scholarly journals Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

2017 ◽  
Author(s):  
Joshua B. Radke ◽  
Danielle Worth ◽  
Dong-Pyo Hong ◽  
Sherri Huang ◽  
William J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Repression ◽  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Cyst Formation ◽  
Tissue Cyst ◽  
Genetic Lineages ◽  
Inflammatory Monocytes ◽  
Bradyzoite Differentiation

AbstractBradyzoite differentiation is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an importantToxoplasmatranscriptional repressor mechanism controlling bradyzoite differentiation that operates exclusively in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the increased expression of bradyzoite mRNAs in replicating tachyzoites, and in two different genetic lineages we confirmed the misexpression of tissue cyst wall components (e.g. BPK1, MCP4, CST1) and the bradyzoite surface antigen SRS9 in the tachyzoite stage. In the murine animal model, the loss of AP2IV-4 had profound biological consequences. Type II prugniaud strain parasites lacking AP2IV-4 were unable to form tissue cysts in brain tissue and the absence of this factor also recruited a potent immune response characterized by increases inflammatory monocytes, IFN-γ and higher numbers of both CD8+ and CD4+ T-cells. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required forToxoplasmato establish a chronic infection in the immune-competent host.Author SummaryTheToxoplasmabiology that underlies the establishment of a chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite-tissue cyst stage. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control formation of the tissue cyst is still poorly understood. A fundamental feature of tissue cyst formation is the expression of bradyzoite-specific genes. Here we show the transcription factor AP2IV-4 directly silences bradyzoite mRNA and protein expression in the acute tachyzoite stage demonstrating that developmental control of tissue cyst formation is as much about when not to express bradyzoite genes as it is about when to activate them. Loosing the suppression of bradyzoite gene expression in the acute tachyzoite stage caused by deleting AP2IV-4 blocked the establishment of chronic disease in healthy animals through the pre-arming of the immune system suggesting a possible strategy for preventing chronicToxoplasmainfections.

scholarly journals Toxoplasma gondii AP2IX-4 Regulates Gene Expression during Bradyzoite Development

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
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.

scholarly journals Targeted Disruption of the GRA2 Locus inToxoplasma gondii Decreases Acute Virulence in Mice

1998 ◽  
Vol 66 (9) ◽  
pp. 4176-4182 ◽  
Author(s):  
Corinne Mercier ◽  
Daniel K. Howe ◽  
Dana Mordue ◽  
Maren Lingnau ◽  
L. David Sibley
Keyword(s):  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Early Death ◽  
Dense Granule ◽  
Positive Serology ◽  
Targeted Disruption ◽  
The Brain

ABSTRACT Following invasion into the host cell, the protozoanToxoplasma gondii secretes a variety of proteins that modify the parasitophorous vacuole. Within the vacuole, the 28-kDa dense granule protein known as GRA2 is specifically targeted to the tubulovesicular network which forms connections with the vacuolar membrane. To investigate the importance of GRA2, we derived from strain RH a mutant T. gondii line in which GRA2 was disrupted by replacement with the marker Ble (selecting for phleomycin resistance). The Δgra2 mutant invaded and grew normally in both fibroblasts and macrophages in vitro; however, it was less virulent during acute infection in mice. The survival rate of mice inoculated with Δgra2 was significantly higher; some infected mice survived the acute infection, whereas all mice infected with the wild-type strain RH succumbed to early death. Chronic infection by Δgra2 was detected by positive serology, immunohistochemical detection of parasites and cysts in the brain, and reisolation of parasites by bioassay at 6 weeks postinfection. Thus, absence of GRA2 partially attenuates the virulence of T. gondii during the acute phase of infection and allows for establishment of chronic infection by the otherwise highly virulent RH strain. These results establish that GRA2 plays an important role during in vivo infection and provide a potential model for examining acute pathogenesis by T. gondii.

scholarly journals The Toxoplasma gondii Rhoptry Kinome Is Essential for Chronic Infection

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Barbara A. Fox ◽  
Leah M. Rommereim ◽  
Rebekah B. Guevara ◽  
Alejandra Falla ◽  
Miryam Andrea Hortua Triana ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Toxoplasma Gondii ◽  
Virulence Factors ◽  
Chronic Infection ◽  
Parasitophorous Vacuole ◽  
Acute Infection ◽  
Host Cells ◽  
Type Ii ◽  
Host Manipulation ◽  
Parasite Survival

ABSTRACT    Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the Δ rop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5 , ROP17 , ROP18 , ROP35 , or ROP38 / 29 / 19 ( ROP38 , ROP29 , and ROP19 ) severely reduced cyst burdens. Δ rop5 and Δ rop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Δ rop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δ rop35 or Δ rop38 / 29 / 19 strain. Resistance to host IRGs was not affected in the Δ rop17 , Δ rop35 , or Δ rop38 / 29 / 19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondii . IMPORTANCE Reactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections.

scholarly journals Nonreplicating, Cyst-Defective Type II Toxoplasma gondii Vaccine Strains Stimulate Protective Immunity against Acute and Chronic Infection

2015 ◽  
Vol 83 (5) ◽  
pp. 2148-2155 ◽  
Author(s):  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Chronic Infection ◽  
Acute Infection ◽  
Cyst Formation ◽  
Type I ◽  
Type Ii ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Monophosphate Decarboxylase ◽  
Uracil Auxotroph

Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection byToxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80genetic background by targeting the deletion of the orotidine 5′-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion ofOMPDCinduced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80Δompdcmutants stimulated a fully protective CD8+T cell-dependent immunity that prevented acute infection by type I and type II strains ofT. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdcmutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine.

scholarly journals Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ–mediated host defenses

2016 ◽  
Vol 213 (9) ◽  
pp. 1779-1798 ◽  
Author(s):  
Gabrielle Gay ◽  
Laurence Braun ◽  
Marie-Pierre Brenier-Pinchart ◽  
Julien Vollaire ◽  
Véronique Josserand ◽  
...  
Keyword(s):  
Gene Expression ◽  
Toxoplasma Gondii ◽  
Acute Infection ◽  
Protective Role ◽  
Immune Gene ◽  
Nucleosome Remodeling ◽  
Inflammatory Monocytes ◽  
Ifn Γ ◽  
Immune Pathology ◽  
Toxoplasma Gondii Infection

An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii–targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ–stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1+ inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ–mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism.

scholarly journals Toxoplasma gondiiAP2IX-4 regulates gene expression during bradyzoite development

2017 ◽  
Author(s):  
Sherri Huang ◽  
Michael J. Holmes ◽  
Joshua B. Radke ◽  
Dong-Pyo Hong ◽  
Ting-Kai Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Animal Health ◽  
Protozoan Parasite ◽  
Cyst Formation ◽  
Tissue Cyst ◽  
Alkaline Media ◽  
Alkaline Stress ◽  
Stress Induction ◽  
Reduced Frequency ◽  
Current Therapies

ABSTRACTToxoplasma gondiiis a protozoan parasite of great importance to human and animal health. In the host, this obligate intracellular parasite persists as a tissue cyst form that is invisible 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 immune compromised patients. Transcriptional changes accompany conversion of the rapidly replicating tachyzoites into the encysted bradyzoites, 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 many bradyzoite mRNAs dramatically increased beyond 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 also 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.

scholarly journals Opposing transcriptional mechanisms regulateToxoplasmadevelopment

2016 ◽  
Author(s):  
Dong-Pyo Hong ◽  
Joshua B. Radke ◽  
Michael W. White
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cyst Formation ◽  
Tissue Cyst ◽  
Luciferase Reporter ◽  
Alkaline Stress ◽  
Developmental Pathway ◽  
Reciprocal Effect ◽  
Gene Target ◽  
Term Survival

ABSTRACTTheToxoplasmabiology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the role of two alkaline-stress induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased tissue cyst formation demonstrating these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified of AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker, BAG1, and mechanistic experiments determined that like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specific binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest the AP2IX-9 transcriptional repressor and AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permitToxoplasmato better adapt to different tissue environments and select a suitable host cell for long term survival of the dormant tissue cyst.IMPORTANCEToxoplasmainfections are life-long due to the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control formation of the tissue cyst is still poorly understood. Significant changes in gene expression are associated with tissue cyst development and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 mechanisms are not well defined and the operating principles of ApiAP2 mechanisms are poorly understood. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.

scholarly journals Opposing Transcriptional Mechanisms Regulate Toxoplasma Development

mSphere ◽  
2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Dong-Pyo Hong ◽  
Joshua B. Radke ◽  
Michael W. White
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Immune System ◽  
Molecular Basis ◽  
Cyst Formation ◽  
Molecular Composition ◽  
Tissue Cyst ◽  
Developmental Pathway ◽  
Clinical Consequences ◽  
Cyst Development

ABSTRACT Toxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway. The Toxoplasma biology that underlies human chronic infection is developmental conversion of the acute tachyzoite stage into the latent bradyzoite stage. We investigated the roles of two alkaline-stress-induced ApiAP2 transcription factors, AP2IV-3 and AP2IX-9, in bradyzoite development. These factors were expressed in two overlapping waves during bradyzoite development, with AP2IX-9 increasing expression earlier than AP2IV-3, which peaked as AP2IX-9 expression was declining. Disruption of the AP2IX-9 gene enhanced, while deletion of AP2IV-3 gene decreased, tissue cyst formation, demonstrating that these factors have opposite functions in bradyzoite development. Conversely, conditional overexpression of FKBP-modified AP2IX-9 or AP2IV-3 with the small molecule Shield 1 had a reciprocal effect on tissue cyst formation, confirming the conclusions of the knockout experiments. The AP2IX-9 repressor and AP2IV-3 activator tissue cyst phenotypes were borne out in gene expression studies that determined that many of the same bradyzoite genes were regulated in an opposite manner by these transcription factors. A common gene target was the canonical bradyzoite marker BAG1, and mechanistic experiments determined that, like AP2IX-9, AP2IV-3 regulates a BAG1 promoter-luciferase reporter and specifically binds the BAG1 promoter in parasite chromatin. Altogether, these results suggest that the AP2IX-9 transcriptional repressor and the AP2IV-3 transcriptional activator likely compete to control bradyzoite gene expression, which may permit Toxoplasma to better adapt to different tissue environments and select a suitable host cell for long-term survival of the dormant tissue cyst. IMPORTANCE Toxoplasma infections are lifelong because of the development of the bradyzoite tissue cyst, which is effectively invisible to the immune system. Despite the important clinical consequences of this developmental pathway, the molecular basis of the switch mechanisms that control tissue cyst formation is still poorly understood. Significant changes in gene expression are associated with tissue cyst development, and ApiAP2 transcription factors are an important mechanism regulating this developmental transcriptome. However, the molecular composition of these ApiAP2 complexes and the operating principles of ApiAP2 mechanisms are not well defined. Here we establish that competing ApiAP2 transcriptional mechanisms operate to regulate this clinically important developmental pathway.

scholarly journals Repression of ESR1 through Actions of Estrogen Receptor Alpha and Sin3A at the Proximal Promoter

2009 ◽  
Vol 29 (18) ◽  
pp. 4949-4958 ◽  
Author(s):  
Stephanie J. Ellison-Zelski ◽  
Natalia M. Solodin ◽  
Elaine T. Alarid
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Estrogen Receptor Alpha ◽  
Proximal Promoter ◽  
Receptor Alpha ◽  
Expression Of Genes ◽  
Activation Of Transcription

ABSTRACT Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ERα has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ERα represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ERα gene, ESR1. Upon estrogen treatment, ERα is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ERα-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ERα is identified. These data support a model of repression wherein actions of ERα and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression.

Sign in / Sign up

Export Citation Format

Share Document