# Identification of the mechanism by which Toxoplasma activates the NLRP1 inflammasome

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2020 · $235,500

## Abstract

Toxoplasma gondii is an extremely successful intracellular pathogen that causes lifelong chronic infections in
almost all warm-blooded animals and severe disease in fetuses and immunocompromised individuals. The
Lewis rat is the only known animal that has sterilizing immunity against the parasite and is therefore a unique
animal model that can help us understand how the immune system can defeat T.gondii. NLRP1 inflammasome
activation in Lewis rat macrophages upon T.gondii infection induces pyroptosis resulting in loss of the
parasite’s replication niche and clearance of infection. The mechanism by which NLRP1 inflammasomes detect
T.gondii infection and become activated remains poorly understood. The murine NLRP1 inflammasome is
activated by ubiquitination and proteasomal degradation of its auto-inhibitory N-terminal polypeptide, which
liberates the C-terminal polypeptide for Caspase-1 recruitment and inflammasome assembly. Therefore, E3
ubiquitin ligases from pathogens and/or host play a critical role in ubiquitination of NLRP1 and inflammasome
activation. We previously identified three T.gondii secreted effector proteins that are required for NLRP1
inflammasome activation in Lewis rat macrophages, however, none of these effectors possess predicted
ubiquitin ligase function. Immunoprecipitation identified several host E3 ubiquitin ligases that interact with these
T.gondii effectors. In addition, we have found that the murine NLRP1 inflammasome activator, Val-boroPro, an
inhibitor of the cytosolic serine dipeptidase DPP8/9, induces pyroptosis in Lewis rat macrophages but not in
macrophages from rat strains resistant to Toxoplasma-induced pyroptosis. Thus, Val-boroPro phenocopies
T.gondii infection in Lewis macrophages. Murine NLRP1 inflammasome activation triggered by inhibition of
DPP8/9 requires ubiquitination of NLRP1 via unknown E3 ubiquitin ligases. Thus, we hypothesize that the
Lewis E3 ubiquitin ligases interacting with our T.gondii effectors, or other host E3 ubiquitin ligases, serve as
checkpoints for NLRP1 inflammasome activation in Lewis rat macrophages in response to T.gondii infection
and DPP8/9 inhibition. This hypothesis will be tested by pursuing two specific aims. In our first aim we will
evaluate the role of the Lewis rat E3 ubiquitin ligases interacting with our T.gondii effectors in T.gondii- and
Val-boroPro-induced pyroptosis of Lewis rat macrophages, and in binding to and ubiquitination of Lewis rat
NLRP1. In our second aim we will use an unbiased gain-of-function CRISPR/Cas9 screen to identify the host
E3 ubiquitin ligases involved in Lewis rat NLRP1 inflammasome activation triggered by T.gondii infection and
DPP8/9 inhibition. This proposal will advance the field by: 1) identifying the mechanism by which T.gondii
activates the Lewis rat NLRP1 inflammasome, which could lead to novel insights into controlling toxoplasmosis
in humans as human NLRP1 polymorphisms are associated with severity of congenital toxoplasmosis; 2)
unco...

## Key facts

- **NIH application ID:** 9952058
- **Project number:** 1R21AI151081-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** JEROEN SAEIJ
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $235,500
- **Award type:** 1
- **Project period:** 2020-01-21 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9952058

## Citation

> US National Institutes of Health, RePORTER application 9952058, Identification of the mechanism by which Toxoplasma activates the NLRP1 inflammasome (1R21AI151081-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9952058. Licensed CC0.

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