs, early cytokine responses, antibody recognition, antigen expression, parasite growth and invasion of PIGJ mutant parasites that lack a GPI sidechain. Our findings reveal intact cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants. In contrast, the scavenger receptor CD36 shows enhanced binding to parasites that lack GPI sidechains, potentially explaining the tropism for macrophages observed early in infection. Galecitn-3, which binds to GIPL of T. gondii, exhibits enhanced binding to PIGJ mutants and infection of galectin-3 deficient mice results in their protection from lethal outcomes, suggesting the enhanced virulence of sidechain deficient parasites is galectin-3 dependent. Loss of the sidechain has no bearing on parasite numbers early in the infection, suggest a breakdown of tolerance is occurring, the exact mechanism for which is still unclear. In contrast, we see increased tissue cysts in the brains of PIGJ mutant infected mice, indicating some advantage over the wildtype is involved. Finally, vaccination of TLR2 or TLR4 deficient mice are susceptible to challenge with virulent T. gondii strains. Collectively, our data point to an important role for the GPI and its sidechain in host-parasite interactions, and that the presence of the sidechain prevents T. gondii pathogenesis promoting survival of its host.

Toxoplasma gondii is the only apicomplexan parasite known to possess a well-developed reactive oxygen species (ROS) scavenging system which includes an endogenous catalase and complete glutathione and thioredoxin pathways. Compared against other apicomplexan parasites, T. gondii is uniquely equipped with a wide array of ROS scavenging systems. These ROS scavenging enzymes are key to neutralizing both endogenous and host cell generated ROS. By virtue of having more ROS scavenger systems, T. gondii may be capable of tolerating a wider variety of intracellular conditions, which is then reflected in its wide host cell tropism. Conversely, these ROS scavenging systems may also be the key to eliminating parasitic infections as their components are unique to the parasite. Several treatment options are able to selectively inhibit the activity of parasite ROS scavengers, and thereby treat various apicomplexan parasitic infections. Of ROS scavenger targeting therapeutic strategies, auranofin was thought to specifically inhibit pathogen thioredoxin reductase. To determine if thioredoxin reductase is still the target of auranofin in T. gondii, we isolated drug resistant mutants. Auranofin resistant T. gondii parasites, harbor mutations in T. gondii’s mitochondrial superoxide dismutase (TgSOD2). These parasites showed reduced accumulation of ROS upon exposure to auranofin. However, the L201P mutation of TgSOD2 is not sufficient to confer resistance to auranofin. Our work indicated that auranofin likely induces parasite death by apoptosis: evidenced by parasite shrinkage and exposure of phosphatidylserine (PS) residues on the parasite surface detected by Annexin V. To understand the role of TgSOD2 throughout the parasite’s life, we generated inducible knockdown mutants using an auxin-inducible degron system that allowed us to explore the role of TgSOD2 in parasite replication and replication fitness (Chapter Three). During stages of extracellular stress, TgSOD2 transcription is upregulated. TgSOD2 depletion leads to reduction in replication fitness as these parasites showed decreased plaque formation when compared to their parental line. At the mitochondrial level, depletion of TgSOD2 results in aberrant mitochondrial morphology as well as reduction of the parasite’s mitochondrial membrane potential. Through a proximal biotinylation approach, we found that TgSOD2 localizes adjacent to complexes IV and V of T. gondii mitochondrial electron transport chain, suggesting that these may be sites of ROS generation or ROS sensitive enzyme subunits. Our studies are the first ones to demonstrate the role of TgSOD2 in parasite life and mitochondrion maintenance. Lastly, Chapter Four describes the future lines of inquiry opened by the findings presented in Chapters Two and Three. Based on the studies presented, TgSOD2 is likely implicated in the formation or maintenance of the ATP synthase complex. Furthermore, inhibition of TgSOD2 may sensitize the parasite to further ROS damage. Future studies ought to investigate the link between TgSOD2 and ATP synthase, then evaluate the viability of dual inhibition as a therapeutic strategy for T. gondii infection clearance.

Toxoplasma gondii (T. gondii) is one of the most prolific parasites in the world where it is estimated that 30% of the human population is infected. The encystment of the parasite in CNS-neurons results in a lifelong chronic infection. Within the brain, a pro-inflammatory response is essential to prevent parasite reactivation, however, an anti-inflammatory response is also required to prevent inflammation-induced mortality. This suggests a balanced immune response to the pathogen. Infection in the immunocompetent experience asymptomatic infection, further suggesting a tightly regulated immune response in the CNS. However, it is unclear what are the signals or cell-mediators that regulate this delicately balanced immune response. In addition, parasite recrudescence leads to lethal toxoplasmic encephalitis if left untreated in immunocompromised individuals. However, it is unclear what developmental pathways the parasite undergoes upon reawakening from its dormant state. This dissertation investigates these unknowns in the follow ways:Chapter 1 is an introduction to T. gondii infection and the effects the parasite has on the host. Topics covered in this section include the life cycle and the immune response to T. gondii. This encompasses evasion strategies by the parasite, and signals that are required for protective immune cells to traverse the blood brain barrier. This chapter ends on describing the role astrocytes play in the CNS, and the implications of T. gondii infection on glutamate regulation.Chapter 2 describes the attempts that have been made to study recrudescence of tissue cysts. This chapter poses a new ex vivo model of bradyzoite-initiated development that better preserves the key attributes of the parasite’s intermediate life cycle. In chapter 3, we discuss elements found in the CNS-microenvironment during chronic T. gondii infection, and consider how tissue-derived signals, such as high extracellular glutamate, influence protective CD8 T cells. Chapter 4 investigates if a potential mechanism for the cause of GLT-1 downregulation in astrocyte is due to direct invasion. Finally, chapter 5 reviews topics discussed in previous sections, and highlights my contributions to the fields of neuroimmunology and parasitology. Furthermore, this chapter emphasizes work that still needs to be done and unanswered questions in the field.

Immune defense against the ubiquitous brain-resident parasite Toxoplasma gondii requires a balanced immune response that ultimately results in chronic infection of the host. Monocytes play a key role in host defense and are critical for controlling T. gondii in the periphery. Monocytes also enter the brain and protect against Toxoplasmic encephalitis. However, the dynamic and regional relationship between brain infiltration of the parasite and host monocytes is unknown. In addition, the ability of T. gondii to disseminate through the brain is poorly understood. In this dissertation, we report the mobilization of inflammatory and patrolling monocytes into the blood and brain during T. gondii infection of mice and define their regional localization. We also describe previously unappreciated effects of T. gondii infection on the blood-brain barrier (BBB) and the novel observation that infected cells can shuttle T. gondii at the BBB and in the brain parenchyma.Many of these questions were addressed using longitudinal analysis of mice using two-photon, intravital imaging of the brain microvasculature through cranial windows. This imaging approach revealed that CCR2-RFP monocytes were recruited to the blood-brain barrier (BBB) within two weeks of T. gondii infection, exhibited distinct rolling and crawling behavior at the BBB, and accumulated within the vessel lumen before entering the parenchyma. Optical clearing of intact T. gondii-infected brains and light sheet microscopy coupled with computational alignment of brains to the Allen annotated reference atlas indicated consistent patterns of monocyte infiltration during T. gondii infection, specifically in the olfactory tubercle and pontine areas. These data provide novel insights into the localization of monocytes in the brain during CNS infection and raise the provocative hypothesis that regional neuroinflammation may contribute to the behavioral phenotypes observed during T. gondii infection of mice. At the BBB, we observed profound vascular remodeling in T. gondii-infected mice expressing the tight junction protein claudin-5 fused to eGFP in endothelial cells. We detected increased tortuosity of individual endothelial cells and reorganization of the vascular network. In addition, two-photon imaging revealed T. gondii within highly motile cells travelling along cerebral blood vessels. These parasite-infected cells were highly dynamic and traveled at speeds greater than 5 µm/min. As a result, infected cell motility resulted in significantly greater displacement of T. gondii in the brain than individual parasites migrating using their own motility. These data revealed that the infection of motile cells enhanced the dissemination of T. gondii in the brain. Collectively, this work highlights inherent trade-offs in T. gondii immunity, revealing the delicate balance between mounting a protective monocyte immune response while suggesting it may also contribute to parasite-induced pathology and spread.

Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii traverses endothelial barriers to enter tissues and establish chronic infections that underlie the most severe manifestations of toxoplasmosis. Despite a growing appreciation for the importance of endothelial infection in T. gondii pathogenesis, the molecular interactions occurring at this host-pathogen interface remain poorly defined. The research presented here examines T. gondii infection in the context of human primary endothelial cells in static conditions and, for the first time, under the physiological conditions of shear stress found in the bloodstream. We demonstrate that T. gondii infection of primary human umbilical vein endothelial cells (HUVEC) altered cell morphology and dysregulated barrier function, increasing permeability to low molecular weight polymers. Altered cell morphology coincided with parasite-induced remodeling of the three main components of the cytoskeleton: actin microfilaments, intermediate filaments, and microtubules. By conducting a global transcriptome analysis of infected endothelial cells, we identified gene expression changes associated with mechanotransduction and show that T. gondii infection activated Hippo signaling. Infected endothelial cells exposed to shear forces exhibit disrupted planar cell polarity and fragmented Golgi. RNA-Seq analysis on infected endothelial cells exposed to shear suggests that infection alters expression of genes associated with cell proliferation and lipid metabolism. Collectively, this work reveals novel interactions occurring at the interface between endothelial cells and T. gondii to provide insights into processes linked to parasite dissemination and pathogenesis.

Cryptosporidium parvum and Toxoplasma gondii are obligate intracellular parasites transmitted by ingestion of resilient walled structures called oocysts. Infection is self-limiting in adults with normal immune systems. However, severe disease can occur in immunocompromised individuals, or those without cellular immunity. Cryptosporidium is a leading cause of infant mortality in developing countries, due to diarrhea. There are no human vaccines and no broad effective drug treatments. Several vaccine candidates have been described: the glycoproteins Gp900, Gp40, and Gp15 and the protein Cp23, the immuno-dominant-antigen. Details about modifications to these proteins have not previously been reported. Using mass spectrometry, we identified 16 Cryptosporidium N-glycosylated proteins, including Gp900 and a possible oocyst wall protein. The observed N-glycan structures exhibited only two compositions: HexNAc2Hex5 and HexNAc2Hex6; these glycoforms had a single extended arm. The simplicity of Cryptosporidium N-glycans contrasts with the complexity of host N-glycans. Four heavily O-glycosylated proteins included Gp900, Gp40, Gp15, and a novel mucin-like protein, Gp20. Single O-HexNAc residues modified Ser/Thr in low density regions of Gp15 and Gp900, while attachment of O-HexNAc residues on tandem Ser/Thr repeats of Gp20 and Gp40 approached saturation. Identification of N-acetylgalactosamine (GalNAc) as the HexNAc released from proteins suggests that most Cryptosporidium O-glycans resemble the immunogenic Tn antigen (O-GalNAc). The immunodominant antigen Cp23, while not glycosylated, was discovered to be N-myristoylated and S-palmitoylated on the first and second residues, respectively. This is the first identification in Cryptosporidium of these modifications. Information about the N-glycans, O-glycans, and lipid modifications may be useful for design of better serodiagnostic reagents and more effective vaccines. To date, there are no vaccines against Toxoplasma infection, and the only available pharmaceutical therapies are expensive. In the second study, a novel O-fucose modification was discovered on nuclear pore-associated proteins including nucleoporins. This observation has profound implications on how the organism may regulate trafficking in/out of the nucleus by employing a system parallel to that described for O- linked N-acetylglucosamine in other organisms. In summary, the new details regarding the vaccine candidates of Cryptosporidium and the discovery of the novel O-fucose modifications in T. gondii provide information that could prove useful for development of effective drugs and vaccines.

Toxoplasma gondii is an Apicomplexan obligate intracellular protozoan parasite, which is able to infect virtually all warm-blooded animals. According to the Centers for Disease Control approximately 60 million people in the U.S. are currently infected with T. gondii. Treatments for toxoplasmosis are limited and generally consist of a combination of pharmaceuticals. Based on the prevalence and limited options for treatment new drug targets should be explored. Cell cycle proteins often present themselves as good drug targets. TgCDK1 and TgCYC2 are cell cycle proteins. TgCDK1 is a cyclin dependent kinase and TgCYC2 is thought to be a cyclin protein. TgCDK1 and TgCYC2 are associated with the G2 to M phase regulation of the cell cycle. Inhibition of the cell cycle would prevent the cell from proliferating ultimately eliminating T. gondii from the host. TgCDK1 and TgCYC2 proteins must first be purified and a kinase assay has to be run in order to determine the function of these two proteins. Experimental approaches to the purification and characterization of these proteins refines the search for drug targets.

Toxocariasis and toxoplasmosis are two of the most common zoonotic parasites of people worldwide, causing ocular and neurological disease. The probability of being infected with both pathogens is increased due to shared modes of transmission. These parasites behave the same way in all paratenic hosts, which include mice and people, and so mice represent an excellent model for investigating human infection. During secondary infection of mice with Toxocara canis, many larvae become trapped in the host's liver, preventing onward migration to other body tissues such as the eye. This dissertation aimed to elucidate the immunomodulatory effect of Toxoplasma gondii infection on T. canis migratory arrest by first identifying the adaptive immune cells and humoral components contributing to trapping and then the changes elicited by co-infection. In my research, I show that CD4+ cells are critical for directing the generation of antigenspecific antibodies during primary T. canis infection, but they play no direct role in larval trapping during secondary infection. Passive immunization using purified IgG from infected mice enables naïve recipients to trap larvae in the liver, suggesting humoral immunity is the key player. I then explored the effect of concomitant infection with T. gondii on T. canis migration. Strikingly, I show that mice pre-infected with T. gondii trap far fewer larvae than mice infected only with T. canis. This corresponds to a partial shift from IgG1 antibody production to the IgG2a isotype. Reciprocal modulation by T. canis of cell-mediated immunity to T. gondii is also observed, as evidenced by a reduction in the abundance of activated CD8+ T lymphocytes in coinfected mice compared to T. gondii mono-infected mice. Finally, I examined factors with potential influence on T. canis migration. I show that male BALB/c mice have an enhanced ability to trap larvae relative to females. In contrast, no difference in larval distribution or viability is detected in eosinophil-deficient dblGATA1-/- mice on a C57BL/6 background compared to wild type. This dissertation provides insight into host immunity against larval toxocariasis and the immune cross-regulation induced by two parasites inhabiting the same host.

Toxoplasma gondii – an obligate intracellular parasite – has a complex multistep mechanism for host cell invasion with a pivotal role played by calcium signaling. However, the coordination amongst the players in all the key steps of this signaling pathway, essential for parasitic life cycle of Toxoplasma, is still not entirely known. Given the evolutionary relationship between ciliates and apicomplexans, this work evaluates the functions of three orthologous proteins in cell signaling of alveolates leading to calcium-dependent exocytosis of vesicles such as trichocysts and micronemes. The proteins investigated are calmodulin (CaM) which is a calcium sensor, calcineurin (CN), a protein phosphatase, and parafusin-related protein 1 (PRP1), the Toxoplasma ortholog of ciliate protein parafusin. In the ciliate, CaM activates CN upon rise in intracellular calcium. Activated CN then leads to the dephosphorylation of the secretory vesicle scaffolding protein called parafusin. Parafusin dephosphorylation dissociates it from the calcium-dependent secretory vesicles, which then discharge their contents. As expected upon conditional depletion, we found CaM to be essential for Toxoplasma tachyzoites and CN to be significant for the lytic cycle. Surprisingly, the microneme secretion remained normal in the CN depleted parasites, although there was significant reduction in the attachment of the parasite to the host cell. We also found that PRP1 is dispensable for Toxoplasma lytic cycle, despite its absence affecting the microneme secretion induced by the calcium ionophore, A23187. However the secretion defect is not uniform and remains comparable to the wild type when it is induced using other pharmacological agents like ethanol, zaprinast and propranolol. Therefore despite phylogenetic conservation of the three proteins in alveolates, this work demonstrates their involvement in distinct functional aspects in Toxoplasma compared to other ciliates. Alongside the limited understanding of the molecular mechanism, our knowledge about the cellular sensors in Toxoplasma is also scarce. Our laboratory has previously identified a double C2 (DOC2) domain containing protein called TgDOC2 and demonstrated its role in overall microneme secretion. However, TgDOC2 does not regulate the dosed microneme release associated with varied steps of the egress-invasion trajectory. In general, DOC2 domain containing proteins work in coordination with each other to execute their cellular function, mostly in calcium-dependent manner. We therefore, wanted to expand our knowledge of this domain containing proteins in the parasite. In this work, we investigated a conserved apicomplexan protein called FER2 containing multiple DOC2 domains. Conditional depletion of FER2 appeared detrimental due to significant loss of invasion and attachment of the parasites. FER2 depleted parasites have normal microneme secretion, which is currently the only known calcium-dependent secretory organelle in Toxoplasma, but abrogated the release of rhoptries, the second secretory organelle to be released during invasion. Altogether this work extends the importance of calcium signaling in Toxoplasma gondii and brings into light the novel aspects of some parasitic proteins which are significant in reconstructing our understanding of the signaling pathway in this parasite.

Toxoplasma gondii is an apicomplexan parasite that infects approximately thirty percent of the human population. As there is a need for new therapies, it is important to investigate Toxoplasma metabolic pathways to determine their role in parasite survival. In my first project, I used metabolic oligosaccharide engineering to profile glycosylated proteins in Toxoplasma tachyzoites. This approach uses metabolic incorporation of unnatural sugars into Toxoplasma proteins. My studies identified a variety of parasite proteins that are modified by glycans and validated modification of SAG1, a surface antigen in tachyzoite stage parasites. As β-elimination reduced the modified sugar signal, Ac4GlcNAz likely targets O-linked glycans in Toxoplasma. In a second study, I characterized a novel cytochrome P450 enzyme (CYP) in Toxoplasma. TgCYP localizes to the tachyzoite mitochondrion and has an unexpectedly restricted substrate: to date, it has only been shown to act on acetone. Although tgcyp null parasites have a modest growth defect when grown in competition with wild-type parasites, the null parasites have a robust growth advantage when 2% acetone is added to the culture conditions, suggesting that expressing TgCYP is deleterious under certain conditions.

Bioorthogonal chemical reporters are small functional groups that can be metabolically incorporated into biomolecules by the cell’s native metabolic machinery. These moieties can be covalently modified with probes for detection or isolation. I applied this strategy to profile glycoproteins in Toxoplasma gondii, a prevalent intracellular parasite that infects nearly one-third of the world's population and may cause life-threatening conditions in immunocompromised patients. Using the bioorthogonal chemical reporter strategy, I identified a large, diverse set of glycosylated proteins in T. gondii including some previously unannotated proteins likely involved in modulating host-parasite interactions.In addition to glycosylated structures, parasites and host cells exchange numerous other metabolites. Simultaneous monitoring of these communication pathways requires multiple cell-compatible reactions that can be used concurrently, or “mutually orthogonal” bioorthogonal chemistries. Unfortunately, the existing toolbox of bioorthogonal chemistries is rather sparse, limiting our ability to look at multiple biomolecules in tandem. As part of Prescher lab, I have contributed to the development of novel chemical reporters for simultaneous profiling of host-pathogen crosstalk. My labmates and I established a new bioorthogonal chemical reaction—a cycloaddition between 1,3-disubstituted cyclopropenes and tetrazines—that can be used in living systems. Excitingly, this reaction can be used in tandem with common azide-alkyne chemistries for multi-component imaging. We also demonstrated that 1,3-disubstituted cyclopropenes can be used concurrently with isomeric cyclopropenes—3,3- cyclopropenes—in biological labeling applications. Both of these molecules harbor unique reaction preferences, enabling them to be used simultaneously. This was noteworthy result, since the structures of these cyclopropenes only differ by the placement of a single methyl group. I also developed another orthogonal chemical reporter for use in multi-component applications: methylene cyclopropane. This molecule exhibits unique cycoaddition reactivities and is poised to join the ranks of useful chemical reporters.

The obligate intracellular parasite Toxoplasma gondii exhibits closed mitosis, as chromosome segregation occurs with the confines of the nuclear envelope. Distinct structural changes are absent during mitosis, as the nucleolus is maintained and condensation of chromosomes is largely restricted. Moreover, the centromeres are clustered and remain persistently associated with the centrocone (spindle pole). To elucidate the process of chromosome segregation during mitosis in the parasite, the role of kinetochores and microtubules was examined. Localization studies of the functionally conserved kinetochore proteins TgNuf2 and TgNdc80 revealed that clustered kinetochores colocalize with clustered centromeres at the centrocone throughout the cell cycle. Pharmacological disruption of microtubules resulted in partial loss of clustering, which indicates spindle microtubules are necessary, but not strictly required for this process. Furthermore, the generation of a conditional TgNuf2 knockdown revealed this kinetochore protein is essential for chromosome segregation but dispensable for clustering of centromeres, which remain associated with the centrocone. Moreover, in the absence of TgNuf2 the centrosome behaves normally, but looses its association with the centrocone. Further analysis of this phenotype revealed that the centrocone is devoid of spindle microtubules following depletion of this essential kinetochore protein. Examination of tubulin localization dynamics through parasite development showed that the initiation of spindle microtubules occurs at the basal region of the nucleus prior to centrosome duplication. Furthermore, acetylation of α-tubulin, a posttranslational modification associated with microtubule stability, was confirmed to be specifically associated with stabilization of the spindle microtubules following comigration of the centrocone and centrosome to the apical end of the nucleus. Collectively, these data demonstrate that the persistent association of clustered centromeres with the centrocone is independent of spindle microtubules. These discoveries are contributing unprecedented details to chromosome anchoring and segregation during the cell cycle in this protozoan parasite.

Toxoplasma gondii is a protozoan parasite of wide geographic and host range. Although many infections in humans are asymptomatic, the potential exists for severe disease. The parasite is highly successful given its ability to establish life -long infections. To achieve this, the parasite must establish a delicate balance between activation and suppression of the host immune response. The cytokine IFN[gamma] is essential for host resistance to the parasite . Therefore, Toxoplasma actively manipulates the IFN[gamma] pathway in order to ensure parasite survival. It has become clear that Toxoplasma can evade the host IFN[gamma] response in a variety of cell types. However, the exact mechanism by which this occurs remains unclear. Furthermore, evasion of the IFN[gamma] response has not been studied in the dendritic cell, an immune cell type important during in vivo infection. I show that Toxoplasma can block STAT1 binding to IFN[gamma]-responsive gene promoters in dendritic cells, preventing transcription. However, STAT1 activation is not impaired. In fact, I show that Toxoplasma alone causes STAT1 phosphorylation and nuclear translocation, as well as consensus sequence binding. Phosphorylation and consensus sequence binding occur synergistically with IFN[gamma]. However, the parasite induces formation of aberrant STAT1 DNA-binding complexes. Further work shows that Toxoplasma-induced STAT1 phosphorylation and/or synergy with IFN[gamma] occur independently of the parasite kinases ROP16, ROP18, ROP21 as well as the parasitetriggered host MyD88-, GiPCR-, and PI3K-dependent signaling pathways. Furthermore, inhibition of STAT1 transcriptional activity occurs in a parasite strain-independent manner. Synergistic levels of phospho-STAT1 correlate with parasite mediated inhibition of IFN[gamma] induced Socs1 expression. Yet, the parasite alone induces modest Socs1 expression in a ROP16dependent manner, which may participate in regulation of other pathways such as TLR signaling. From this work it is clear that the impacts of Toxoplasma on dendritic cell STAT1 signaling are complex. The parasite induces STAT1 phosphorylatio n, but how this occurs and whether this STAT1 participates in the transcription of a unique subset of genes remains to be determined. However, it is clear that the parasite can block STAT1 chromatin binding and transcription in dendritic cells in response to IFN[gamma], permitting evasion of this important host immune response.

As an immune-privileged site, the brain represents a particularly challenging area when it comes to controlling infection and inflammation. Toxoplasma gondii provides us with a model of a chronic infection in the CNS that represents an efficient and balanced response from the healthy immune system. The effector functions of immune cells coupled with the ability to effectively migrate to and within the brain to sites of infection is essential in controlling the replication of the parasite in the brain. Impairment of this immune response results in parasite reactivation and fatal encephalitis, as seen in AIDS patients. Immune-compromised patients are currently subject to lifelong drug therapies that do not result in cyst clearance, presenting a constant danger of reactivation. Therefore, the identification of new mechanisms of cyst control is particularly important as it may lead to new and effective therapies. This dissertation examines two novel mechanisms of parasite control in the CNS during chronic infection. Previously published data has shown that a reticular network of fibers is present in Toxoplasma infected brains and absent in naïve brains which T cells migrate along. SPARC (secreted protein acidic and rich in cysteine) is a multifunctional glycoprotein that has been shown to be involved in the production and maintenance of similar fiber networks. Here, data will be presented demonstrating upregulation of SPARC in the brain following infection and association of this molecule with a seminal fibrous network in the CNS as well as with cysts and migrating cells. Furthermore, a requirement for SPARC in protective immunity and matrix assembly will be demonstrated in vivo, using infection studies with SPARC-null mice and live imaging in the brain. The exact mechanisms and cells involved in the clearance and control of cysts from the brain remain poorly defined. Data presented here will demonstrate the presence of a population of alternatively activated macrophages (AAMϕ) in the infected brain that maintain control of the cyst burden through the secretion of the chitolytic enzyme, acidic mammalian chitinase (AMCase). This mechanism is demonstrated in vitro by both chemical and genetic inhibition of the enzyme and the requirement for protective immunity is shown through infection of AMCase-null mice. These mechanisms of parasite control during chronic T. gondii infection represent two novel areas of study that provide hopeful therapeutic targets in combating this chronic infection.

Apicomplexans are a large group of obligate intracellular parasites including Plasmodium falciparum, the causative agent of human malaria, and Toxoplasma gondii, an important pathogen of immunocompromised individuals. T. gondii is capable of replicating in a broad range of host cells and establishes its infection by actively invading its host cells. This invasion process is mediated by the rhoptries, unique apical secretory organelles that inject a complex of proteins into the host cell to facilitate penetration through formation of a tight-junction interface between the parasite and host called the moving junction (MJ). During the invasion process, a parasitophorus vacuole is created within which the parasite resides, evading destruction by host lysosomes. Sequestered within this vacuole, the parasite is free to divide by a unique process of internal budding known as endodyogeny wherein two daughter parasites form within a mother cell. Following repeated rounds of replication, the host is disrupted and the parasites egress out to re-invade neighboring cells, beginning this lytic cycle anew. A better understanding of these unique processes of host cell invasion and parasite replication are needed as the pathology caused by T. gondii and other apicomplexans is dependent on these processes. In the first section of this dissertation, I describe the discovery and functional analysis of a Toxoplasma palmitoyl acyl transferase (TgDHHC7) that localizes to the surface of the rhoptries. Remarkably, conditional disruption of this enzyme results in a loss of apical tethering of the rhoptries and a complete block in their function, allowing for a definitive establishment of their role in invasion but not replication or egress. Palmitoylation by membrane-resident PATs is a well-characterized mechanism for recruiting proteins to target membrane systems. Therefore, it is likely that TgPAT1 facilitates apical tethering of rhoptries by recruiting one or more proteins to the cytosolic face of the rhoptry membrane which then serve to mediate docking. Indeed, knockdown of the palmitoylated rhoptry armadillo-repeat protein TgARO recapitulates loss of TgDHHC7, showing this protein is also required for rhoptry tethering and strongly suggesting it is a target of TgDHHC7.During invasion, a complex of the rhoptry neck proteins RON2/4/5/8 localizes to the MJ where it is thought to provide a stable anchoring point for host penetration. This complex is also believed to serve as a molecular filter that restricts access of host plasma membrane proteins to the nascent parasitophorus vacuole, protecting it from lysosomal fusion. During the initiation of invasion, the preformed MJ/RON complex is injected into the host cell where RON2 spans the plasma membrane while RON4/5/8 localize to its cytosolic face. While an important interaction between a parasite surface-bound adhesin, AMA1, and RON2 outside of the host cell has been elucidated, little is known about the interactions and role of the MJ/RONs present within the host cytosol. In the second section, I provide a comprehensive analysis of RON5. Using a conditional knockdown approach, I show RON5 is critical for the organization of the MJ RON complex and that disruption of this complex results in a block in rhoptry secretion and host invasion, demonstrating the importance of MJ RONs for host entry. Furthermore, domain analysis of RON5 using functional complementation reveals that a C-terminal region of RON5 is critical for RON2 stability and invasion, defining the first functionally important domain in RON5.Apicomplexan parasites undergo complex life cycles, employing unique forms of internal budding for their replication. The simplest example of these is the Toxoplasma binary division system known as endodyogeny, wherein two daughter parasites are assembled within an intact mother cell. Internal budding is facilitated through the de novo construction of an inner membrane complex (IMC), a series of flattened vesicles and underlying cytoskeletal features that provides the scaffold for daughter cell assembly within the cytosol. Little is known regarding the molecular mechanisms that orchestrate internal budding. The final section of this work identifies the apicomplexan-specific IMC Sub-compartment Protein (ISP) family of IMC proteins in Toxoplasma. These proteins are organized into distinct sub-compartments within the IMC and this arrangement was found to depend on coordinated myristoylation and palmitoylation of conserved N-terminal residues in each protein as well as a unique hierarchical targeting mechanism. Interestingly, while a replicating parasite typically produces two daughters per round of division, disruption of ISP2 results in the assembly of aberrant numbers of daughters with a corresponding loss in parasite fitness indicating a role for this family in the control of budding. Together, these studies provide valuable new insights into host cell invasion and parasite division in this important opportunistic pathogen.

The apicomplexan parasite Toxoplasma gondii divides rapidly and asexually through a unique process of internal daughter budding. The physical infrastructure for this process is the cytoskeleton, which is composed of subpellicular microtubules, flattened vesicles (alveoli), and a meshwork of intermediate filament-like proteins. This meshwork is composed of a family of 14 inner membrane complex (IMC) proteins that were identified based on the presence of a repeat sequence shared across the Alveolata, the alveolin-repeat. All 14 proteins were cloned as YFP fusions to study their subcellular localization and antibodies were generated against several representative IMC proteins. Each IMC displays unique spatio-temporal dynamics throughout development, but four physically distinct localizations were identified: eight IMCs localize to the alveoli, four IMCs localize to a structure known as the basal complex, IMC11 localizes to the apical cap in mature parasites, and IMC15 localizes primarily to the centrosomes and early buds. IMC15 is of particular interest because its appearance before membrane occupation and recognition nexus 1 (MORN1) in the early bud suggests that it is the first cytoskeletal component to associate with the buds. A conditional knockdown of this protein using the destabilization domain (DD) reveals IMC15 has a strong affinity for the centrosomes that overcomes targeting of the DD fusion protein to the proteasome and the presence of IMC15 in the early bud may not be necessary for the division process. Conditional knockdowns using a tetracycline repressible promoter reveal that a minimal amount of IMC15 is sufficient for parasite survival. In order to further characterize IMC15, dominant negative constructs based on mutating putative palmitoylation sites or overexpression of deletion constructs are being pursued. Collectively, the IMC family is being incorporated into the temporal and spatial dynamics of cytoskeletal development through the creation of a comprehensive timeline of daughter bud assembly. These findings are contributing unprecedented detail to the cell division process.

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects a wide range of mammalian and avian hosts, including up to one third of the human population. Immunocompetent individuals clear acute infection with the parasite, however chronic infection is always established and persists for the life of the infected host. Reactivation of chronic infection in immunocompromised individuals, and vertical transmission from mother to fetus during acute infection can cause devastating neural pathology. Acute and chronic infection are associated with two distinct forms of the parasite: tachyzoites, which are fast replicating and responsible for acute infection and bradyzoites, which are slow replicating and establish chronic tissue cysts. During the course of infection, tachyzoites differentiate into bradyzoites. While tachyzoites can infect and replicate within virtually any nucleated cell, bradyzoite cysts typically only develop within neural and muscle tissue. Despite the critical importance of bradyzoite development to T. gondii pathogenesis, the factors responsible for this tissue tropism are unclear. Specifically, there are few defined molecular characteristics of the host cell that have been shown to regulate parasite growth and differentiation as bradyzoites. Using an optimized in vitro system of bradyzoite induction, we have identified several new mechanisms by which T. gondii stage conversion is regulated in vitro. First, we have shown that enhancement of host cell glycolysis can support continued tachyzoite growth under metabolic stress conditions and thus inhibit bradyzoite conversion in a cell-intrinsic manner. Second, we have shown that cell lines that are intrinsically resistant to conversion, either basally or due to the induction of glycolysis, surprisingly release soluble mediators that inhibit conversion in trans. Finally, we have defined a new metabolic function for host Akt in T. gondii differentiation. These results suggest two new hypotheses as to how growth and differentiation of T. gondii may be regulated in vivo. One, the preferential encystment seen in highly glycolytic tissues may be a result of the ability of these tissues to sustain enhanced tachyzoite growth and increased parasite load, and if conversion from tachyzoites to bradyzoites is spontaneously occurring at some level in vivo, increased parasite load may lead to a higher incidence of cyst development in these tissues. Two, cells may broadly be releasing inhibitory mediators, making many tissues inhospitable to conversion, and thus restricting cyst development to particular tissues. While the in vivo significance of these hypotheses remains to be investigated, they provide a new metabolic framework within which the mechanisms that regulate tachyzoite to bradyzoite conversion in vivo can be investigated.

The complex interplay between infectious agents and host defenses in both the innate and adaptive compartments of the immune system determines the outcome of host-pathogen interactions. Bacteria use virulence strategies to invade biological barriers, but active processes of host epithelial cells may also contribute to the endocytosis of microbial particles. To focus on the latter, the uptake of fixed bacterial particles by the inflamed intestinal epithelium was modeled in vitro by Caco-2BBe cells conditioned with TNF-&alpha and an agonist antibody to the lymphtoxin-&beta receptor. Staphylococcus and Yersinia were readily endocytosed, compared with scant uptake of the enteric and pulmonary pathogens Shigella , Salmonella, and Klebsiella. Endocytosed S. aureus, but not Yersinia, was often associated with cytoplasmic claudin-4 vesicles, suggesting that cytokine treatment upregulated at least two distinct endocytic pathways. Treatment induced epithelial redistribution of &beta1 integrin; consistent with this effect, &beta1 integrin blockade reduced uptake of bacterial particles in epithelial layers. Together, these data indicate that cells of the inflamed mucosa selectively enhance endocytic sampling of bacteria from the lumen, perhaps as a mechanism for enhancing the antigen-specific repertoire of the mucosal lymphocyte population. Other biological barriers to infection form a bulwark between immune cells in circulation and tissues at high risk for damage secondary to inflammation; these include the brain and eye. Infection of these tissues with the intracellular protozoan parasite Toxoplasma gondiiinduces a proinflammatory adaptive response. This is accompanied in the brain by the emergence of localized extracellular fiberlike networks along which parasite-specific T cells migrate. To identify molecules associated with tissue remodeling in the infected brain, matrix metalloprotease transcripts were measured. Substantial post-infection upregulation of MMP-8 and MMP-10 together with their endogenous inhibitor TIMP-1 was observed. Intracellular staining of brain mononuclear cells showed MMP-8 and -10 to be expressed by CD4+ and CD8+ cells; these populations, along with CNS-resident microglia and astrocytes, upregulated TIMP-1 during chronic infection. T. gondii burden was significantly reduced in the brains of TIMP-1 null animals. Together, these findings identify a role for specific MMPs and TIMP-1 in matrix remodeling and pathogen clearance associated with migration of brain-infiltrating leukocytes during Toxoplasma infection.

The retina and the choroids are richly vascularised structures and can therefore be colonised by germs via the haematogenous route in the course of a systemic infectious disease. The germs responsible for this type of infection can be fungi, viruses, bacteria and parasites. Ocular candidiasis is outstanding amongst these colonisations because of its frequency; it can manifest itself as an endophthalmitis with a slow and hidden course. The so-called ocular histoplasmosis syndrome, although it is infrequent in our setting, is an important cause of choroidal neovascularisation. The viruses that most frequently affect the retina are of the herpes type and can produce devastating symptoms in immunoincompetent patients, named acute retinal necrosis syndrome. Retinitis due to cytomegalovirus is more frequent in immunodepressed patients, as in the case of AIDS, but it must also be contemplated in patients with lymphoma and immunomodulatory treatment. The most frequent bacterial diseases that affect the retina are syphilis and tuberculosis. Disease due to cat scratches, caused by a borrelia, can produce a neuroretinitis. Toxoplasmosis is the most common of the infectious diseases caused by a parasite and gives rise to chorioretinitis. Toxocariasis, also caused by a parasite, is second in importance, giving rise to choroidal granulomas and retinal tractions.