Biology subjects

Mayoral, J.

Publications and source records attributed to Mayoral, 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↗

bioRxiv2021

Dense granule protein, GRA64 interacts with host cell ESCRT proteins during Toxoplasma gondii infection

The intracellular parasite Toxoplasma gondii adapts to diverse host cell environments within a replicative compartment that is heavily decorated by secreted proteins. In attempts to identify novel parasite secreted proteins that influence host cell activity, we identified and characterized a trans-membrane dense granule protein dubbed GRA64 (TGME49_202620). We found that GRA64 is on the parasitophorous vacuolar membrane (PVM) and is partially exposed to the host cell cytoplasm in both tachyzoite and bradyzoite parasitophorous vacuoles. Using co-immunoprecipitation and proximity-based biotinylation approaches, we demonstrate that GRA64 appears to interact with certain components of the host Endosomal Sorting Complexes Required for Transport (ESCRT). Genetic disruption of GRA64 does not affect acute Toxoplasma virulence in mice nor encystation as observed via tissue cyst burdens in mice during chronic infection. However, ultrastructural analysis of {Delta}gra64 tissue cysts using electron tomography revealed enlarged vesicular structures underneath the cyst membrane, suggesting a role for GRA64 in organizing the recruitment of ESCRT proteins and subsequent intracystic vesicle formation. This study uncovers a novel host-parasite interaction that contributes to an emerging paradigm in which specific host ESCRT proteins are recruited to the limiting membranes (PVMs) of tachyzoite and bradyzoite vacuoles formed during acute and chronic Toxoplasma infection. IMPORTANCEToxoplasma gondii is a widespread foodborne parasite that causes congenital disease and life-threatening complications in immune compromised individuals. Part of this parasites success lies in its ability to infect diverse organisms and host cells, as well as to persist as a latent infection within parasite constructed structures called tissue cysts. In this study, we characterized a protein secreted by T. gondii into its parasitophorous vacuole during intracellular infection, which we dub GRA64. On the vacuole, this protein is exposed to the host cell and interacts with specific host ESCRT proteins. Parasites lacking the GRA64 protein exhibit ultrastructural changes in tissue cysts during chronic infection. This study lays the foundation for future studies on the mechanics and consequences of host ESCRT-parasite protein interactions.

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

Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export

ABSTRACTToxoplasma gondii is a highly successful parasite that infects a significant portion of the human population. As an intracellular parasite, T. gondii thrives within many different cell types due to its residence in the parasitophorous vacuole, a specialized and heavily modified compartment in which parasites divide. Within this vacuole, numerous secreted proteins facilitate functions that optimize intracellular survival. We characterized one such protein, TgPPM3C, which is predicted to contain a domain belonging to the PP2C class of serine/threonine phosphatases and is secreted by both tachyzoites and differentiating bradyzoites into the vacuolar lumen. Genetic deletion of TgPPM3C established that parasites lacking this predicted phosphatase exhibit a minor growth defect in vitro, are avirulent during acute infection in mice, and form fewer cysts in mouse brain during chronic infection. A label-free phosphoproteomic approach was utilized to identify putative TgPPM3C substrates and demonstrated several secreted proteins with altered phosphorylation status in the absence of TgPPM3C. Altered phosphorylation status was seen in MYR1, a protein essential to the process of protein effector export from the parasitophorous vacuole into the host cell, and in GRA16 and GRA28, two exported effector proteins. Defects were seen in the export of GRA16 and GRA28, but not the effector TgIST, in the TgPPM3C knockout strain. Parasites lacking TgPPM3C also exhibited defects in host c-Myc induction, a process influenced by effector export. Phosphomimetic mutations of GRA16 serine residues recapitulated export defects, implicating de-phosphorylation as an important process in facilitating the export of GRA16. These findings provide an example of the emerging critical role that phosphatases play in regulating the complex environment of the T. gondii parasitophorous vacuole.View Full Text

microbiology↗