Novel Approaches Reveal that Toxoplasma gondii Bradyzoites within Tissue Cysts Are Dynamic and Replicating EntitiesIn Vivo

Abstract

Despite their critical role in chronic toxoplasmosis, the biology of Toxoplasma gondii bradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative “occupancy” was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replication in vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cysts in vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage.

Using metabolomics to dissect host–parasite interactions

“…Loss of CDPK2 expression leads to dysregulation of amylopectin turnover and the accumulation of massively enlarged amylopectin granules, bradyzoite death and absence of chronic infections in mice. Metabolic and post-translational control of amylopectin turnover may thus be required to sustain metabolic homeostasis and to allow bradyzoites to switch to active growth in response to changes in nutrient levels and/or immune mediators in infected tissues or upon reactivation of an acute infection [41 ].…”

“…gondii parasites are also thought to enter into a metabolically quiescent stage when they form large intracellular tissue cysts, each containing hundreds of bradyzoites, in brain, muscle and eye tissues following the development of a protective immune response [41 ]. While little is known about the physiological state and metabolism of encysted bradyzoites, recent studies suggest that individual cysts are physiologically variable [41 ], and that key pathways such as those involved in cyst wall production [48] and the synthesis and turnover of large cytoplasmic amylopectin granules are constitutively active [49]. Interestingly, amylopectin synthesis and turnover is regulated by several enzymes which, in turn, are coordinately regulated by the Ca 2+ -dependent protein kinase-2 (CDPK2) [49].…”

“…This strategy is utilized by T. gondii bradyzoites [41 ], Plasmodium vivax hypnozoites [42] and Leishmania amastigotes [43], which reside within vacuolar compartments in neuronal and muscle cells, liver cells, and different phagocytic cells, respectively. It is often difficult (or impossible) to generate physiologically equivalent parasite stages in culture and analysis of the metabolism of these stages is technically challenging.…”

Neurophysiological Changes Induced by Chronic <em>Toxoplasma gondii</em> Infection

“…3-D imaging analysis found cysts predominantly in neuronal processes such as dendritic spines rather than soma [61]. The formation of these, up to 70μm in diameter and increasing in size as infection progresses, may reduce neuronal functionality [6]. Indeed, dendritic spine length and density of infected mice was significantly reduced compared to uninfected control animals [62,63].…”

“…The bradyzoites become encysted, forming the chronic stage of infection which can persist for the lifetime of the host. Historically considered a latent stage and only associated with pathogenesis in the immunocompromised host, recent data has demonstrated that bradyzoites continue to replicate [6,7]. However, as the host-parasite interactions are delineated the chronic stages have become more clinically significant [8].…”