Assays for Monitoring Toxoplasma gondii Infectivity in the Laboratory Mouse

Wang, Qiuling; Sibley, L. David

doi:10.1007/978-1-4939-9857-9_5

Cited by 15 publications

(27 citation statements)

References 45 publications

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“…Total cyst burden was then extrapolated. Statistical significance of cyst burden between GRA55-HA, Δgra55, and GRA55c experiments was calculated by two-sample 2-tailed t-test [77].…”

Section: Mouse Brain Cyst Quantitationmentioning

confidence: 99%

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

et al. 2020

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Toxoplasma gondii is an obligate intracellular parasite which is capable of establishing lifelong 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 bradyzoiteupregulated 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.

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“…Total cyst burden was then extrapolated. Statistical significance of cyst burden between GRA55-HA, Δgra55, and GRA55c experiments was calculated by two-sample 2-tailed t-test [77].…”

Section: Mouse Brain Cyst Quantitationmentioning

confidence: 99%

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

et al. 2020

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“…with 200 μl of sterile PBS containing freshly harvested tachyzoites of indicated T. gondii strains. Survivors were sacrificed at the indicated days post-infection and tested for sero-conversion using the Toxocell latex kit and by ELISA [67].…”

Section: Animal Strains and Infection Conditionsmentioning

confidence: 99%

“…C57BL/6 MEFs confluently grown in 6 well-plates in the absence or presence of 100 U/ml IFNγ were infected with 200 freshly harvested tachyzoites of T. gondii wt and ko strains. 7 days post infection, monolayers were thoroughly washed with PBS and plaque numbers determined after staining with 0,1% crystal violet [67].…”

Section: Plaque Assaysmentioning

confidence: 99%

ROP39 is an Irgb10-specific parasite effector that modulates acute Toxoplasma gondii virulence

et al. 2023

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Toxoplasma gondii (T. gondii) is zoonotic apicomplexan parasite that is an important cause of clinical disability in humans. On a global scale, one third of the human population is infected with T. gondii. Mice and other small rodents are believed to be responsible for transmission of T. gondii to the domestic cat, its definitive host. Interferon-inducible Immunity-Related GTPases (IRG proteins) are important for control of murine T. gondii infections. Virulence differences between T. gondii strains are linked to polymorphic rhoptry proteins (ROPs) that cooperate to inactivate individual IRG family members. In particular, the pseudokinase ROP5 isoform B is critically important in laboratory strains of mice. We identified T. gondii ROP39 in complex with ROP5B and demonstrate its contribution to acute T. gondii virulence. ROP39 directly targets Irgb10 and inhibits homodimer formation of the GTPase leading to an overall reduction of IRG protein loading onto the parasitophorous vacuolar membrane (PVM). Maintenance of PVM integrity rescues the parasite from IRG protein-mediated clearance in vitro and in vivo. This study identifies a novel T. gondii effector that is important for specific inactivation of the IRG resistance system. Our data reveal that yet unknown T. gondii effectors can emerge from identification of direct interaction partners of ROP5B.

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“…The central nervous system (CNS) is the tissue par excellence to study the development of cysts during the chronic phase of the infection but the T. gondii tropism towards immune privileged organs also involves ocular or muscle tissues ( Jiang et al., 2020 ; Yang et al., 2020 ; Dubey, 2021 ). Cysts can be quantified and even measured (diameter) by microscopic observation of fresh/unstained ( Dubey et al., 2012 ) and immunostained fixed brain sections or brain homogenates ( Masatani et al., 2020 ; Wang and Sibley, 2020 ). An indirect way to quantify the presence of cysts in the mouse brain is to measure the parasite burden in the CNS from 3 weeks pi by quantitative PCR ( Fernández-Escobar et al., 2020 ; Fernández-Escobar et al., 2021 ; Salman et al., 2021 ).…”

Section: Complementary Approaches On the Determination Of Virulence I...mentioning

confidence: 99%

Unifying Virulence Evaluation in Toxoplasma gondii: A Timely Task

Calero‐Bernal

Fernández‐Escobar

Katzer

et al. 2022

Front. Cell. Infect. Microbiol.

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Toxoplasma gondii, a major zoonotic pathogen, possess a significant genetic and phenotypic diversity that have been proposed to be responsible for the variation in clinical outcomes, mainly related to reproductive failure and ocular and neurological signs. Different T. gondii haplogroups showed strong phenotypic differences in laboratory mouse infections, which provide a suitable model for mimicking acute and chronic infections. In addition, it has been observed that degrees of virulence might be related to the physiological status of the host and its genetic background. Currently, mortality rate (lethality) in outbred laboratory mice is the most significant phenotypic marker, which has been well defined for the three archetypal clonal types (I, II and III) of T. gondii; nevertheless, such a trait seems to be insufficient to discriminate between different degrees of virulence of field isolates. Many other non-lethal parameters, observed both in in vivo and in vitro experimental models, have been suggested as highly informative, yielding promising discriminatory power. Although intra-genotype variations have been observed in phenotypic characteristics, there is no clear picture of the phenotypes circulating worldwide; therefore, a global overview of T. gondii strain mortality in mice is presented here. Molecular characterization has been normalized to some extent, but this is not the case for the phenotypic characterization and definition of virulence. The present paper proposes a baseline (minimum required information) for the phenotypic characterization of T. gondii virulence and intends to highlight the needs for consistent methods when a panel of T. gondii isolates is evaluated for virulence.

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Assays for Monitoring Toxoplasma gondii Infectivity in the Laboratory Mouse

Cited by 15 publications

References 45 publications

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

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

ROP39 is an Irgb10-specific parasite effector that modulates acute Toxoplasma gondii virulence

Unifying Virulence Evaluation in Toxoplasma gondii: A Timely Task

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