Date of Award
Doctor of Philosophy (PhD)
Toxoplasma gondii is an obligate intracellular parasite that can infect a wide range of mammalian hosts. T. gondii can cause severe disease in immunocompromised individuals as well as pregnant women, in which congenital toxoplasmosis can lead to spontaneous abortion and fetal blindness. T. gondii infects up to roughly one-third of the global human population and is the causative agent of toxoplasmosis. Given this health burden of toxoplasmosis and that antibiotics currently used for the treatment of infection lead to strong side effects, identification of parasite-specific targets are necessary for novel therapeutic strategies.
T. gondii parasites contain a dynamic and active endolysosomal system, including the lysosome-like vacuolar compartment/plant-like vacuole (VAC/PLV), which plays an important role in maintaining intracellular propagation and parasite virulence. The VAC has been shown to be important during both the acute and chronic stages of infection, for the digestion of endocytosed host materials and the turnover of autophagosomes, respectively. In addition, the VAC contributes to the proteolytic maturation of invasion effector proteins, which are critical for completion of the parasite lytic cycle (attachment, invasion, replication, and egress). So far, it has been reported that the VAC houses acidic hydrolases along with an ortholog of the malarial chloroquine resistance transporter (TgCRT).
Herein, we investigated the native role of this transmembrane transporter, by generating parasites lacking TgCRT (∆crt) and found that the mutant parasites display several phenotypes, including down-regulation of several endolysosomal proteases, defective trimming of microneme proteins, and decreased ability to invade host cells. Most strikingly, ∆crt also exhibited a swollen VAC that abnormally colocalizes with the neighboring endosome-like compartment (ELC). We hypothesize that this disrupted organization of the endolysosomal system alters the physiology of the normally acidified VAC.
In this study, we sought to quantify the acidity of the VAC by introducing the pH-sensitive GFP biosensor, pHluorin2 (PHL2), into WT, ∆crt, and complement (∆crtCRT) parasites. Here, we establish the use of PHL2 for the measurement of the VAC, as well as the cytosol, in both the extracellular and intracellular stages of T. gondii. We observed a consistent increase in the ratio of emission fluorescence intensity readings at 405 nm and 485 nm (I405/I485) within the VAC of ∆crt, indicating that the lumen of the ∆crt VAC is less acidic than in WT. Furthermore, we complemented ∆crt individually with three different P. falciparum CRT isoforms (PfCRT-3D7, -Dd2, -L272F) and determined that they successfully localized to the T. gondii VAC/PLV, indicating potential conserved function.
A Toxoplasma ortholog of the cathepsin L protease (TgCPL), which is found within the VAC, plays important roles during parasite infection. Additionally, Toxoplasma expresses a cathepsin B-like protease and two putative lysosomal cathepsin C-like proteases (TgCPC1 and TgCPC2) during acute infection. TgCPC1 and TgCPC2, were previously identified as dense granule proteins but limited studies have been done to characterize their function. Therefore, we sought out to better characterize both TgCPC1 and TgCPC2 and determine their importance in the lytic cycle of T. gondii. Here, we show that the majority of TgCPC1 was found localized to the ELC, while TgCPC2 localized to the rhoptries. Furthermore, parasites lacking TgCPC1 (∆cpc1) had defects in invasion, egress, and motility as a result of defective secretion and maturation of multiple microneme proteins. In contrast, there were no noticeable defects observed within the lytic cycle of parasites lacking TgCPC2 (∆cpc2); however, this mutant displayed a loss of the mature form of the rhoptry protein TgROP1, as well as altered accumulation of the pro-form of other ROPs. Additionally, we utilized quantitative PCR to demonstrate that TgCPC1 and TgCPC2 do not seem to exhibit a compensatory interaction with one another. Taken together, our findings indicate that TgCPC1 and TgCPC2 function independently to fulfill their distinct roles in microneme protein processing and regulation of rhoptry protein stability, respectively. Collectively, this body of work provides further understanding of the importance of the organization, physiology, and function of the T. gondii endolysosomal system.
Thornton, Leah Brock, "Functional Characterization of the Toxoplasma gondii Endolysosomal System" (2022). All Dissertations. 3068.
Available for download on Friday, August 11, 2023