Abstract
Toxoplasma gondii has evolved different developmental stages for disseminating during acute infection (i.e., tachyzoites) and establishing chronic infection (i.e., bradyzoites). Calcium ion (Ca2+) signaling tightly regulates the lytic cycle of tachyzoites by controlling microneme secretion and motility to drive egress and cell invasion. However, the roles of Ca2+ signaling pathways in bradyzoites remain largely unexplored. Here, we show that Ca2+ responses are highly restricted in bradyzoites and that they fail to egress in response to agonists. Development of dual-reporter parasites revealed dampened Ca2+ responses and minimal microneme secretion by bradyzoites induced in vitro or harvested from infected mice and tested ex vivo. Ratiometric Ca2+ imaging demonstrated lower Ca2+ basal levels, reduced magnitude, and slower Ca2+ kinetics in bradyzoites compared with tachyzoites stimulated with agonists. Diminished responses in bradyzoites were associated with downregulation of Ca2+-ATPases involved in intracellular Ca2+ storage in the endoplasmic reticulum (ER) and acidocalcisomes. Once liberated from cysts by trypsin digestion, bradyzoites incubated in glucose plus Ca2+ rapidly restored their intracellular Ca2+ and ATP stores, leading to enhanced gliding. Collectively, our findings indicate that intracellular bradyzoites exhibit dampened Ca2+ signaling and lower energy levels that restrict egress, and yet upon release they rapidly respond to changes in the environment to regain motility.
Highlights
Toxoplasma gondii is an obligate intracellular parasite, capable of infecting most warm-blooded animals and frequently causing human infections [1]
Bradyzoites secreted 143 much less MIC2-Gaussia Luciferase (Gluc) protein compared to tachyzoites in response to Ca2+ agonists, zaprinast and ionomycin as shown by Gaussia luciferase assays performed on excretory-secretory antigens (ESA) fractions collected following stimulation (Figure 145 2D)
We observed significant reduction in the relative expression level of TgSERCA, TgA1, TgPMCA1, TgA2, Ca2+/H+ exchanger and TgTRPPL-2 in bradyzoites compared to tachyzoites (Figure 6G). These findings indicate that bradyzoites have lower levels of stored Ca2+, which is associated with the overall downregulation of Ca2+-related pumps and channels. 288 Calcium signaling plays a critical role in gliding motility of bradyzoites To test whether dampened Ca2+ signaling would still be sufficient to drive gliding motility of bradyzoites, we treated BAG1-mCherry GCaMP6f expressing cysts cultured in vitro with trypsin to liberate bradyzoites (Figure 7A)
Summary
Toxoplasma gondii is an obligate intracellular parasite, capable of infecting most warm-blooded animals and frequently causing human infections [1]. The ingestion of tissue cysts in undercooked meat or shed oocysts by infected cats are the major transmission routes of T. gondii [2,3]. Following oral ingestion of bradyzoites within tissue cysts, or sporozoites within oocysts, the parasite migrates across the intestinal epithelial barrier and disseminates throughout the body as the actively proliferating tachyzoite form that infects many cell types but primarily traffics in monocytes [4]. In response to immune pressure, the parasite differentiates to asynchronously growing bradyzoites within cysts that can persist as chronic infections in muscle and brain tissues [5,6,7]. Tachyzoites are adapted for rapid proliferation and dissemination due to an active lytic cycle that is controlled at numerous stages by intracellular calcium ion (Ca2+) signaling [8].
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