TgLaforin, a glucan phosphatase, reveals the dynamic role of storage polysaccharides inToxoplasma gondiitachyzoites and bradyzoites
Authors: Murphy Robert D. , Troublefield Cortni A. , Miracle Joy S. , Young Lyndsay E.A. , Tripathi Aashutosh , Brizzee Corey O. , Dhara Animesh , Patwardhan Abhijit , Sun Ramon C. , Vander Kooi Craig W. , Gentry Matthew S. , Sinai Anthony P. 
Published: September 2023

ABSTRACTThe 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), 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. TheT. gondiigenome 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 chronicT. gondiiinfections.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 ofToxoplasma 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.