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

Abstract

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.