Three Toxoplasma gondii dense granule proteins are required for induction of Lewis rat macrophage pyroptosis

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

Characterization of NLRP1B Inflammasome Activation

An inflammasome is a multi-protein complex that consists of a sensor protein, caspase-1, and often an adaptor protein ASC. An inflammasome assembles in response to microbial or danger signals and the assembly leads to autoproteolysis of pro-caspase-1 and, consequently, to processing of the inflammatory cytokines pro-IL-1β and pro-IL-18 and to a type of cell death called pyroptosis. NLRP1B is a cytosolic protein that forms an inflammasome in response to anthrax lethal toxin or reduction in cytosolic ATP levels. NLRP1B detects anthrax lethal toxin when the toxin cleaves an amino-terminal fragment from the protein. The mechanism by which NLRP1B senses reduction in cytosolic ATP is unknown. In chapter 3, I addressed whether the amino-terminal region of NLRP1B also serves as a sensor of reduced ATP levels. I found that an NLRP1B mutant lacking the amino- terminal region exhibited some spontaneous activity and failed to be further activated by lethal toxin, but was still activated by metabolic inhibitors, indicating that the amino-terminal region is not the sole sensing domain of NLRP1B. Comparison of NLRP1B alleles that differed in their response to metabolic inhibitors, but not to lethal toxin, led to the finding that a repeated sequence in the FIIND facilitated detection of ATP depletion. NLRP1B might sense ATP depletion as a way of sensing microbial infections. In chapter 4, I show that an infection with an intracellular pathogen, Listeria monocytogenes caused a reduction of cytosolic ATP levels and activation of the NLRP1B inflammasome in a reconstituted system. NLRP1B activation was dependent on Listeriaâ s expression of a protein required for vacuolar escape, listeriolysin O. Furthermore, I show that infection with Listeria monocytogenes and Shigella flexneri also caused reduction of ATP and activation of NLRP1B in the murine RAW264.7 macrophage cell line.Ph.D.2019-12-19 00:00:0

Distinct regions of NLRP1B are required to respond to anthrax lethal toxin and metabolic inhibition

10.1128/IAI.02167-14Infection and Immunity8293697-370

Pathogen perception by NLRs in plants and animals: Parallel worlds

Intracellular NLR (Nucleotide‐binding domain and Leucine‐rich Repeat‐containing) receptors are sensitive monitors that detect pathogen invasion of both plant and animal cells. NLRs confer recognition of diverse molecules associated with pathogen invasion. NLRs must exhibit strict intramolecular controls to avoid harmful ectopic activation in the absence of pathogens. Recent discoveries have elucidated the assembly and structure of oligomeric NLR signalling complexes in animals, and provided insights into how these complexes act as scaffolds for signal transduction. In plants, recent advances have provided novel insights into signalling‐competent NLRs, and into the myriad strategies that diverse plant NLRs use to recognise pathogens. Here, we review recent insights into the NLR biology of both animals and plants. By assessing commonalities and differences between kingdoms, we are able to develop a more complete understanding of NLR function