Factors Influencing Tissue Cyst Yield in a Murine Model of Chronic Toxoplasmosis

Troublefield, Cortni A.; Miracle, Joy S.; Murphy, Ray; Donkin, Ryan; Sinai, Anthony P.

doi:10.1128/iai.00566-22

Cited by 2 publications

(5 citation statements)

References 66 publications

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“…The capacity of ∆TgLaf cysts to initiate new infections was assessed in vivo. Consistent with other studies (7,68), infection with tissue cysts is associated with limited symptomology and low overall mortality. Similar to what was observed with tachyzoite-initiated infections, the ∆TgLaf parasites consistently presented with lower cyst burdens relative to WT cyst infections.…”

Section: Despite Glucan Metabolism Being Historically Viewed As Being...supporting

confidence: 90%

“…To determine if the loss of TgLaforin impacted the overall viability/infectivity of in vivo tissue cysts, we examined the disease progression in WT, ∆TgLaf, and COMP infected animals following injection of 20 tissue cysts i.p. Consistent with prior data, infection with tissue cysts results in markedly lower pathology and consequent mortality during the acute phase for WT as well as both the ∆TgLaf and COMP lines (Figure 7A, B) (68). The death rate from cyst infection did not differ statistically among the three lines.…”

Section: ∆Tglaf Tissue Cysts Can Reestablish Infections In Naïve Micesupporting

confidence: 90%

“…To test this hypothesis, equal numbers of male and female CBA/J mice were infected with 100 tachyzoites intraperitoneally (i.p.) and monitored daily using a previously developed five-stage body index score to track the severity of symptoms associated with a tachyzoite infection over the course of 28 days (68).…”

Section: Loss Of Tglaforin Results In Attenuated Virulence and Cyst F...mentioning

confidence: 99%

“…Type II ME49∆HXGPRT ("WT"-the parental line utilized to generate all other lines in this study): This line was generated in a previous study using CRISPR/Cas9 targeting of TgHXGPRT and selection with 6-Tx (68).…”

Section: Generation Of T Gondii Mutant Linesmentioning

confidence: 99%

“…Mice were then monitored and assigned a body index score daily. Monitoring frequency increased to twice a day once symptomatic throughout the course of infection as previously described (68). When symptomatic, mice were administered a gel diet and wet chow on the cage floor and given 0.25-0.5 mL saline solution subcutaneously as needed.…”

Section: Mouse Infectionmentioning

confidence: 99%

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TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides inToxoplasma gondiitachyzoites and bradyzoites

Murphy,

Troublefield,

Miracle

et al. 2023

Preprint

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The asexual stages ofToxoplasma gondiiare defined by the rapidly growing tachyzoite during the acute infection and by the slow growing bradyzoite housed within tissue cysts during the chronic infection. These stages represent unique physiological states, each with distinct glucans reflecting differing metabolic needs. A defining feature ofT. gondiibradyzoites is the presence of insoluble storage glucans known as amylopectin granules (AGs) that are believed to play a role in reactivation, but their functions during the chronic infection remain largely unexplored. More recently, the presence of storage glucans has been recognized in tachyzoites where their precise function and architecture have yet to be fully defined. Importantly, theT. gondiigenome encodes activities needed for glucan turnover: a glucan phosphatase (TgLaforin; TGME49_205290) and a glucan kinase (TgGWD; TGME49_214260) that catalyze a cycle of reversible glucan phosphorylation required for glucan degradation by amylases. The expression of these enzymes in tachyzoites supports the existence of a storage glucan, evidence that is corroborated by specific labeling with the anti-glycogen antibody IV58B6. Disruption of reversible glucan phosphorylation via a CRISPR/Cas9 knockout (KO) of TgLaforin revealed no growth defects under nutrient-replete conditions in tachyzoites. However, the growth of TgLaforin-KO tachyzoites was severely stunted when starved of glutamine, even under glucose replete conditions. The loss of TgLaforin also resulted in the attenuation of acute virulence in mice accompanied by a lower cyst burden. Defective cyst formation due to profound changes in AG morphology was also observed in TgLaforin-KO parasites, bothin vitroandin vivo. Together, these data demonstrate the importance of glucan turnover across theT. gondiiasexual cycle. These findings, alongside our previously identified class of small molecules that inhibit TgLaforin, implicate reversible glucan phosphorylation as a legitimate target for the development of new drugs against chronicT. gondiiinfections.

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Section: Despite Glucan Metabolism Being Historically Viewed As Being...supporting

confidence: 90%

Section: ∆Tglaf Tissue Cysts Can Reestablish Infections In Naïve Micesupporting

confidence: 90%

Section: Loss Of Tglaforin Results In Attenuated Virulence and Cyst F...mentioning

confidence: 99%

Section: Generation Of T Gondii Mutant Linesmentioning

confidence: 99%

Section: Mouse Infectionmentioning

confidence: 99%

See 3 more Smart Citations

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides inToxoplasma gondiitachyzoites and bradyzoites

Murphy,

Troublefield,

Miracle

et al. 2023

Preprint

Self Cite

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Dynamics of amylopectin granule accumulation during the course of the chronicToxoplasmainfection is linked to intra-cyst bradyzoite replication

Tripathi,

Donkin,

Miracle

et al. 2024

Preprint

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The contribution of amylopectin granules (AG), comprised of a branched chain storage homopolymer of glucose, to the maintenance and progression of the chronic Toxoplasma gondii infection has remained undefined. Here we describe the role of AG in the physiology of encysted bradyzoites by using a custom developed imaging-based application AmyloQuant that permitted quantification of relative levels of AG within in vivo derived tissue cysts during the initiation and maturation of the chronic infection. Our findings establish that AG are dynamic entities, exhibiting considerable heterogeneity among tissue cysts at all post infection time points examined. Quantification of relative AG levels within tissue cysts exposes a previously unrecognized temporal cycle defined by distinct phases of AG accumulation and utilization over the first 6 weeks of the chronic phase. This AG cycle is temporally coordinated with overall bradyzoite mitochondrial activity implicating amylopectin in the maintenance and progression of the chronic infection. In addition, the staging of AG accumulation and its rapid utilization within encysted bradyzoites was associated with a burst of coordinated replication. As such our findings suggest that AG levels within individual bradyzoites, and across bradyzoites within tissue cysts may represent a key component in the licensing of bradyzoite replication, intimately linking stored metabolic potential to the course of the chronic infection. This extends the impact of AG beyond the previously assigned role that focused exclusively on parasite transmission. These findings force a fundamental reassessment of the chronic Toxoplasma infection, highlighting the critical need to address the temporal progression of this crucial stage in the parasite life cycle.

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Factors Influencing Tissue Cyst Yield in a Murine Model of Chronic Toxoplasmosis

Cited by 2 publications

References 66 publications

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides inToxoplasma gondiitachyzoites and bradyzoites

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides inToxoplasma gondiitachyzoites and bradyzoites

Dynamics of amylopectin granule accumulation during the course of the chronicToxoplasmainfection is linked to intra-cyst bradyzoite replication

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