Biology subjects

de Moraes de Siqueira, C.

Publications and source records attributed to de Moraes de Siqueira, C..

3 recordsLinked to original sources

bioRxiv2026

Toxoplasma gondii GRA66 prevents premature egress driven by the host phospholipase RARRES3, independently of RNF213

Toxoplasma gondii replicates inside a host-derived parasitophorous vacuole (PV), and interferon-gamma (IFN{gamma}) induces host restriction factors that target this compartment. Previous CRISPR screens identified the dense granule protein GRA66 and the GRA57/GRA70/GRA71 complex as required for parasite fitness in IFN{gamma}-stimulated human cells, but the host pathways they oppose were unknown. Here, we show that GRA66, a PV membrane (PVM)-associated protein predicted to be an N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D, is required to prevent premature egress driven by the host phospholipase and acyltransferase RARRES3. Loss of GRA66 caused premature parasite egress, host cell death, and impaired replication in human cells. These phenotypes persisted in cells lacking RNF213, the E3 ubiquitin ligase that dominates IFN{gamma}-dependent Toxoplasma restriction in human cells, and{Delta} gra66 vacuoles recruited less RNF213 and ubiquitin than wild type, indicating an RNF213-independent mechanism rather than an exaggerated RNF213 response. Complementation with a catalytic-site mutant of the GRA66 zinc-binding motif failed to restore any of these phenotypes, indicating a requirement for its predicted enzymatic activity. Deleting RARRES3 rescued the premature egress of {Delta}gra66 and catalytic-mutant parasites but not of{Delta} gra70 parasites, whereas the replication defect persisted, revealing a second, RARRES3-independent consequence of GRA66 loss. RARRES3 was recruited to the PVM and intravacuolar network after IFN{gamma} stimulation, and structural modeling supported assignment of GRA66 to the NAPE-phospholipase D family with an intact di-zinc active site. These findings define a lipid-centered, RNF213-independent arm of human cell-autonomous immunity and identify the parasite effector required to withstand it.

microbiology↗

bioRxiv2025

Toxoplasma effector TgWIP hijacks dendritic cell actin and motility via Nck1/Grb2 and the WAVE complex

The intracellular parasite Toxoplasma gondii enhances its dissemination to distant organs by hijacking infected leukocytes via a Trojan Horse mechanism. Upon infecting dendritic cells (DCs), Toxoplasma induces a hypermigratory phenotype characterized by podosome dissolution and formation of F-actin stress fibers. We previously showed that these cytoskeletal changes depend on the effector protein Toxoplasma WAVE complex-interacting protein (TgWIP) secreted from parasites to infected leukocytes. Here, we identify the host adaptor proteins Non-catalytic region of tyrosine kinase adaptor protein 1 and 2 (Nck1/2) and Growth factor receptor-bound protein 2 (Grb2) as direct TgWIP interactors. TgWIP mainly uses two distinct proline-rich regions (PRRs) to interact with Nck1 and Grb2. Mutating these PRRs abrogates TgWIP binding to Nck1 and Grb2 and diminishes podosome dissolution and DC hypermotility. Furthermore, we show that TgWIP directly interacts with the actin nucleation promoting factor WAVE Regulatory Complex (WRC) via a WRC-interacting receptor sequence (WIRS). Disrupting this interaction also influences actin cytoskeletal remodeling and DC hypermotility. Collectively, our data reveal that TgWIP directly interacts with multiple actin regulators, including Nck1, Grb2, and the WRC, to remodel the actin cytoskeleton of the host cells, elucidating a key mechanism that Toxoplasma exploits to enhance host cell migration and dissemination.

microbiology↗

bioRxiv2025

Unveiling the Unique Structure and Singular Function of the Histone Deacetylase 2 (TgHDAC2) of Toxoplasma gondii

Histone deacetylases (HDACs) are enzymes traditionally recognized for their role in removing acetyl groups from lysines on histones. However, recent findings have revealed that many HDACs also target non-histone proteins. In Toxoplasma gondii, although we identified TgHDAC2, an enzyme annotated as a class I HDAC, we found that its substrates are non-histone proteins. Notably, TgHDAC2 possesses two unique peptide insertions within its HDAC domain, whose structural and functional roles were previously unknown. Using cross-linking mass spectrometry (XLMS), we resolved the three-dimensional structure of TgHDAC2, while biophysical analyses demonstrated that these insertions do not compromise the proteins stability but play an important part in its function. Localization studies revealed differential expression of TgHDAC2 throughout the cell cycle, with prominent enrichment around daughter cells during mitosis and cytokinesis. Its deletion severely disrupts parasite replication, suggesting a critical role in cell cycle regulation. RNA sequencing of TgHDAC2 knockout parasites highlighted significant downregulation of genes involved in membrane composition, cytoskeletal organization, and cell signaling pathways, further supporting its role in modifying non-histone proteins. Collectively, our results suggest that TgHDAC2 acts as a deacetylase for non-histone proteins, modulating cytoskeletal and membrane proteins critical for T. gondii cell cycle progression and replication. IMPORTANCEToxoplasma gondii is an obligate intracellular parasite and a significant global public health concern. It is estimated that up to one-third of the worlds population may be infected, depending on the region, with even higher prevalence rates in South America due to the circulation of atypical and more virulent strains. Understanding the biology of this parasite and identifying novel therapeutic targets is therefore critical, as current treatments are outdated and ineffective against the chronic phase of toxoplasmosis. In this study, we identified a novel lysine deacetylase that plays an essential role in T. gondii replication, highlighting its potential as a promising therapeutic target.

microbiology↗