A Nucleotide Sugar Transporter Involved in Glycosylation of the Toxoplasma Tissue Cyst Wall Is Required for Efficient Persistence of Bradyzoites

Caffaro, Carolina E.; Koshy, Anita A.; Liu, Li; Zeiner, Gusti M.; Hirschberg, Carlos B.; Boothroyd, John C.

doi:10.1371/journal.ppat.1003331

Cited by 67 publications

(74 citation statements)

References 34 publications

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“…To create the ME49⌬gra25 parasites, we started with a parental type II ME49-luc⌬hxgprt (ME49) strain that expresses firefly luciferase but lacks the hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene (21). The TGME49_290700 (GRA25) gene was deleted from ME49 and replaced with HXGPRT by homologous recombination using the pTKO2 vector (21) in which the HXGPRT is flanked by ϳ1 kb and ϳ1.5 kb, respectively, of genomic sequence amplified by PCR from the 5= and 3= regions flanking the TGME49_290700 open reading frame (ORF). The 5=-flanking region was amplified using 5=-GCGCGGTACCGACCTAGA GAGCATTCAACGC-3= (forward) and 5=-GCGCGAATTCGATAACCG ACGAAGACCAGGAGAG-3= (reverse) primer sequences and was cloned into the KpnI and EcoRI restriction sites of pTKO2 (21).…”

Section: Methodsmentioning

confidence: 99%

“…The TGME49_290700 (GRA25) gene was deleted from ME49 and replaced with HXGPRT by homologous recombination using the pTKO2 vector (21) in which the HXGPRT is flanked by ϳ1 kb and ϳ1.5 kb, respectively, of genomic sequence amplified by PCR from the 5= and 3= regions flanking the TGME49_290700 open reading frame (ORF). The 5=-flanking region was amplified using 5=-GCGCGGTACCGACCTAGA GAGCATTCAACGC-3= (forward) and 5=-GCGCGAATTCGATAACCG ACGAAGACCAGGAGAG-3= (reverse) primer sequences and was cloned into the KpnI and EcoRI restriction sites of pTKO2 (21). The 3=-flanking region was amplified using 5=-GGGGAAGCTTGAAAACGGTGCGCGG AAGG-3= (forward) and 5=-GCGCGCTAGCCAGATTCCCCAGACGGA TTTG-3= (reverse) primer sequences and was cloned into HindIII and NheI restriction sites of the pTKO2 vector.…”

Section: Methodsmentioning

confidence: 99%

“…To transfer (complement) the GRA25 locus back into the knockout (KO) strain, the HXGPRT marker was first removed using Cre recombinase as previously described (21). Briefly, a plasmid expressing Cre recombinase was transiently transfected into the ME49⌬gra25 strain to catalyze recombination at the loxP sites flanking the HXGPRT marker in the pGRA25_KO vector.…”

Section: Methodsmentioning

confidence: 99%

“…3B and C). To generate GRA25-complemented strains in the ME49⌬gra25 background, the HXGPRT selectable marker, which is flanked by loxP sites, was first removed by transiently transfecting parasites with a CRE recombinase-expressing plasmid and selecting for loss of HXGPRT using 6-thioxanthine as described previously (21). Single clones of the resulting ME49⌬gra25 strain were then transfected with the GRA25 II gene (also from type II ME49, as indicated by the subscript "II") driven by its own pro-moter.…”

Section: Qtl Mapping Reveals a Region On Toxoplasma Chromosome IX Thamentioning

confidence: 99%

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GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response

et al. 2014

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The obligate intracellular parasite Toxoplasma gondii is able to infect a broad range of hosts and cell types due, in part, to the diverse arsenal of effectors it secretes into the host cell. Here, using genetic crosses between type II and type III Toxoplasma strains and quantitative trait locus (QTL) mapping of the changes they induce in macrophage gene expression, we identify a novel dense granule protein, GRA25. Encoded on chromosome IX, GRA25 is a phosphoprotein that is secreted outside the parasites and is found within the parasitophorous vacuole. In vitro experiments with a type II ⌬gra25 strain showed that macrophages infected with this strain secrete lower levels of CCL2 and CXCL1 than those infected with the wild-type or complemented control parasites. In vivo experiments showed that mice infected with a type II ⌬gra25 strain are able to survive an otherwise lethal dose of Toxoplasma tachyzoites and that complementation of the mutant with an ectopic copy of GRA25 largely rescues this phenotype. Interestingly, the type II and type III versions of GRA25 differ in endogenous expression levels; however, both are able to promote parasite expansion in vivo when expressed in a type II ⌬gra25 strain. These data establish GRA25 as a novel virulence factor and immune modulator.T oxoplasma gondii is an obligate intracellular parasite with the remarkable ability to infect a broad range of mammalian and avian hosts. During infection in the mouse, a natural Toxoplasma host, macrophages are known to play a key role in mounting an immune response to infection via the secretion of cytokines such as interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-␣) (1). The secretion of these cytokines leads to the activation of NK cells and CD4 ϩ and CD8 ϩ T cells (2-4). Although macrophages can themselves be targets of parasite infection, they also can control parasite dissemination through cell-intrinsic, antimicrobial strategies, such as the production of nitric oxide (NO) and reactive oxygen species (ROS) and, in mice, the activation of immunityrelated GTPases (IRGs) (5, 6). Recent studies have shown that inflammatory monocytes, in particular, are recruited to the site of infection by the cytokine CCL2 (monocyte chemotactic protein-1 [MCP-1]) (7, 8). As has been described for infection with several pathogens (9), CCL2 is essential for control of parasite spread and survival of infection in both intraperitoneal (i.p.) and oral Toxoplasma infections (7,8).The strains of Toxoplasma that predominate in Europe and North America, termed types I, II, and III, differ in a wide range of phenotypes, including how they interface with the immune response. The tachyzoite stage of the parasite interacts with the host through secretion of polymorphic proteins from specialized organelles termed rhoptries, which house "ROP" proteins, and dense granules, which contain "GRA" proteins. One such polymorphic protein is the tyrosine kinase ROP16. Upon injection into the host cell, it directly phosphorylates STAT3 and STAT6, and in cells infec...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Qtl Mapping Reveals a Region On Toxoplasma Chromosome IX Thamentioning

confidence: 99%

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GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response

et al. 2014

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“…T. gondii CST1 deletion strains lead to a reduced cyst burden in mice, suggesting that CST1 plays a critical role in conversion to or survival of the bradyzoite form (142). Similarly, T. gondii strains containing deletions in the nucleotide-sugar transporter (TgNST1) can no longer interact with lectins and display severe defects in persistence during chronic infection (143). Analysis of the bradyzoite transcriptome for potentially secreted proteins identified two proteins, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, which colocalize with DBA on the cyst wall (144)(145)(146).…”

Section: Molecular Analysis Of Bradyzoitesmentioning

confidence: 99%

Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections

Pittman

Knoll

2015

Microbiol Mol Biol Rev

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SUMMARYToxoplasma gondiirepresents one of the most common parasitic infections in the world. The asexual cycle can occur within any warm-blooded animal, but the sexual cycle is restricted to the feline intestinal epithelium.T. gondiiis acquired through consumption of tissue cysts in undercooked meat as well as food and water contaminated with oocysts. Once ingested, it differentiates into a rapidly replicating asexual form and disseminates throughout the body during acute infection. After stimulation of the host immune response,T. gondiidifferentiates into a slow-growing, asexual cyst form that is the hallmark of chronic infection. One-third of the human population is chronically infected withT. gondiicysts, which can reactivate and are especially dangerous to individuals with reduced immune surveillance. Serious complications can also occur in healthy individuals if infected with certainT. gondiistrains or if infection is acquired congenitally. No drugs are available to clear the cyst form during the chronic stages of infection. This therapeutic gap is due in part to an incomplete understanding of both host and pathogen responses during the progression ofT. gondiiinfection. While many individual aspects ofT. gondiiinfection are well understood, viewing the interconnections between host and parasite during acute and chronic infection may lead to better approaches for future treatment. The aim of this review is to provide an overview of what is known and unknown about the complex relationship between the host and parasite during the progression ofT. gondiiinfection, with the ultimate goal of bridging these events.

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Toxoplasma gondii: Bradyzoite Differentiation In Vitro and In Vivo

Mayoral

Cristina

Carruthers

et al. 2019

Methods in Molecular Biology

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Toxoplasma gondii, a member of the Apicomplexa, is known for its ability to infect an impressive range of host species. It is a common human infection that causes significant morbidity in congenitally infected children and immunocompromised patients. This parasite can be transmitted by bradyzoites, a slowly-replicating life stage found within intracellular tissue cysts, and oocysts, the sexual life cycle stage that develops in domestic cats and other Felidae. T. gondii bradyzoites retain the capacity to revert back to the quickly-replicating tachyzoite life stage, and when the host is immune compromised unrestricted replication can lead to significant tissue destruction. Bradyzoites are refractory to currently available Toxoplasma treatments. Improving our understanding of bradyzoite biology is critical for the development of therapeutic strategies to eliminate latent infection. This chapter describes a commonly used protocol for the differentiation of T. gondii tachyzoites into bradyzoites using human foreskin fibroblast cultures and a CO 2limited alkaline cell media, which results in a high proportion of differentiated bradyzoites for further study. Also described are methods for purifying tissue cysts from chronically infected mouse brain using isopycnic centrifugation and a recently developed approach for measuring bradyzoite viability.

show abstract

A Nucleotide Sugar Transporter Involved in Glycosylation of the Toxoplasma Tissue Cyst Wall Is Required for Efficient Persistence of Bradyzoites

Cited by 67 publications

References 34 publications

GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response

GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response

Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections

Toxoplasma gondii: Bradyzoite Differentiation In Vitro and In Vivo

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