Modified Protection against Toxoplasma gondii Lethal Infection and Brain Cyst Formation by Vaccination with SAG2 and SRS1.

The persistence of Toxoplasma gondii tissue cysts in organs of cats (definitive host) and rodents (intermediate hosts) was studied. Nine cats, 12 rats, and 12 mice were fed T. gondii oocysts and their organs were digested in pepsin and then bioassayed for bradyzoites in mice. Of 9 cats killed 37 or 51 days after feeding 102 (2 cats), 103 (3 cats) or 104 (4 cats) oocysts of the VEG strain, tissue cysts were found in each cat; in the tongue of 9, in the heart of 5, in the brain of 4, and in the eyes of 1 cat. The dose had no effect on the distribution of tissue cysts in cats. Twelve rats were each fed 105 oocysts of the VEG strain of T. gondii and killed 21, 29, 64 or 237 days later. At each time-period, 11 tissues of 3 rats were pooled and bioassayed in mice. Tissue cysts were found in the brain, skeletal muscle, heart and kidneys of rats at each killing time; in the lungs, intestines, and mesenteric lymph nodes in 3 of 4 instances; in the tongue, liver, and eyes in 2 instances and in the spleen in 1 instance. Also, using the same procedures and sampling the same 11 tissues as used for rats, tissue cysts were seen in all organs except in the tongue and liver of 3 mice killed on day 82 after feeding the VEG strain. In 9 mice (3 with each strain) fed oocysts of the ME-49, GT-1, or P89 T. gondii strain and killed 62–130 days later, tissue cysts were found consistently only in the brain. Thus, in rats and mice, most tissue cysts were found in the brain and rarely in the tongue. This was in marked contrast to the distribution of tissue cysts in cats.

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Prime-Boost Vaccination with Toxoplasma Lysate Antigen, but Not with a Mixture of Recombinant Protein Antigens, Leads to Reduction of Brain Cyst Formation in BALB/c Mice

PLoS ONE ◽

10.1371/journal.pone.0126334 ◽

2015 ◽

Vol 10 (5) ◽

pp. e0126334 ◽

Cited By ~ 16

Author(s):

Angelika Wagner ◽

Irma Schabussova ◽

Bärbel Ruttkowski ◽

Roman Peschke ◽

Józef Kur ◽

...

Keyword(s):

Recombinant Protein ◽

Cyst Formation ◽

Protein Antigens ◽

Brain Cyst ◽

Boost Vaccination

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Analysis of Noncanonical Calcium-Dependent Protein Kinases in Toxoplasma gondii by Targeted Gene Deletion Using CRISPR/Cas9

Infection and Immunity ◽

10.1128/iai.01173-15 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1262-1273 ◽

Cited By ~ 35

Author(s):

Shaojun Long ◽

Qiuling Wang ◽

L. David Sibley

Keyword(s):

Toxoplasma Gondii ◽

Protein Kinases ◽

Gene Deletion ◽

Cyst Formation ◽

Content Type ◽

Calcium Dependent ◽

Targeted Gene Deletion ◽

Dependent Protein ◽

Calcium Dependent Protein Kinases

Calcium-dependent protein kinases (CDPKs) are expanded in apicomplexan parasites, especially inToxoplasma gondiiwhere 14 separate genes encoding these enzymes are found. Although previous studies have shown that several CDPKs play a role in controlling invasion, egress, and cell division inT. gondii, the roles of most of these genes are unexplored. Here we developed a more efficient method for gene disruption using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) that was modified to completely delete large, multiexonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system, we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the functions of noncanonical CDPKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growthin vitroor infection in the mouse model. The exception to this was CDPK6, loss of which leads to reduced plaquing, fitness defect in a competition assay, and reduced tissue cyst formation in chronically infected mice. Our findings highlight the utility of CRISPR/Cas9 for rapid serial gene deletion and also suggest that additional models are needed to reveal the functions of many genes inT. gondii.

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Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii

Biochimie ◽

10.1016/s0300-9084(03)00076-2 ◽

2003 ◽

Vol 85 (3-4) ◽

pp. 353-361 ◽

Cited By ~ 47

Author(s):

Alexandra Coppin ◽

Florence Dzierszinski ◽

Sandra Legrand ◽

Marlène Mortuaire ◽

David Ferguson ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Cyst Formation ◽

Tissue Cyst ◽

Developmentally Regulated ◽

And Storage ◽

Storage Polysaccharide

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Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development

mSphere ◽

10.1128/msphere.00054-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 22

Author(s):

Sherri Huang ◽

Michael J. Holmes ◽

Joshua B. Radke ◽

Dong-Pyo Hong ◽

Ting-Kai Liu ◽

...

Keyword(s):

Gene Expression ◽

Toxoplasma Gondii ◽

Cyst Formation ◽

Tissue Cyst ◽

Stress Induction ◽

Content Type ◽

A Cell ◽

Transitional Forms ◽

Mouse Model Of Infection ◽

Cyst Development

ABSTRACT Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite. Toxoplasma gondii is a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst that is imperceptible to the immune response and unaffected by current therapies. The tissue cysts facilitate transmission through predation and give rise to chronic cycles of toxoplasmosis in immunocompromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, and yet the mechanisms underlying these alterations in gene expression are not well defined. Here we show that AP2IX-4 is a nuclear protein exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had no discernible effect on tachyzoite replication but resulted in a reduced frequency of tissue cyst formation following alkaline stress induction—a defect that is reversible by complementation. AP2IX-4 has a complex role in regulating bradyzoite gene expression, as the levels of many bradyzoite mRNAs dramatically increased beyond those seen under conditions of normal stress induction in AP2IX-4 knockout parasites exposed to alkaline media. The loss of AP2IX-4 also resulted in a modest virulence defect and reduced cyst burden in chronically infected mice, which was reversed by complementation. These findings illustrate that the transcriptional mechanisms responsible for tissue cyst development operate across the intermediate life cycle from the dividing tachyzoite to the dormant bradyzoite. IMPORTANCE Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

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