# Extragenic adaptation to the loss of a deubiquitinase affecting the T. gondii cell cycle and development

> **NIH NIH R21** · UNIVERSITY OF KENTUCKY · 2021 · $191,250

## Abstract

Project Summary
The cell cycle of protozoan parasite Toxoplasma gondii, an important opportunistic pathogen in HIV-AIDS
patients, is fundamentally distinct from that of typical eukaryotes. Replication in the actively growing
tachyzoites in acute infection and within bradyzoites associated with chronic infection occurs by an internal
budding process termed endodyogeny. With endodyogeny, each mother parasite produces 2 daughters per
cycle. In contrast, the sexual cycle of the parasite, a stage restricted to the feline gut, occurs by a hybrid of
schizogony and endopolygeny which are typically associated with the malaria parasite and members of the
genus Sarcocystis respectively. These cell cycle architectures are associated with the generation of multiple
progeny per replicative cycle. We recently characterized a cell cycle regulated deubiquitnase TgOTUD3A
(TgGT1_258780) the targeted disruption of which relaxed the restriction of tachyzoites replication to
endodyogeny (2 progeny per cycle) to exhibit characteristics of both schizogony and endopolygeny-often
within the same clonal vacuole. These mutant parasites generate 3,4 or 5 progeny per cycle to define the
multi-daughter phenotype. In addition, over 70% of TgOTUD3A tachyzoites ectopically express markers
associated with bradyzoites and induce the expression of genes typically associated with merozoties, the
stage associated with entry into the sexual cycle in cats. Complementation of the TgOTUD3A-KO failed to
restore any of these phenotypes, with whole genome sequencing revealing no credible mutations at the
genomic level. This suggested that a compensatory adaptation had occurred. Examination of the other
TgOTU family members revealed a selective transcriptional upregulation of two closely related family
member, TgOTUD1B (TgGT1_237894) and TgOTUD1C (TgGT1_323200). It is likely that TgOTUD1A
(TgGT1_207650) is similarly upregulated. This presents the possibility that one or more of the cell cycle and
developmental phenotypes attributed to the loss of TgOTUD3A may in fact be due to the upregulation of the
Clade D1 TgOTU members. To address this specific question we propose to functionally characterize the
Clade D1 TgOTU members and establish the consequence of both their ablation and regulated
overexpression in the wild type and TgOTUD3A-KO background. In doing so we expect to gain insights into
how these deubiquitinases govern the selection of cell cycle architecture and control aspects of key
developmental transitions cementing an emerging association between these fundamental processes.

## Key facts

- **NIH application ID:** 10129275
- **Project number:** 5R21AI150085-02
- **Recipient organization:** UNIVERSITY OF KENTUCKY
- **Principal Investigator:** ANTHONY P. SINAI
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $191,250
- **Award type:** 5
- **Project period:** 2020-03-20 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10129275

## Citation

> US National Institutes of Health, RePORTER application 10129275, Extragenic adaptation to the loss of a deubiquitinase affecting the T. gondii cell cycle and development (5R21AI150085-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10129275. Licensed CC0.

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