ABSTRACT Upon invasion of Lewis rat macrophages, Toxoplasma rapidly induces programmed cell death (pyroptosis), which prevents Toxoplasma replication, possibly explaining the resistance of the Lewis ...rat to Toxoplasma. Using a chemical mutagenesis screen, we identified Toxoplasma mutants that no longer induced pyroptosis. Whole-genome sequencing led to the identification of three Toxoplasma parasitophorous vacuole-localized dense granule proteins, GRA35, GRA42, and GRA43, that are individually required for induction of Lewis rat macrophage pyroptosis. Macrophage infection with Δgra35, Δgra42, and Δgra43 parasites led to greatly reduced cell death rates and enhanced parasite replication. Lewis rat macrophages infected with parasites containing a single, double, or triple deletion of these GRAs showed similar levels of cell viability, suggesting that the three GRAs function in the same pathway. Deletion of GRA42 or GRA43 resulted in GRA35 (and other GRAs) being retained inside the parasitophorous vacuole instead of being localized to the parasitophorous vacuole membrane. Despite having greatly enhanced replication in Lewis rat macrophages in vitro, Δgra35, Δgra42, and Δgra43 parasites did not establish a chronic infection in Lewis rats. Toxoplasma did not induce F344 rat macrophage pyroptosis, but F344 rats infected with Δgra35, Δgra42, and Δgra43 parasites had reduced cyst numbers. Thus, these GRAs determined parasite in vivo fitness in F344 rats. Overall, our data suggest that these three Toxoplasma dense granule proteins play a critical role in establishing a chronic infection in vivo, independently of their role in mediating macrophage pyroptosis, likely due to their importance in regulating protein localization to the parasitophorous vacuole membrane. IMPORTANCE Inflammasomes are major components of the innate immune system and are responsible for detecting various microbial and environmental danger signals. Upon invasion of Lewis rat macrophages, the parasite rapidly activates the NLRP1 inflammasome, resulting in pyroptosis and elimination of the parasite’s replication niche. The work reported here revealed that Toxoplasma GRA35, GRA42, and GRA43 are required for induction of Lewis rat macrophage pyroptosis. GRA42 and GRA43 mediate the correct localization of other GRAs, including GRA35, to the parasitophorous vacuole membrane. These three GRAs were also found to be important for parasite in vivo fitness in a Toxoplasma-susceptible rat strain, independently of their role in NLRP1 inflammasome activation, suggesting that they perform other important functions. Thus, this study identified three GRAs that mediate the induction of Lewis rat macrophage pyroptosis and are required for pathogenesis of the parasite.
Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic ...hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871–base ...pair designer eukaryotic chromosome, synlll, which is based on the 316,617–base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Toxoplasma gondii is an obligate ...intracellular pathogen capable of chronically infecting nearly all warm-blooded animals, including humans. The chronic stage is characterized by the presence of semi-dormant cysts in brain and muscle tissues. These cysts are crucial in the success of Toxoplasma as they are orally infectious and allow for the transmission of the parasite between hosts. As the host immune response drives cyst formation, the establishment of this chronic infection relies on the parasite's ability to find a balance between activation of a host immune response and evasion of parasiticidal mechanisms. This balance is achieved through the modulation of host cell processes by parasite proteins secreted from specialized secretory organelles known as rhoptries and dense granules. Here, we report that Toxoplasma activates the inflammasomes in mice and rats. The inflammasomes are a set of cytoplasmic pattern recognition receptors (PRRs). Activation of the inflammasomes results in caspase-1 activation and the cleavage and release of the pro-inflammatory cytokines, Interleukin (IL)-1beta and IL-18. IL-1p is an important mediator of local inflammation and neutrophil recruitment. IL- 18 induces Interferon (IFN)-gamma, which is a critical cytokine in the control of Toxoplasma. A form of cell death, termed pyroptosis, can accompany inflammasome activation. The NLRP3 inflammasome is activated in mouse macrophages, leading to the secretion of IL-1beta in vitro. The NLRP1 and NLRP3 inflammasomes play a major role in mouse survival and control of parasite replication in vivo. The NLRPI inflammasome is activated in infected macrophages from rats that are able to completely clear infection. Toxoplasma infection leads to the secretion of active IL-Ibeta and IL-18. Activation of the NLRP1 inflammasome leads to pyroptosis, a programmed form of cell death. Pyroptosis prevents parasite replication within the host cell and likely promotes clearance by nearby immune cells. Using a chemical mutagenesis screen, we identified three Toxoplasma dense granule proteins (GRAs), GRA18, GRA27 and GRA28, essential for NLRP1 inflammasome activation and pyroptosis in rat macrophages. Our work has identified Toxoplasma gondii as a novel activator of the rodent inflammasomes and demonstrated host cell death as a mechanism to control parasite replication. We have also identified three novel parasite proteins required for this activation, providing insight into interactions between parasite and host, which may aid in the treatment of human infection.
by Kimberly M. Cirelli.
Ph. D.
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