Martorelli di Genova, B. | Search bioRxiv

bioRxiv2023

Pyrimidine salvage in Toxoplasma gondii as a target for new treatment

Toxoplasmosis is a common protozoan infection that can have severe outcomes in the immunocompromised and during pregnancy, but treatment options are limited. Recently, nucleotide metabolism has received much attention as a target for new antiprotozoal agents and here we focus on pyrimidine salvage by Toxoplasma gondii as a drug target. Whereas uptake of [3H]-cytidine and particularly [3H]-thymidine was at most marginal, [3H]-uracil and [3H]-uridine were readily taken up. Kinetic analysis of uridine uptake was consistent with a single transporter with a Km of 3.3 {+/-} 0.8 {micro}M, which was inhibited by uracil with high affinity (Ki = 1.15 {+/-} 0.07 {micro}M) but not by thymidine or 5-methyluridine, showing that the 5-Me group is incompatible with uptake by T. gondii. Conversely, [3H]-uracil transport displayed a Km of 2.05 {+/-} 0.40 {micro}M, not significantly different from the uracil Ki on uridine transport, and was inhibited by uridine with a Ki 2.44 {+/-} 0.59 {micro}M, also not significantly different from the experimental uridine Km. The reciprocal, complete inhibition, displaying Hill slopes of approximately [~]1, strongly suggest that uridine and uracil share a single transporter with similarly high affinity for both, and we designate it uridine/uracil transporter 1 (TgUUT1). While TgUUT1 excludes 5-methyl substitutions, the smaller 5F substitution was tolerated as 5F-uracil inhibited uptake of [3H]-uracil with a Ki of 6.80 {+/-} 2.12 {micro}M (P > 0.05 compared to uracil Km). Indeed, we found that 5F-Uridine, 5F-uracil and 5F,2-deoxyuridine were all potent antimetabolites against T. gondii with EC50 values well below that of the current first line treatment, sulfadiazine. In vivo evaluation also showed that 5F-uracil and 5F,2-deoxyuridine were similarly effective as sulfadiazine against acute toxoplasmosis. Our preliminary conclusion is that TgUUT1 mediates potential new anti-toxoplasmosis drugs with activity superior to the current treatment.

microbiology↗

bioRxiv2023

Organoid-based in vitro systems to model Cryptosporidium parvum infection in 2D and 3D

Many advances have been made recently in our understanding of Cryptosporidiums asexual cycle and sexual differentiation. However, the process of fertilization, which is required for transmission of infectious oocysts, is not well understood. Typical cancer cell-based culture only allows robust exploration of asexual cycle and sexual differentiation of Cryptosporidium. To facilitate exploration of sexual reproduction in C. parvum we developed an organoid-based culture system that supports Cryptosporidiums full life cycle and a novel fertilization reporter. Organoid derived monolayers (ODMs) supported fertilization and oocyst production and maintained the infection for up to 3 weeks. ODM derived oocysts were infectious in vivo. Fertilization was confirmed by successfully mating two strains of C. parvum and with a novel fertilization switch reporter. The fertilization switch reporter utilizes a DiCre system in which cre fragments are expressed under the control of sexual stage promoters resulting in a rapamycin-inducible switch in fluorescent protein expression from mCherry to mNeonGreen after fertilization that is spatially and temporally controlled. This results in mCherry positive parasites in the first generation and offspring that express mNeonGreen. In vivo validation of the fertilization switch reporter demonstrated the precision and efficiency of the fertilization switch reporter and confirmed excision of the mCherry gene sequence only after rapamycin treatment. The start of a second generation of parasites was also shown in the ODMs and rarely in HCT8s. Use of this reporter in ODMs can help investigate the Cryptosporidium lifecycle post sexual differentiation in a physiologically relevant in vitro system. ImportanceOrganoid derived monolayers provide an opportunity to elucidate previously inaccessible aspects of Cryptosporidiums biology. This system overcomes the disadvantages of previous organoid-based methods for Cryptosporidium culture. It is faster and simpler than previously described systems, uses defined media to increase reproducibility and consistency, enables real-time observation, supports parasite fertilization and oocyst production, and provides a physiologically relevant tissue culture system to facilitate studies of Cryptosporidium cell biology. The ODM system could facilitate the study of host-pathogen interactions, Cryptosporidium-host specificity, or innate or cellular immune responses to Cryptosporidium infection stimulated in the intestinal epithelium. The fertilization switch reporter could be used to test factors or drugs that may have potential to interfere with Cryptosporidiums sexual reproduction. Organoid-based cell cultures in combination with the fertilization switch reporter could increase our understanding of sexual reproduction in Cryptosporidium, leading to vital information for the development of sexual reproduction inhibitors or vaccines that could shorten disease duration and prevent transmission.

microbiology↗