Biology subjects

Miracle, J. S.

Publications and source records attributed to Miracle, J. S..

3 recordsLinked to original sources

bioRxiv2024

Dynamics of amylopectin granule accumulation during the course of the chronic Toxoplasma infection is linked to intra-cyst bradyzoite replication.

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.

microbiology↗

bioRxiv2023

TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides in Toxoplasma gondii tachyzoites and bradyzoites

The asexual stages of Toxoplasma gondii are 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 of T. gondii bradyzoites is the presence of insoluble storage glucans known as amylopectin granules (AGs), the function of which remains largely unexplored during the chronic infection. The presence of storage glucans has more recently been established in tachyzoites, a finding corroborated by specific labeling with the anti-glycogen antibody IV58B6. The T. gondii genome encodes activities needed for glucan turnover inlcuding: 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. Disruption of TgLaforin in tachyzoites had no impact on growth under nutrient-replete conditions. Growth of TgLaforin-KO tachyzoites was however severely stunted when starved of glutamine despite being glucose replete. Loss of TgLaforin attenuated acute virulence in mice and was accompanied by a lower tissue cyst burden, without a direct impact on tissue cyst size. Quantification of relative AG levels using AmyloQuant, an imaging based application, revealed the starch-excess phenotype associated with the loss of TgLaforin is heterogeneous and linked to an emerging AG cycle in bradyzoites. Excessive AG accumulation TgLaforin-KO bradyzoites promoted intra-cyst bradyzoite death implicating reversible glucan phosphorylation as a legitimate target for the development of new drugs against chronic T. gondii infections. ImportanceStorage of glucose is associated with a projected need for future metabolic potential. Accumulation of glucose in insoluble amylopectin granules (AG) is associated with encysted forms of Toxoplasma gondii. AG which are not observed in rapidly growing tachyzoites do appear to possess glycogen, a soluble storage glucan. Here we address the role of reversible glucan phosphorylation by targeting TgLaforin, a glucan phosphatase and key component of reversible glucan phosphorylation controlling AG and glycogen turnover. Loss of TgLaforin fundamentally alters tachyzoite metabolism making them dependent on glutamine. These changes directly impact acute virulence resulting in lowering tissue cyst yields. The effects of the loss of TgLaforin on AG levels in encysted bradyzoites is heterogenous, manifesting non-uniformly with the progression of the chronic infection. With the loss of TgLaforin culminating with the death of encysted bradyzoites, AG metabolism presents a potential target for therapeutic intervention, the need for which is acute.

microbiology↗

bioRxiv2022

Factors influencing tissue cyst yield in a murine model of chronic toxoplasmosis

Recent advances into the unique biology of Toxoplasma tissue cysts and the bradyzoites they house necessitates optimization of tissue cyst recovery from infected mouse brains. Here, we present data from 68 tissue cyst purifications of Type II ME49 tissue cysts in CBA/J mice performed over a period of two years. The effects if infecting with both tissue culture tachyzoites as well as ex vivo tissue cysts were assessed. Significant mortality was restricted to tachyzoite infections with female mice being more susceptible. Infection with tissue cysts was associated with both lower overall symptomology and mortality exhibiting no sex bias. Cumulatively, host sex did not impact overall tissue cyst yields, although, tachyzoite initiated infections generated significantly higher yields compared to tissue cyst-initiated infections. Notably, serial passage of tissue cysts was accompanied with a decreasing trend for subsequent cyst recovery. The time of tissue cyst harvest, a potential reflection of bradyzoite physiological state, had no significant impact on subsequent cyst yield at the selected time points. In aggregate, the data reveal the considerable heterogeneity associated with tissue cyst yield making the design of adequately powered experiments critical. This is particularly the case for drug studies where overall tissue cyst burden currently serves as the primary and often sole metric of efficacy, as the data presented here demonstrate that cyst recovery between preparations of untreated animals can mirror the reported effects of drug treatment.

microbiology↗