Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

Radke, Joshua B.; Worth, Danielle; Hong, David D.; Huang, Sherri; Sullivan, William J.; Wilson, Emma H.; White, Michael

doi:10.1371/journal.ppat.1007035

Cited by 88 publications

(71 citation statements)

References 63 publications

(130 reference statements)

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“…Neurons are the primary target cells for Toxoplasma cyst formation within distal neuronal processes (40,41). Bradyzoite differentiation is triggered by changes in nutrition or cellular conditions (42)(43)(44), which drives the transcriptional program that leads to the production of bradyzoite-stage proteins (45,46) and the formation of the cyst wall and cyst structure (32). The cyst wall is a protective structure that safely encloses bradyzoites until their transmission to a new host after oral ingestion of cysts (14).…”

Section: Discussionmentioning

confidence: 99%

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

Guevara

Fox

Bzik

2020

mSphere

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The glycosylated mucin domain of the Toxoplasma gondii cyst wall glycoprotein CST1 is heavily stained by Dolichos biflorus agglutinin, a lectin that binds to N-acetylgalactosamine. The cyst wall is also heavily stained by the chitin binding lectin succinylated wheat germ agglutinin (s-WGA), which selectively binds to N-acetylglucosamine-decorated structures. Here, we tracked the localization of N-acetylglucosamine-decorated structures that bind to s-WGA in immature and mature in vitro cysts. s-WGA localization was observed at the cyst periphery 6 h after the differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 and at all later times after differentiation, s-WGA was localized in a continuous staining pattern at the cyst wall. Coinciding with the maturation of the cyst matrix by day 3 of cyst development, s-WGA also localized in a continuous matrix pattern inside the cyst. s-WGA localized in both the outer and inner layer regions of the cyst wall and in a continuous matrix pattern inside mature 7-and 10-day-old cysts. In addition, s-WGA colocalized in the cyst wall with CST1, suggesting that N-acetylglucosamineand N-acetylgalactosamine-decorated molecules colocalized in the cyst wall. In contrast to CST1, GRA4, and GRA6, the relative accumulation of the molecules that bind s-WGA in the cyst wall was not dependent on the expression of GRA2. Our results suggest that GRA2-dependent and GRA2-independent mechanisms regulate the trafficking and accumulation of glycosylated molecules that colocalize in the cyst wall. IMPORTANCE Chronic Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N-acetylglucosamine-decorated structures. N-Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall.

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Section: Discussionmentioning

confidence: 99%

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

Guevara

Fox

Bzik

2020

mSphere

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“…Apicomplexan genomes encode a family of putative transcription factors that are characterized by the possession of one or more DNA binding AP2 domains [16]. ApiAP2 transcription factors were shown to control expression profiles during T. gondii differentiation [17][18][19]. ApiAP2s were also shown to cooperatively control the expression of virulence factors [20,21] in T. gondii indicating that this family of transcription factors may control cell cycle dependent expression profiles as also suggested for P. berghei [22].…”

Section: Introductionmentioning

confidence: 67%

“…Interestingly, much like other genes directly regulated by TgAP2IX-5 ( Figure 4E), these genes showed an expression peak during the late S phase ( Figure S9B). TgAP2IV-4, a known repressor of developmentally regulated genes, is expressed during the S/M phase [18]. These data suggest that TgAP2IX-5 directly activates other TF during the S phase that may in turn control the late S and M expression program.…”

Section: Tgap2ix-5 Impacts the Expression Of Cell-cycle Regulated Genesmentioning

confidence: 77%

“…These TF may be responsible for the expression of the transcripts that were identified as downregulated in RNA-seq and that peak during the late S and M phase. Although no data is available for TgAP2III-2, TgAP2XII-2 and TgAP2XII-9, TgAP2IV-4 was shown to be a repressor of the bradyzoite differentiation expression program [18]. The control of the expression of a repressor of differentiation by a TF that also controls the continuation of cell cycle may provide the missing link between cell cycle and differentiation.…”

Section: Discussionmentioning

confidence: 99%

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A single master regulator controls asexual cell cycle division patterns inToxoplasma gondii

Khelifa

Guillen

Lesage

et al. 2020

Preprint

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All apicomplexan parasites have complex life cycles exhibiting division characterized by a tightly regulated cell cycle control, resulting in the emergence of daughter parasites in possession of a single nucleus and a complete set of organelles. Apicomplexa have evolved efficient and distinctive strategies for intracellular replication where the timing of emergence of the daughter cells, a process termed "budding", is a decisive element. However, the molecular mechanisms that provide the proper timing of parasite budding remain unknown.Using Toxoplasma gondii as a model Apicomplexa, we identified a master regulator that controls the timing of the budding process. We show that an ApiAP2 transcription factor, TgAP2IX-5, controls cell cycle events downstream of centrosome duplication including organelle division and segregation. TgAP2IX-5 binds to the promoter of hundreds of genes and controls the activation of the budding-specific cell cycle expression program. We show that TgAP2IX-5 regulates the expression of specific transcription factors that are necessary for the completion of the budding cycle. TgAP2IX-5 acts as a licensing factor that ensures that asexual proliferation continues by promoting the inhibition of the differentiation pathway at each round of the cell cycle. Therefore, TgAP2IX-5 is a master regulator that controls both cell cycle and developmental pathways.

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“…Other studies have implicated AP2s in regulating a developmental transition 16 . Radke et al 17 recently characterized a T. gondii AP2 and showed that this molecule acts as a repressor of bradyzoite development. Our results showed that egress assay was partially affected by parasite proliferation (Fig 4B).…”

Section: Discussionmentioning

confidence: 99%

Gene silencing in Cryptosporidium: A rapid approach to identify novel targets for drug development

Castellanos-González

Martinez-Traverso

et al. 2019

Preprint

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24 25 26 27 2 28Abstract: 29 Background: Cryptosporidiosis is a major cause of diarrheal disease. However, the only drug 30 approved for cryptosporidiosis does not work well in high risk populations. Therefore, novel 31 drugs are urgently needed. Then, the identification of novel is necessary to develop new 32 therapies against this parasite. Recently, we have developed a rapid method to block gene 33 expression in Cryptosporidium by using pre-assembled complexes of Cryptosporidium 34 antisense RNA and human protein with slicer activity (Argonaute 2). We hypothesized that 35 structural proteins, proteases, enzymes nucleotide synthesis and transcription factors are 36 essential for parasite development, thus in this work we knock down expression of 4 selected 37 genes: Actin, Apicomplexan DNA-binding protein (AP2), Rhomboid protein 1 (Rom 1) and 38 nucleoside diphosphate kinase (NDK) and elucidated its role during invasion, proliferation and 39 egress of Cryptosporidium. 40 41 Methods: We used protein transfection reagents (PTR) to introduce pre-assembled complexes 42 of antisense RNA and human Argonaute 2 into Cryptosporidium parvum oocysts, the complexes 43 blocked expression of Actin (Act), Transcription factor AP2 (AP2), nucleoside diphosphate 44 kinase (DKN), and rhomboid protein 1 (Rom1). After gene silencing, we evaluated parasite 45 reduction using In vitro models of excystation, invasion, proliferation and egress. We evaluated 46 the potency of ellagic acid, a nucleoside diphosphate kinase inhibitor for anti-cryptosporidial 47 activity using a model of in vitro infection with human HCT-8 cells. 48 49 Results: Silencing of Act, AP2, NDK and Rom1 reduce significantly invasion, proliferation and 50 egress of Cryptosporidium. We showed that silencing of NDK markedly inhibited 51 Cryptosporidium proliferation. This was confirmed by demonstration that ellagic acid reduced 52 the number of parasites at micro molar concentrations (EC 50 =15-30 µM) without showing any 53 toxic effect on human cells. 54 55 Conclusions: Overall the results confirmed the usefulness RNA silencing can be used to 56 identify novel targets for drug development against Cryptosporidium. We identified ellagic acid 57 (EA), a nucleoside diphosphate kinase inhibitor also blocks Cryptosporidium proliferation. Since 58 EA is a dietary supplement approved for human use, then this compound should be studied as 59 a potential treatment for cryptosporidiosis. 3 60 Author summary 61 The World Health Organization reports diarrhea kills around 760,000 children under five every 62 year. Cryptosporidium infection is a leading cause of diarrhea morbidity and mortality. Current 63 therapies to treat this infection are suboptimal, therefore novel treatments are urgently needed. 64 We used genetic tools to identify novel targets for drug development, thus in this work we 65 evaluated the role of 4 genes during Cryptosporidium infection. We demonstrated that silencing 66 of nucleoside-diphosphate kinase (NDK) drastically reduced invasion, proliferation and e...

show abstract

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

Cited by 88 publications

References 63 publications

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

A single master regulator controls asexual cell cycle division patterns inToxoplasma gondii

Gene silencing in Cryptosporidium: A rapid approach to identify novel targets for drug development

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