Biology subjects

Guerra, A. J.

Publications and source records attributed to Guerra, A. J..

4 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↗

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↗

bioRxiv2021

The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii

Iron is essential to living cells, acting as a cofactor in a number of essential enzymes in metabolism; however, iron requires proper storage or it can be dangerous to the cell. In both yeast and plants, iron is stored in a vacuole through the action of a vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii, a pathogen of medical and veterinary importance. Here, we assess the role of VIT in T. gondii. We show that deletion of VIT causes a slight growth defect in vitro, however leads to hypersensitivity in the presence of excess iron, confirming its essential role in iron detoxification in the parasite. In the absence of VIT, parasites contain less iron and are at a growth disadvantage when moving into an iron-depleted environment. We show parasite VIT expression is regulated by environmental iron levels at both the transcript and protein level, and by altering the distribution of VIT within the cell. In the absence of VIT, we find that the T. gondii responds by altering expression of genes with a role in iron metabolism and by increasing the activity of the antioxidant protein catalase. We also show that iron detoxification has an important role both in parasite survival within macrophages and in pathogenesis in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

cell biology↗

bioRxiv2018

Structural basis of Toxoplasma gondii Perforin-Like Protein 1 membrane interaction and activity during egress

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APC{beta} domain within the CTD reveals an unusual double-layered {beta}-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the {beta}-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.\n\nAuthor SummaryToxoplasma gondii has a complex life cycle that involves active invasion of the host cell, the formation of a replicative compartment, and egress from the replicative niche. T. gondii encodes a pore-forming protein, TgPLP1, that contains a C-terminal domain that is crucial for efficient exit from both the parasite containing vacuole and the host cell. However, the mechanism by which TgPLP1 recognizes and binds to the appropriate membrane is unclear. Here we use a combination of biochemistry, structural biology, and parasitology to identify the a preference of TgPLP1 for specific lipids and show that a loop within the structure of the C-terminal domain inserts into the membrane and is necessary for egress from the parasite containing vacuole. Our study sheds light into the determinants of membrane binding in TgPLP1 which may inform the overall mechanism of pore formation in similar systems

microbiology↗