The in vitro model of tissue cyst formation in Toxoplasma gondii

ABSTRACT Toxoplasma gondii is a protozoan parasite that causes lifelong chronic infection that can reactivate in immunocompromised individuals. Upon infection, the replicative stage (tachyzoite) converts into a latent tissue cyst stage (bradyzoite). Like other apicomplexans, T. gondii possesses an extensive lineage of proteins called ApiAP2s that contain DNA-binding domains first characterized in plants. The function of most ApiAP2s is unknown. We previously found that AP2IX-4 is a cell cycle-regulated ApiAP2 expressed only in dividing parasites as a putative transcriptional repressor. In this study, we purified proteins interacting with AP2IX-4, finding it to be a component of the recently characterized microrchidia (MORC) transcriptional repressor complex. We further analyzed AP2XII-2, another cell cycle-regulated factor that associates with AP2IX-4. We monitored parallel expression of AP2IX-4 and AP2XII-2 proteins in tachyzoites, detecting peak expression during S/M phase. Unlike AP2IX-4, which is dispensable in tachyzoites, loss of AP2XII-2 resulted in a slowed tachyzoite growth due to a delay in S-phase progression. We also found that AP2XII-2 depletion increased the frequency of bradyzoite differentiation in vitro. These results suggest that multiple AP2 factors collaborate to ensure proper cell cycle progression and tissue cyst formation in T. gondii. IMPORTANCE Toxoplasma gondii is a single-celled parasite that persists in its host by converting into a latent cyst stage. This work describes a new transcriptional factor called AP2XII-2 that plays a role in properly maintaining the growth rate of replicating parasites, which contributes to signals required for development into its dormant stage. Without AP2XII-2, Toxoplasma parasites experience a delay in their cell cycle that increases the frequency of latent cyst formation. In addition, we found that AP2XII-2 operates in a multisubunit complex with other AP2 factors and chromatin remodeling machinery that represses gene expression. These findings add to our understanding of how Toxoplasma parasites balance replication and dormancy, revealing novel points of potential therapeutic intervention to disrupt this clinically relevant process.

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Toxoplasma gondii AP2XII-2 contributes to proper progression through S-phase of the cell cycle

10.1101/2020.06.09.143586 ◽

2020 ◽

Author(s):

Sandeep Srivastava ◽

Michael W. White ◽

William J. Sullivan

Keyword(s):

Cell Cycle ◽

Toxoplasma Gondii ◽

Cell Cycle Progression ◽

Transcriptional Repressor ◽

Protozoan Parasite ◽

Cyst Formation ◽

S Phase ◽

Tissue Cyst ◽

Binding Domains

AbstractToxoplasma gondii is a protozoan parasite that causes lifelong chronic infection that can reactivate in immunocompromised individuals. Upon infection, the replicative stage (tachyzoite) converts into a latent tissue cyst stage (bradyzoite). Like other apicomplexans, T. gondii possesses an extensive linage of proteins called ApiAP2s that contain plant-like DNA-binding domains. The function of most ApiAP2s is unknown. We previously found that AP2IX-4 is a cell cycle-regulated ApiAP2 expressed only in dividing parasites as a putative transcriptional repressor. In this study, we purified proteins interacting with AP2IX-4, finding it to be a component of the recently characterized microrchidia (MORC) transcriptional repressor complex. We further analyzed AP2XII-2, another cell cycle-regulated factor that associates with AP2IX-4. We monitored parallel expression of AP2IX-4 and AP2XII-2 proteins in tachyzoites, detecting peak expression during S/M phase. Unlike AP2IX-4, which is dispensable in tachyzoites, loss of AP2XII-2 resulted in a slowed tachyzoite growth due to a delay in S-phase progression. We also found that AP2XII-2 depletion increased the frequency of bradyzoite differentiation in vitro. These results suggest that multiple AP2 factors collaborate to ensure proper cell cycle progression and tissue cyst formation in T. gondii.ImportanceToxoplasma gondii is a single-celled parasite that causes opportunistic infection due to its ability to convert into a latent cyst stage. This work describes a new transcriptional factor called AP2XII-2 that plays a role in properly maintaining the growth rate of replicating parasites, which contributes to signals required for development into its dormant stage. Without AP2XII-2, Toxoplasma parasites experience a delay in their cell cycle that increases the frequency of latent cyst formation. In addition, we found that AP2XII-2 operates in a multi-subunit complex with other AP2 factors and chromatin remodeling machinery that represses gene expression. These findings add to our understanding of how Toxoplasma parasites balance replication and dormancy, revealing novel points of potential therapeutic intervention to disrupt this clinically relevant process.

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Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and impact on neuron biology.

10.1101/2021.03.15.435467 ◽

2021 ◽

Author(s):

Thomas Mouveaux ◽

Emmanuel Roger ◽

Alioune Gueye ◽

Fanny Eysert ◽

Ludovic Huot ◽

...

Keyword(s):

Toxoplasma Gondii ◽

In Vitro Model ◽

Brain Cell ◽

Brain Cells ◽

Specific Response ◽

Cell Infection ◽

Vitro Model ◽

The Brain

Toxoplasma gondii is a eukaryotic parasite that form latent cyst in the brain of immunocompetent individuals. The latent parasites infection of the immune privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons long-term infection are unknown. It has long been known that T. gondii specifically differentiate into a latent form (bradyzoite) in neurons, but how the infected neuron is responding to the infection remain to be elucidated. We have established a new in vitro model resulting in the production of fully mature bradyzoites cysts in brain cells. Using dual, host and parasite, RNA-seq we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal specific pathways are strongly affected, with synapse signaling being particularly affected, especially glutamatergic synapse. The establishment of this new in vitro model allows to investigate both the dynamics of the parasite differentiation and the specific response of neurons to the long term infection by this parasite.

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Bradyzoite Pseudokinase 1 Is Crucial for Efficient Oral Infectivity of the Toxoplasma gondii Tissue Cyst

Eukaryotic Cell ◽

10.1128/ec.00343-12 ◽

2013 ◽

Vol 12 (3) ◽

pp. 399-410 ◽

Cited By ~ 35

Author(s):

Kerry R. Buchholz ◽

Paul W. Bowyer ◽

John C. Boothroyd

Keyword(s):

Toxoplasma Gondii ◽

Acid Treatment ◽

Cyst Formation ◽

Oral Infection ◽

Tissue Cyst ◽

Content Type ◽

Oral Transmission

ABSTRACTThe tissue cyst formed by the bradyzoite stage ofToxoplasma gondiiis essential for persistent infection of the host and oral transmission. Bradyzoite pseudokinase 1 (BPK1) is a component of the cyst wall, but nothing has previously been known about its function. Here, we show that immunoprecipitation of BPK1 fromin vitrobradyzoite cultures, 4 days postinfection, identifies at least four associating proteins: MAG1, MCP4, GRA8, and GRA9. To determine the role of BPK1, a strain ofToxoplasmawas generated with thebpk1locus deleted. This BPK1 knockout strain (Δbpk1) was investigatedin vitroandin vivo. No defect was found in terms ofin vitrocyst formation and no difference in pathogenesis or cyst burden 4 weeks postinfection (wpi) was detected after intraperitoneal (i.p.) infection withΔbpk1tachyzoites, although the Δbpk1cysts were significantly smaller than parental or BPK1-complemented strains at 8 wpi. Pepsin-acid treatment of 4 wpiin vivocysts revealed that Δbpk1parasites are significantly more sensitive to this treatment than the parental and complemented strains. Consistent with this, 4 wpi Δbpk1cysts showed reduced ability to cause oral infection compared to the parental and complemented strains. Together, these data reveal that BPK1 plays a crucial role in thein vivodevelopment and infectivity ofToxoplasmacysts.

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Hammondia hammondihas a developmental programin vitrothat mirrors its stringent two host life cycle

10.1101/148270 ◽

2017 ◽

Author(s):

Sarah L. Sokol ◽

Abby S. Primack ◽

Sethu C. Nair ◽

Zhee S. Wong ◽

Maiwase Tembo ◽

...

Keyword(s):

Life Cycle ◽

Toxoplasma Gondii ◽

Specific Growth ◽

Cyst Formation ◽

Host Cells ◽

Tissue Cyst ◽

Life Stages ◽

New Host ◽

Host Life

AbstractHammondia hammondiis the nearest relative ofToxoplasma gondii,but unlikeT. gondiiis obligately heteroxenous. We have comparedH. hammondiandT. gondiidevelopmentin vitroand identified multipleH. hammondi-specific growth states. Despite replicating slower thanT. gondii,H. hammondiwas resistant to pH-induced tissue cyst formation early after excystation. However, in the absence of stressH. hammondispontaneously converted to a terminally differentiated tissue cyst stage whileT. gondiidid not. CulturedH. hammondicould infect new host cells for up to 8 days following excystation, and this period was exploited to generate stably transgenicH. hammondi. Coupled with RNAseq analyses, our data clearly show thatH. hammondizoites grow as stringently regulated life stages that are fundamentally distinct fromT. gondiitachyzoites and bradyzoites.

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An in vitro model for investigation of vitamin A effects on wound healing

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000692 ◽

2021 ◽

pp. 1-6

Author(s):

Hoda Keshmiri Neghab ◽

Mohammad Hasan Soheilifar ◽

Gholamreza Esmaeeli Djavid

Keyword(s):

Wound Healing ◽

Endothelial Cell ◽

Vitamin A ◽

In Vitro Model ◽

Cellular Proliferation ◽

Endothelial Cell Migration ◽

Normal Fibroblast ◽

Anti Inflammatory ◽

Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inﬂammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inﬂammation responses.

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