The role of novel Toxoplasma dense granule proteins in establishing infection and modulating host interactions

Toxoplasma gondii is an obligate intracellular parasite that can infect any warm-blooded mammal, making it one of the most successful parasites in the world. It is estimated to infect approximately one-third of the global human population and can cause life threatening complications in immunocompromised individuals and congenitally infected neonates. During the acute infection, rapidly multiplying T. gondii tachyzoites must balance between avoiding clearance to ensure the infection and limiting parasite replication to avoid killing the host. While most tachyzoites are eliminated during the acute infection by the host’s immune response, a fraction converts into slow growing bradyzoites that are encased by a cyst wall that constitute the chronic infection. These infectious tissue cysts remain largely undetected from the host’s immune surveillance.The ability of T. gondii to survive intracellularly and establish a productive infection relies significantly on its capacity to regulate host cell functions. One way the parasite achieves this is through the secretion of dense granule proteins (GRAs) that are involved in remodeling the parasitophorous vacuole (PV), obtaining nutrients from the host, manipulating the host cell cycle, and modulating the immune response. Because of their diverse functions during infection, the goal of this work is to identify and characterize novel GRAs in the acute and chronic infection to better understand the array of effectors this parasite uses to colonize its mammalian hosts. This work first describes a proximity labeling experiment to identify several novel T. gondii GRAs, GRA55-59, that are expressed in bradyzoites. Deletion of these GRAs does not affect parasite fitness in vitro. Further characterization of GRA55 reveals that its deletion does not impact virulence during the acute infection but does reduce brain cyst burden in infected mice. This suggests that GRA55 is an important secreted protein for the establishment or maintenance of the chronic infection in mice. The next section identifies the dense granule protein GRA83 and reveals that this effector is secreted into the PV and plays a role in modulating a key element of the host’s innate immune response. Disruption of GRA83 impacts proinflammatory cytokine production and increases T. gondii’s virulence during the acute infection. This also results in a significantly higher brain cyst burden during the chronic infection in mice. Therefore, this effector functions in a pro-host manner to limit unrestricted parasite growth. Lastly, this work describes the novel secreted effector, GRA84, that is exported to the host cell nucleus. GRA84 is dependent on the aspartyl protease (ASP5) and the MYR translocon to traverse the PV barrier and reach the host cell nucleus. Disruption of GRA84 does not substantially impact in vitro parasite growth or the chronic infection in mice. Most interestingly, this work demonstrates that GRA84 is proteolytically cleaved for maturation in its N-terminus and that this processing event is essential its ability to translocate across the vacuolar membrane to reach its destination in the host nucleus. Together, this thesis identifies and characterizes an array of novel GRA proteins that regulate Toxoplasma infection, which is likely to aid in the development of new treatments or therapeutics that can ultimately clear this widespread human infection

The Toxoplasma gondii effector GRA83 modulates the hosts innate immune response to regulate parasite infection.

Toxoplasma gondiis propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasites ability to efficiently invade and regulate components of the hosts infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasmas ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasites diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogens tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cells response to limit infection

Proximity biotinylation reveals novel secreted dense granule proteins of Toxoplasma gondii bradyzoites.

Toxoplasma gondii is an obligate intracellular parasite which is capable of establishing life-long chronic infection in any mammalian host. During the intracellular life cycle, the parasite secretes an array of proteins into the parasitophorous vacuole (PV) where it resides. Specialized organelles called the dense granules secrete GRA proteins that are known to participate in nutrient acquisition, immune evasion, and host cell-cycle manipulation. Although many GRAs have been discovered which are expressed during the acute infection mediated by tachyzoites, little is known about those that participate in the chronic infection mediated by the bradyzoite form of the parasite. In this study, we sought to uncover novel bradyzoite-upregulated GRA proteins using proximity biotinylation, which we previously used to examine the secreted proteome of the tachyzoites. Using a fusion of the bradyzoite upregulated protein MAG1 to BirA* as bait and a strain with improved switch efficiency, we identified a number of novel GRA proteins which are expressed in bradyzoites. After using the CRISPR/Cas9 system to characterize these proteins by gene knockout, we focused on one of these GRAs (GRA55) and found it was important for the establishment or maintenance of cysts in the mouse brain. These findings highlight new components of the GRA proteome of the tissue-cyst life stage of T. gondii and identify potential targets that are important for maintenance of parasite persistence in vivo

Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins.

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. While the molecular tools are highly developed for the apicomplexan Toxoplasma gondii, the closely related parasite Neospora caninum lacks efficient tools for genetic manipulation. To enable efficient homologous recombination in N. caninum, we targeted the Ku heterodimer DNA repair mechanism in the genomic reference strain, Nc-Liverpool (NcLiv), and show that deletion of Ku80 results in a destabilization and loss of its partner Ku70. Disruption of Ku80 generated parasites in which genes are efficiently epitope tagged and only short homology regions are required for gene knockouts. We used this improved strain to target novel nonessential genes encoding dense granule proteins that are unique to N. caninum or conserved in T. gondii. To expand the utility of this strain for essential genes, we developed the auxin-inducible degron system for N. caninum using parasite-specific promoters. As a proof of concept, we knocked down a novel nuclear factor in both N. caninum and T. gondii and showed that it is essential for survival of both parasites. Together, these efficient knockout and knockdown technologies will enable the field to unravel specific gene functions in N. caninum, which is likely to aid in the identification of targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. IMPORTANCE Neospora caninum is a parasite with veterinary relevance, inducing severe disease in dogs and reproductive disorders in ruminants, especially cattle, leading to major losses. The close phylogenetic relationship to Toxoplasma gondii and the lack of pathogenicity in humans drives an interest of the scientific community toward using N. caninum as a model to study the pathogenicity of T. gondii. To enable this comparison, it is important to develop efficient molecular tools for N. caninum, to gain accuracy and save time in genetic manipulation protocols. Here, we have developed base strains and protocols using the genomic reference strain of N. caninum to enable efficient knockout and knockdown assays in this model. We demonstrate that these tools are effective in targeting known and previously unexplored genes. Thus, these tools will greatly improve the study of this protozoan, as well as enhance its ability to serve as a model to understand other apicomplexan parasites