Biology subjects

Olafsson, E. B.

Publications and source records attributed to Olafsson, E. B..

5 recordsLinked to original sources

bioRxiv2026

Toxoplasma GRA8 engages the host ESCRT accessory protein ALG-2 and is necessary for parasite metabolic integrity

The endosomal sorting complex required for transport (ESCRT) is a hetero-multimeric membrane-remodeling machinery essential for endosomal sorting, intraluminal vesicle formation, cytokinetic abscission, and membrane repair. ESCRT is also hijacked by some pathogens including the protozoan Toxoplasma gondii, which subverts it at the parasitophorous vacuole membrane to support parasite ingestion of host cytosolic proteins. Although the ESCRT accessory protein, ALG-2 is recruited to the parasitophorous vacuole, nothing was known about how this happens. Herein we identify the dense granule protein TgGRA8 as a key effector that recruits ALG-2 and ALIX to the parasitophorous vacuole. We show that TgGRA8 directly binds ALG-2 via conserved ALG-2-binding elements like those found in other ALG-2 interacting proteins including ALIX and SEC31A. Biochemical assays show high-affinity, Ca{superscript 2}-dependent TgGRA8-ALG-2 binding, and structural modeling suggests TgGRA8 may assemble multivalently to coordinate multiple ALG-2 dimers, stabilizing ALG-2/ALIX recruitment through a non-canonical bridging mechanism. Conservation of these motifs among tissue cyst-forming coccidians implies a lineage-linked adaptation that supports infection by these parasites. Metabolomics further indicates that TgGRA8 loss disrupts amino acid, purine, and central carbon metabolism, like those seen in other ingestion deficient mutants. Together, these findings uncover a conserved, multivalent strategy by which Toxoplasma engages host ALG-2 to organize ESCRT at the parasitophorous vacuole, thereby coupling nutrient acquisition to metabolic fitness and exposing a novel agent for probing ESCRT biology.

cell biology↗

bioRxiv2026

A pooled image-based CRISPR screen identifies EAF1 as a T. gondii modulator of ESCRT subversion.

Intracellular pathogens remodel host cells by redirecting cellular machinery to the host-pathogen interface. The protozoan parasite Toxoplasma gondii co-opts host ESCRT proteins at the parasitophorous vacuole membrane (PVM), where they support budding of host-derived vesicles into the vacuole. Yet the parasite effectors that direct this process remain largely unknown. Here we developed spaCR (spatial phenotype analysis of CRISPR-Cas9 screens), a pooled image-based screening framework that combines deep-learning classification of single-cell spatial phenotypes with well-level barcode genotyping and regression-based deconvolution of gene effects. Screening T. gondii secretory proteins recovered the known TSG101 recruiter GRA14 and identified an uncharacterized effector, ESCRT-association factor 1 (EAF1), that promoted TSG101 recruitment to the PVM. Targeted deletion confirmed its role across Type I and II lineages, and IP-MS recovered ESCRT-I and ESCRT-III components, including TSG101. Together, these findings identify EAF1 as an ESCRT-associated parasite effector and establish spaCR as a general framework for linking CRISPR perturbations to spatial phenotypes.

microbiology↗

bioRxiv2024

Metabolic Adaptability and Nutrient Scavenging in Toxoplasma gondii: Insights from Ingestion Pathway-Deficient Mutants

The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, Toxoplasma endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host ESCRT machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the Plant Vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if Toxoplasma uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide CRISPR screen in wild-type (WT) parasites and {Delta}gra14, {Delta}cpl, and {Delta}crt mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that {Delta}cpl and {Delta}crt mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, TCA cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathways potential role in host resource scavenging. Interestingly, ingestion mutants showed an exacerbated growth defect when grown in amino acid-depleted media, suggesting a role for the Toxoplasma ingestion pathway during nutrient scarcity. ImportanceToxoplasma gondii is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which Toxoplasma scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study highlights the function of the ingestion pathway in sustaining parasite metabolites and contributes to parasite replication under amino acid limiting conditions. This work advances our understanding of the metabolic adaptability of Toxoplasma.

microbiology↗

bioRxiv2021

Toxoplasma gondii subverts the host ESCRT machinery for parasite uptake of host cytosolic proteins

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole, the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of GRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PVM. These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission. Author summaryIntracellular pathogens exploit their host to gain the resources necessary to sustain infection; however, precisely how the intracellular parasite Toxoplasma gondii acquires essential nutrients from its host remains poorly understood. Previous work showed that T. gondii is capable of internalizing host derived cytosolic proteins and delivering them to its lysosome-like compartment for degradation. However, the mechanism by which the material is trafficked across the membrane delimiting the replicative vacuole in which the parasite resides remained unclear. Here, we report a role for the parasite effector protein TgGRA14 in the recruitment of the host ESCRT machinery for the uptake of host cytosolic proteins. Important human pathogens have developed strategies for exploiting the host ESCRT machinery for intracellular subsistence. Our study sheds lights on the strategy used by a eukaryotic pathogen in subverting the host ESCRT machinery for the internalization of resources from its host cells.

microbiology↗

bioRxiv2020

Convergent Met and voltage-gated Ca2+ channel signaling on Ras-Erk MAPK drives migratory activation of dendritic cells parasitized by Toxoplasma gondii

Ras-Erk MAPK signaling controls many of the principal pathways involved in metazoan cell motility, drives metastasis of multiple cancer types and is targeted in chemotherapy. Yet, its putative roles in immune cell functions or in infections have remained elusive. Here, using primary dendritic cells (DCs) in an infection model with the protozoan Toxoplasma gondii, we show that two pathways activated by infection converge on Ras-Erk MAPK signaling to promote migration of parasitized DCs. We identify signaling through the receptor tyrosine kinase Met (also known as HGFR) as a driver of T. gondii-induced DC hypermotility. Further, we show that voltage-gated Ca2+channel (VGCC, subtype CaV1.3) signaling impacts the migratory activation of DCs via calmodulin-calmodulin kinase II. We report that VGCC and Met signaling converge on Ras GTPase to drive Erk1/2 phosphorylation and migratory activation of T. gondii-infected DCs. The data provide a molecular basis for the hypermigratory mesenchymal-to-amoeboid transition (MAT) of parasitized DCs. The emerging concept suggests that parasitized DCs acquire metastasis-like migratory properties to promote infection-related dissemination.

cell biology↗