Biology subjects

Thaprawat, P.

Publications and source records attributed to Thaprawat, P..

6 recordsLinked to original sources

bioRxiv2026

Toxoplasma TgATG2 works with TgATG9 and TgProp1 to drive autophagic flux for parasite extracellular survival and persistence.

Chronic infection by Toxoplasma gondii depends on long-term survival of bradyzoites within tissue cysts, a parasite stage highly resistant to current therapies and a major barrier to eradication. Autophagy has emerged as critical pathway for bradyzoite persistence, yet the core machinery driving autophagosome formation in T. gondii remains poorly defined. Here, we identify TGME49_304630 as TgATG2, a previously uncharacterized, unusually large ATG2-like protein with conserved structural features of lipid-transfer factors. TgATG2 associates with TgATG9 and TgPROP1, key components of the parasite autophagy pathway, supporting its role in a membrane expansion complex required for autophagosome biogenesis. Using independent genetic knockouts, we show that TgATG2 is dispensable for intracellular tachyzoite replication but required for parasite fitness during extracellular stress and, most critically, for bradyzoite autophagy and viability. TgATG2 ablation disrupts autophagic activity in bradyzoites, causing progressive loss of viability and compromised cyst integrity. To overcome limitations of previous indirect assays, we developed a bradyzoite-specific dual-fluorescence TgATG8 reporter that quantitatively measures autophagic flux in T. gondii and confirmed TgATG2 as a major contributor. Importantly, TgATG2-deficient parasites are severely impaired during chronic infection in mice, with reduced brain cyst burdens, abnormal cyst morphology, and markedly diminished ex vivo bradyzoite viability. Together, these findings establish TgATG2 as a central component of the T. gondii autophagy machinery, demonstrate that autophagosome biogenesis is critical for parasite persistence in vivo, and define a molecular vulnerability and quantitative platform for targeting autophagy-dependent parasite survival. IMPORTANCECurrent treatments for toxoplasmosis control acute infection but do not eliminate the long-lived tissue cysts responsible for chronic infection. This persistent stage is clinically important because cysts can reactivate in immunocompromised individuals and may also contribute to long-term disease outcomes. A major barrier to developing cyst-targeting therapies is the limited understanding of how bradyzoites maintain viability for extended periods inside host tissues. This study identifies autophagosome biogenesis as a critical survival process in bradyzoites and defines TgATG2 as a key parasite factor required for this pathway. By linking TgATG2-dependent autophagy to cyst viability and persistence in vivo, our work highlights parasite autophagy as a potential target for eliminating chronic infection. The autophagic flux reporter developed here also provides an important tool for future studies and for screening approaches aimed at discovering inhibitors of bradyzoite survival.

molecular biology↗

bioRxiv2024

Toxoplasma gondii PROP1 is critical for autophagy and parasite viability during chronic infection

Macroautophagy is an important cellular process involving lysosomal degradation of cytoplasmic components, facilitated by autophagy-related proteins (ATGs). In the protozoan parasite Toxoplasma gondii, autophagy has been demonstrated to play a key role in adapting to stress and persistence of chronic infection. Despite limited knowledge about the core autophagy machinery in T. gondii, two PROPPIN family proteins (TgPROP1 and TgPROP2) have been identified with homology to Atg18/WIPI. Prior research in acute stage tachyzoites suggests that TgPROP2 is predominantly involved in a non-autophagic function, specifically apicoplast biogenesis, while TgPROP1 may be involved in canonical autophagy. Here, we investigated the distinct roles of TgPROP1 and TgPROP2 in chronic stage T. gondii bradyzoites, revealing a critical role for TgPROP1, but not TgPROP2, in bradyzoite autophagy. Conditional knockdown of TgPROP2 did not impair bradyzoite autophagy. In contrast, TgPROP1 KO parasites had impaired autolysosome formation, reduced cyst burdens in chronically infected mice, and decreased viability. Together, our findings clarify the indispensable role of TgPROP1 to T. gondii autophagy and chronic infection. ImportanceIt is estimated that up to a third of the human population is chronically infected with Toxoplasma gondii; however, little is known about how this parasite persists long term within its hosts. Autophagy is a self-eating pathway that has recently been shown to play a key role in parasite persistence, yet few proteins that carry out this process during T. gondii chronic infection are known. Here, we provide evidence for a non-redundant role of TgPROP1, a protein important in the early steps of the autophagy pathway. Genetic disruption of TgPROP1 resulted in impaired autophagy and chronic infection of mice. Our results reveal a critical role for TgPROP1 in autophagy and underscore the importance of this pathway in parasite persistence.

microbiology↗

bioRxiv2024

TgATG9 is required for autophagosome biogenesis and maintenance of chronic infection in Toxoplasma gondii

Toxoplasma gondii is a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required for T. gondii chronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cells digestive organelle for degradative recycling. While T. gondii appears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy in atg9{Delta} yeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 in T. gondii after bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintaining T. gondii chronic infection.

microbiology↗

bioRxiv2023

Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors

AbstractsThe parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 ({Delta}eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that {Delta}eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in {Delta}eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

microbiology↗

bioRxiv2021

Protection from lethal Clostridioides difficile infection via intraspecies competition for co-germinant

Clostridioides difficile, a Gram-positive, spore-forming bacterium, is the primary cause of infectious nosocomial diarrhea. Antibiotics are a major risk factor for C. difficile infection (CDI) as they disrupt the gut microbial community, enabling increased germination of spores and growth of vegetative C. difficile. To date the only single-species bacterial preparation that has demonstrated efficacy in reducing recurrent CDI in humans is non-toxigenic C. difficile. Using multiple infection models we determined that pre-colonization with a less virulent strain is sufficient to protect from challenge with a lethal strain of C. difficile, surprisingly even in the absence of adaptive immunity. Additionally, we showed that protection is dependent on high levels of colonization by the less virulent strain and that it is mediated by exclusion of the invading strain. Our results suggest that reduction of amino acids, specifically glycine following colonization by the first strain of C. difficile is sufficient to decrease germination of the second strain thereby limiting colonization by the lethal strain. ImportanceAntibiotic-associated colitis is often caused by infection with the bacterium Clostridioides difficile. In this study we found that reduction of the amino acid glycine by pre-colonization with a less virulent strain of C. difficile is sufficient to decrease germination of a second strain. This finding demonstrates that the axis of competition for nutrients can include multiple life stages. This work is important, as it is the first to identify a possible mechanism through which pre-colonization with C. difficile, a current clinical therapy, provides protection from reinfection. Furthermore, our work suggests that targeting nutrients utilized by all life stages could be an improved strategy for bacterial therapeutics that aim to restore colonization resistance in the gut.

microbiology↗

bioRxiv2020

Acquisition of Host Cytosolic Protein by Toxoplasma gondii Bradyzoites

Toxoplasma gondii is a protozoan parasite that persists in the central nervous system as intracellular chronic stage bradyzoites that are encapsulated by a thick cyst wall. While the cyst wall separates bradyzoites from the host cytosol, it has been posited that small solutes can traverse the cyst wall to sustain bradyzoites. Recently it was found that host cytosolic macromolecules can cross the parasitophorous vacuole and are ingested and digested by actively replicating acute stage tachyzoites. However, the extent to which bradyzoites have an active ingestion pathway remained unknown. To interrogate this, we modified previously published protocols that look at tachyzoite acquisition and digestion of host proteins by measuring parasite accumulation of a host-expressed reporter protein after impairment of an endolysosomal protease (Cathepsin Protease L, CPL). Using two cystogenic parasite strains (ME49 and Pru), we demonstrate that T. gondii bradyzoites can ingest host-derived cytosolic mCherry. Bradyzoites acquire host mCherry within 4 hours of invasion and post-cyst wall formation. This study provides direct evidence that host macromolecules can be internalized by T. gondii bradyzoites across the cyst wall in infected cells.

microbiology↗