Biology subjects

Schultz, T. L.

Publications and source records attributed to Schultz, T. L..

13 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↗

bioRxiv2026

Salmonella Typhi asparaginase-dependent activation of GCN2 promotes bacterial killing in murine macrophages

Many intracellular pathogens stimulate host cell stress by directly or indirectly causing an imbalance in host nutrients; depletion of amino acid pools in particular can act as a danger signal to infected cells. Using a restrictive host model of Salmonella enterica serovar Typhi (S. Typhi) infection, we identify early induction the integrated stress response (ISR) by viable bacteria, but not heat-killed bacteria. Genetic deletion of the amino acid sensing ISR kinase GCN2 (also known as EIF2AK4) prevented early ISR activation during S. Typhi infection, and murine macrophages lacking GCN2 show impaired bacterial clearance and decreased cytokine output. Supplementation of wildtype C57BL/6 murine macrophages with only the non-essential amino acid asparagine was sufficient to suppress S. Typhi-induced ISR activation and deletion of S. Typhi ansB, encoding an asparaginase, prevented ISR activation during infection. Pharmacological inhibition of mammalian target of rapamycin (mTOR), the other major amino acid sensing pathway in eukaryotic cells, prevented GCN2 activation and ISR induction in murine macrophages, indicating an upstream role for mTOR in signaling to GCN2. These findings suggest a role for the ISR in macrophage innate immune responses to S. Typhi infection and highlight a potential difference in nutrient-dependent signaling between the S. Typhi-susceptible human host and the restrictive murine host centered around asparagine, mTOR, and GCN2.

immunology↗

bioRxiv2025

Phenotypic Screening Coupled with AI-Driven Target Deconvolution Identifies α-Terthienyl as a Dual DPP-IV/HSD17β13 Modulator with Efficacy in a Mouse Model of MASLD

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is a highly prevalent condition characterized by fat build-up in the liver and ranges from benign steatosis to progression to metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis and end-stage liver disease including hepatocellular carcinoma, representing a significant cause of chronic liver disease globally.1,2 Current treatment options are limited, primarily relying on lifestyle modifications, highlighting an urgent need for novel therapeutic strategies. MethodsA Cell Painting-style high-content screening phenotypic assay was employed using the PH5CH8 human hepatocyte cell line to identify small molecules capable of modulating induced hepatic steatosis. The Plex Research artificial intelligence (AI) platform was utilized for target deconvolution of the lead hit compound, -terthienyl. In vivo efficacy was assessed in a diet-induced obesity (DIO) C57BL/6J mouse model of MASLD. Biochemical assays and molecular docking simulations were performed to validate predicted target interactions. ResultsPhenotypic screening identified 15 chemical probes/drugs that elicit dose-responsive inhibition of steatosis, and 16 that exacerbate steatosis, which could contribute to worsening of the disease clinically. -terthienyl, a plant-derived natural product, was identified as a potent and non-toxic inhibitor of steatosis in PH5CH8 cells with an EC50 of 106 nM. In vivo, -terthienyl administration to diet-induced obesity (60% fat diet) mice significantly reduced hepatic steatosis histologically, improved glucose tolerance, and favorably modulated serum biomarkers including ALT and AST. AI-driven analysis predicted dipeptidyl peptidase 4 (DPP-IV) and 17-beta hydroxysteroid dehydrogenase 13 (HSD17{beta}13) as potential molecular targets of -terthienyl. Biochemical inhibition of DPP-IV was observed and an oxidized -terthienyl analog inhibited HSD17{beta}13. Molecular docking supported these predictions, indicating binding to DPP-IV and HSD17{beta}13. ConclusionThis study demonstrates the successful application of phenotypic screening integrated with AI-driven target deconvolution to identify compounds and drugs that ameliorate or exacerbate hepatic steatosis. -terthienyl was identified as a novel modulator of hepatic steatosis with in vivo efficacy in a MASLD model. Our findings suggest a dual-target mechanism involving DPP-IV and HSD17{beta}13, potentially engaged by the parent compound and its metabolite, respectively, offering a promising polypharmacological approach for MASLD treatment.

pharmacology and toxicology↗

bioRxiv2025

Toxoplasma induced cytokine release syndrome is critically dependent on the expression of pore-forming Perforin-Like Protein-1

Acute virulence in Toxoplasma gondii is linked to an excessive proinflammatory cytokine cascade during laboratory murine infection. Previous work showed that T. gondii secretes a pore forming protein, PLP1, that is required for efficient cytolytic egress from host cells. Deletion of the PLP1 gene results in defective egress from infected culture cells and a marked reduction in parasite virulence. The goal of the present study was to gain insight into the nature of the attenuated virulence observed in PLP1 knockout compared to wild type (WT) RH parasites. Using in vivo bioluminescence imaging, we show that parasites lacking PLP1 establish an acute infection and disseminate throughout the infected mice. Histological tissue analysis indicates that parasites cause severe pathology, even in the absence of PLP1. However, mice infected with{Delta} plp1 parasites evoke a protective inflammatory response, demonstrated by mouse survival and control of infection. Flow cytometric analysis was used to determine cellular changes occurring during both WT and {Delta}plp1 parasite infection. Parasite control in the{Delta} plp1 infection was associated with earlier activation of myeloid cells and a moderate neutrophil response that, by comparison, becomes the dominant infiltrating cell type of WT infection. Positive disease outcome during{Delta} plp1 parasite infection is also associated with regulated induction of proinflammatory cytokines, including IFN-{gamma} and TNF-, and an earlier IL-10 regulatory response that is dysregulated during WT infection. Together these findings suggest a key role for Toxoplasma PLP1 in promoting a lethal inflammatory immune response during acute infection with a virulent strain of the parasite. Author SummaryPore-forming proteins are virulence determinants expressed by multiple different pathogens, with varied roles including cellular invasion and escape, immune cell destruction, and the hijack of host cell defenses. The pathogen, Toxoplasma gondii, expresses a pore-forming protein PLP1, that is required for cell lysis and acute virulence in mice. Here, we investigate the potential mechanisms by which this pore-forming protein promotes parasite virulence; from parasite replication and dissemination to immunologic outcomes after infection. In vivo infections demonstrate that parasites replicate, disseminate, and stimulate a protective immune response when PLP1 is not expressed. We show that PLP1 expression induces a parasite driven dysregulation of cell populations and cytokine/chemokine responses, resulting in cytokine release syndrome. In a broader context, Toxoplasmas PLP1 is comparable to other pathogen pore-forming proteins that function as virulence determinants by their ability to alter host immune responses.

microbiology↗

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

A Membrane Lipid Signature Unravels the Dynamic Landscape of Group 1 ILCs

In an era where the established lines between cell identities are blurred by intra-lineage plasticity, distinguishing between stable and transitional states becomes imperative. This challenge is particularly pronounced within the Group 1 ILC lineage, where the similarity and plasticity between NK cells and ILC1s obscure their classification and the assignment of their unique contributions to immune regulation. This study exploits the unique property of Asialo-GM1 (AsGM1)--a membrane lipid associated with cytotoxic attributes absent in ILC1s--as a definitive criterion to distinguish between these cells. By prioritizing cytotoxic potential as the cardinal differentiator, our strategic use of the AsGM1 signature achieved precise delineation of NK cells and ILC1s across tissues, validated by RNA-seq analysis. This capability extends beyond steady-state classifications, adeptly capturing the binary classification of NK cells and ILC1s during acute liver injury. By leveraging two established models of NK-to-ILC1 plasticity driven by TGF{beta} and Toxoplasma gondii, we demonstrate the stability of the AsGM1 signature, which sharply contrasts with the loss of Eomes. This signature identified a spectrum of known and novel NK cell derivatives--ILC1-like entities that bridge traditional binary classifications in aging and infection. The early detection of the AsGM1 signature at the immature NK (iNK) stage, preceding Eomes, and its stability, unaffected by transcriptional reprogramming that typically alters Eomes, position AsGM1 as a unique, site-agnostic marker for fate mapping NK-to-ILC1 plasticity. This provides a powerful tool to explore the expanding heterogeneity within the Group 1 ILC landscape, effectively transcending the ambiguity inherent to the NK-to-ILC1 continuum.

immunology↗

bioRxiv2024

Type I interferon governs immunometabolic checkpoints that coordinate inflammation during Staphylococcal infection

Fine-tuned inflammation during infection enables pathogen clearance while minimizing host damage. Inflammation regulation depends on macrophage metabolic plasticity, yet coordination of metabolic and inflammatory programs is underappreciated. Here we show that type I interferon (IFN) temporally guides metabolic control of inflammation during methicillin-resistant Staphylococcus aureus (MRSA) infection. In macrophages, staggered Toll-like receptor and type I IFN signaling permitted a transient respiratory burst followed by inducible nitric oxide synthase (iNOS)-mediated OXPHOS disruption. OXPHOS disruption promoted type I IFN, suppressing other pro-inflammatory cytokines, notably IL-{beta}. iNOS expression peaked at 24h post-infection, followed by lactate-driven iNOS repression via histone lactylation. In contrast, type I IFN pre-conditioning sustained infection-induced iNOS expression and amplified type I IFN. Cutaneous MRSA infection in mice constitutively expressing epidermal type I IFN led to elevated iNOS levels, impaired wound healing, vasculopathy, and lung infection. Thus, kinetically regulated type I IFN signaling coordinates immunometabolic checkpoints that control infection-induced inflammation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/575104v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@977b1eorg.highwire.dtl.DTLVardef@1142c6corg.highwire.dtl.DTLVardef@170776borg.highwire.dtl.DTLVardef@b02c2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

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↗

bioRxiv2023

A Toxoplasma gondii putative arginine transporter localizes to the plant-like vacuolar compartment and controls parasite extracellular survival and stage differentiation

Toxoplasma gondii is a protozoan parasite that infects a broad spectrum of hosts and can colonize many organs and cell types. The ability to reside within a wide range of different niches requires substantial adaptability to diverse microenvironments. Very little is known about how this parasite senses various milieus and adapts its metabolism to survive, replicate during the acute stage, and then differentiate to the chronic stage. Most eukaryotes, from yeast to mammals, rely on a nutrient sensing machinery involving the TORC complex as master regulator of cell growth and cell cycle progression. The lysosome functions as a signaling hub where TORC complex assembles and is activated by transceptors, which both sense and transport amino acids, including the arginine transceptor SLC38A9. While most of the TORC components are lost in T. gondii, indicating the evolution of a distinct nutrient sensing mechanism, the parasites lysosomal plant-like vacuolar compartment (PLVAC) may still serve as a sensory platform for controlling parasite growth and differentiation. Using SLC38A9 to query the T. gondii proteome, we identified four putative amino acid transporters, termed TgAAT1-4, that structurally resemble the SLC38A9 arginine transceptor. Assessing their expression and sub-cellular localization, we found that one of them, TgAAT1, localized to the PLVAC and is necessary for normal parasite extracellular survival and bradyzoite differentiation. Moreover, we show that TgAAT1 is involved in the PLVAC efflux of arginine, an amino acid playing a key role in T. gondii differentiation, further supporting the hypothesis that TgAAT1 might play a role in nutrient sensing. IMPORTANCET. gondii is a highly successful parasite infecting a broad range of warm-blood organisms including about one third of all humans. Although Toxoplasma infections rarely result in symptomatic disease in individuals with a healthy immune system, the incredibly high number of persons infected along with the risk of severe infection in immunocompromised patients and the potential link of chronic infection to mental disorders make this infection a significant public health concern. As a result, there is a pressing need for new treatment approaches that are both effective and well-tolerated. The limitations in understanding how Toxoplasma gondii manages its metabolism to adapt to changing environments and triggers its transformation into bradyzoites have hindered the discovery of vulnerabilities in its metabolic pathways or nutrient acquisition mechanisms to identify new therapeutic targets. In this work, we have shown that the lysosome-like organelle PLVAC, acting through the putative arginine transporter TgAAT1, plays a pivotal role in regulating the parasites extracellular survival and differentiation into bradyzoites.

molecular biology↗

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↗

bioRxiv2020

Orally Bioavailable Endochin-like Quinolone Carbonate Ester Prodrug Reduces Toxoplasma gondii Brain Cysts

Toxoplasmosis is a potentially fatal infection for immunocompromised people and the developing fetus. Current medicines for toxoplasmosis have high rates of adverse effects that interfere with therapeutic and prophylactic regimens. Endochin-like quinolones (ELQs) are potent inhibitors of Toxoplasma gondii proliferation in vitro and in animal models of acute and latent infection. ELQ-316, in particular, was found to be effective orally against acute toxoplasmosis in mice and highly selective for the T. gondii cytochrome b over the human cytochrome b. Despite oral efficacy, the high crystallinity of ELQ-316 limits oral absorption, plasma concentrations and therapeutic potential. A carbonate ester prodrug of ELQ-316, ELQ-334, was created to decrease crystallinity and increase oral bioavailability, which resulted in a six-fold increase in both Cmax (maximum plasma concentration) and AUC (area under the curve) of ELQ-316. The increased bioavailability of ELQ-316, when administered as ELQ-334, resulted in greater efficacy than the equivalent dose of ELQ-316 against acute toxoplasmosis and had similar efficacy against latent toxoplasmosis compared to intraperitoneal administration of ELQ-316. Carbonate ester prodrugs are a successful strategy to overcome the limited oral bioavailability of ELQs for the treatment of toxoplasmosis.

pharmacology and toxicology↗

bioRxiv2019

The IRE1α stress signaling axis is a key regulator of neutrophil antimicrobial effector function

Activation of the endoplasmic reticulum stress sensor, IRE1, is required for effective immune responses against bacterial infection and is associated with human inflammatory diseases where neutrophils are a key immune component. However, the specific role of IRE1 in regulating neutrophil effector function has not been studied. Here we show that infection-induced IRE1 activation licenses neutrophil antimicrobial capacity, including IL-1{beta} production, NET formation, and MRSA killing. Inhibition of IRE1 diminished production of mitochondrial reactive oxygen species (mROS) and decreased CASPASE-2 activation, which both contributed to neutrophil antimicrobial activity. Mice deficient in Caspase-2 were highly susceptible to MRSA infection and failed to form NETs in a subcutaneous abscess. IRE1 activation enhanced calcium influx and citrullination of histone H3 (Cit-H3) independently of mROS production, suggesting that IRE1 coordinates multiple pathways required for NET formation. Our data demonstrate that the IRE1-Caspase-2 axis is a major driver of neutrophil activity against MRSA infection and highlight the importance of IRE1 in neutrophil antibacterial function.\n\nOne Sentence SummaryIRE1 controls neutrophil antimicrobial defenses

immunology↗

bioRxiv2019

A role for Toxoplasma gondii chloroquine resistance transporter in bradyzoite viability and digestive vacuole maintenance

Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosomal-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum CRT has previously been identified in T. gondii (TgCRT). To interrogate the function of TgCRT in chronic stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo. Interestingly, impairing VAC proteolysis in TgCRT deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This work provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection.\n\nIMPORTANCEIndividuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well validated potential targets. Here we show that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasites lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.

microbiology↗