Biology subjects

Huynh, M.-H.

Publications and source records attributed to Huynh, M.-H..

7 recordsLinked to original sources

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↗

bioRxiv2022

A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which in Toxoplasma gondii includes MIC8 and the microneme-associated CRMP complex. Here, we examine the function of the microneme protein CLAMP, uncovering its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP-deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. Nevertheless, SPATR, which appears to act as an accessory factor in Toxoplasma, is essential during Plasmodium falciparum blood stages. Our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

microbiology↗

bioRxiv2021

Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during egress in both wild-type RH and {Delta}plp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in {Delta}plp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin-induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification. Author SummaryToxoplasma gondii is a single celled intracellular parasite that infects many different animals, and it is thought to infect up to one third of the human population. This parasite must rupture out of its replicative compartment and the host cell to spread from one cell to another. Previous studies indicated that a decrease in pH occurs within the replicative compartment near the time of parasite exit from host cells, an event termed egress. However, it remained unknown whether the decrease in pH is directly tied to egress and, if so, what is responsible for the decrease in pH. Here we used a fluorescent reporter protein to directly measure pH within the replicative compartment during parasite egress. We found that pH decreases immediately prior to parasite egress and that this decrease is linked to parasite disruption of membranes. We also identified a family of transporters that release acidic products from parasite use of glucose for energy as contributing to the decrease in pH during egress. Our findings provide new insight that connects parasite glucose metabolism to acidification of its replicative compartment during egress from infected cells.

microbiology↗

bioRxiv2021

Toxoplasma gondii Toxolysin 4 contributes to efficient parasite egress from host cells

Egress from host cells is an essential step in the lytic cycle of T. gondii and other apicomplexan parasites; however, only a few parasite secretory proteins are known to affect this process. The putative metalloproteinase Toxolysin 4 (TLN4) was previously shown to be an extensively processed microneme protein, but further characterization was impeded by the inability to genetically ablate TLN4. Herein we show that TLN4 has the structural properties of an M16 family metalloproteinase, that it possesses proteolytic activity on a model substrate, and that genetic disruption of TLN4 reduces the efficiency of egress from host cells. Complementation of the knockout strain with the TLN4 coding sequence significantly restored egress competency, affirming that the phenotype of the {Delta}tln4 parasite was due to the absence of TLN4. This work identifies TLN4 as the first metalloproteinase and the second microneme protein to function in T. gondii egress. The study also lays a foundation for future mechanistic studies defining the precise role of TLN4 in parasite exit from host cells. IMPORTANCEAfter replicating within infected host cells, the single celled parasite Toxoplasma gondii must rupture out of such cells in a process termed egress. Although it is known that T. gondii egress is an active event that involves disruption of host-derived membranes surrounding the parasite, very few proteins that are released by the parasite are known to facilitate egress. In this study we identify a parasite secretory protease that is necessary for efficient and timely egress, thus laying the foundation for understanding precisely how this protease facilitates T. gondii exit from host cells.

microbiology↗

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↗

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↗

bioRxiv2018

POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

Toxoplasma gondii is a ubiquitous obligate intracellular eukaryotic parasite that causes congenital birth defects, disease of the immunocompromised and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune response of many eukaryotic parasites and is also of great relevance to vaccine design. Here, we demonstrate that MIC2, the motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat domains (TSR). At least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at O-glycosylation sites. We demonstrate that the addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homologue (TgPOFUT2) encoded by TGGT1_273550. While POFUT2 homologues are important for stabilizing motility associated adhesins and host infection in other apicomplexan parasites, in T. gondii loss of TgPOFUT2 has only a modest impact on MIC2 levels and the wider proteome. Consistent with this, both plaque formation and tachyzoite infectivity are broadly similar in the presence or absence of TgPOFUT2. These findings demonstrate that TgPOFUT2 O-glycosylates MIC2 and that this glycan is dispensable in T. gondii tachyzoites.

microbiology↗