Identification of Tissue Cyst Wall Components by Transcriptome Analysis of
            <i>In Vivo</i>
            and In Vitro Toxoplasma gondii Bradyzoites

Buchholz, Kerry R.; Fritz, Heather M.; Chen, Xiucui; Durbin‐Johnson, Blythe; Rocke, David M.; Ferguson, David J. P.; Conrad, Patricia A.; Boothroyd, John C.

doi:10.1128/ec.05182-11

Cited by 106 publications

(139 citation statements)

References 47 publications

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“…Interestingly, the GRA25 transcript is expressed in at least three life cycle stages of the parasite: sporozoites, tachyzoites, and bradyzoites (35,(43)(44)(45), and the protein has likewise been readily detected in the tachyzoite and sporozoite stages (44). We have not investigated the function of GRA25 in the bradyzoite or sporozoite stages, but we did observe that GRA25-deficient parasites are able to form cysts in the brains of chronically infected mice (data not shown).…”

Section: Discussionmentioning

confidence: 86%

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: Discussionmentioning

confidence: 86%

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

et al. 2014

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“…Using cluster analysis of transcripts of higher abundance in H. hammondi than in T. gondii VEG sporozoites along with the T. gondii M4 expression data set (39,40), we identified two gene clusters of interest: (i) genes that were more highly expressed in H. hammondi than in T. gondii VEG oocysts that were poorly expressed in all of the life cycle stages in the T. gondii M4 data set ("Hh specific") and (ii) genes that were more highly expressed in H. hammondi than in T. gondii VEG oocysts that were up-regulated during the tachyzoite-to-bradyzoite transition in the T. gondii M4 data set ("Hh-TgCyst-high"). To identify transcripts with these profiles in an unbiased way, the entire data set was analyzed using Pavlidis template matching (PTM) (47) implemented in TM4 using a P value threshold of 1 ϫ 10 Ϫ5 and setting either the "high" values to the maximum of 1 and the "low" values to a minimum of 0.…”

Section: Methodsmentioning

confidence: 99%

“…A custom script (written in Perl and available upon request) was written to generate the CDF file. Raw CEL files for the H. hammondi and T. gondii VEG oocyst hybridizations, as well as those for the complete life cycle hybridizations for T. gondii strain M4 (GEO accession number GSE32427 [39,40]) were analyzed using the Affy package implemented in R statistical software (41), and all data were normalized ("constant" method) and log 2 transformed using "median polishing" (42). To identify genes with significantly different abundances between species, we used Rank Products (43) with a false-discovery rate of 1/100 as implemented in the MeViewer Module of the TM4 microarray software suite (44).…”

Section: Methodsmentioning

confidence: 99%

Hammondia hammondi Harbors Functional Orthologs of the Host-Modulating Effectors GRA15 and ROP16 but Is Distinguished from Toxoplasma gondii by a Unique Transcriptional Profile

et al. 2014

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Toxoplasma gondii and its nearest extant relative, Hammondia hammondi, are phenotypically distinct despite their remarkable similarity in gene content, synteny, and functionality. To begin to identify genetic differences that might drive distinct infection phenotypes of T. gondii and H. hammondi, in the present study we (i) determined whether two known host-interacting proteins, dense granule protein 15 (GRA15) and rhoptry protein 16 (ROP16), were functionally conserved in H. hammondi and (ii) performed the first comparative transcriptional analysis of H. hammondi and T. gondii sporulated oocysts. We found that GRA15 and ROP16 from H. hammondi (HhGRA15 and HhROP16) modulate the host NF-B and STAT6 pathways, respectively, when expressed heterologously in T. gondii. We also found the transcriptomes of H. hammondi and T. gondii to be highly distinct. Consistent with the spontaneous conversion of H. hammondi tachyzoites into bradyzoites both in vitro and in vivo, H. hammondi high-abundance transcripts are enriched for genes that are of greater abundance in T. gondii bradyzoites. We also identified genes that are of high transcript abundance in H. hammondi but are poorly expressed in multiple T. gondii life stages, suggesting that these genes are uniquely expressed in H. hammondi. Taken together, these data confirm the functional conservation of known T. gondii virulence effectors in H. hammondi and point to transcriptional differences as a potential source of the phenotypic differences between these species.

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“…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). Glycomic and proteomic analyses of the cyst wall and the parasites within it would greatly enhance the understanding of bradyzoite biology.…”

Section: Molecular Analysis Of Bradyzoitesmentioning

confidence: 99%

“…Microarray technology has been an excellent tool for characterizing transcriptomic differences between tachyzoites and bradyzoites in tissue culture (144)(145)(146). Studies of the transcriptome have also compared in vitro and in vivo samples with in vitro tachyzoites, in vivo bradyzoites, and feline-derived oocysts, as well as tissue culture tachyzoites and bradyzoites, developing oocysts, and bradyzoites purified from mouse brains 21 days postinfection (144,147). To gain more insight on in vivo development of T. gondii, host microarrays were performed on peritoneum-derived tachyzoites from different strain types of T. gondii from wild-type and IFN-␥-deleted mice (148)(149)(150).…”

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

102

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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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Identification of Tissue Cyst Wall Components by Transcriptome Analysis of In Vivo and In Vitro Toxoplasma gondii Bradyzoites

Cited by 106 publications

References 47 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

Hammondia hammondi Harbors Functional Orthologs of the Host-Modulating Effectors GRA15 and ROP16 but Is Distinguished from Toxoplasma gondii by a Unique Transcriptional Profile

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

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