17th International Congress on Toxoplasmosis

Professor

Identification of proteins mediating ER-Toxoplasma contact sites

Host-Parasite Interaction & Signalling

The formation of membrane contact sites (MCSs) between intracellular pathogens and host organelles is a common consequence of infection. We previously found that the human parasite Toxoplasma gondii forms contact sites with host mitochondria and drives the shedding of their outer membrane. Here, we investigate the molecules that mediate the MCSs between Toxoplasma and host endoplasmic reticulum (ER). Although these MCSs were first described in 1968, the proteins mediating this contact remain unknown. To this end, we developed a split-GFP based sensor wherein GFP reconstitution indicates successful formation of contact sites between the pathogen and host organelles. To validate our sensor for FACS based CRISPR-Cas9 screening, we first applied it to monitor Toxoplasma-mitochondria MCSs and as expected found that our sensor reconstituted GFP at these contact sites, but failed to do so in the absence of the mitochondrial tether TOM70 or its Toxoplasma counterpart MAF1. To identify the proteins that tether the ER to the parasite vacuole in an unbiased manner, we adapted our sensor to Toxoplasma-ER MCSs and performed a genome-wide loss of function CRISPR screen. Our candidate ER tether suggests a conserved mechanism by which pathogens contact diverse host organelles. Defining the molecules that mediate pathogen-organelle MCSs will enable us to determine their function during infection and enhance our understanding of contact site biology in general.

Oral presentation

Formation of mammalian ER membrane contact sites, via host VAPA and VAPB, with the Toxoplasma PV

Cell Biology

The Toxoplasma parasitophorous vacuole membrane (PVM) forms a barrier protecting the parasite from cellular assaults and serves as a platform for nutrient retrieval from the host cell. The PVM physically associates with many host organelles (e.g., ER and mitochondria), at distances reminiscent of membrane contact sites (MCS &lt; 15 nm). We hypothesize that the parasite establishes functional MCS between the PVM and selected host organelles. In support, our RNAseq data show altered expression of several host lipid transfer proteins located at MCS (e.g., StAR, OSBP2, NPC1) in Toxoplasma-infected cells, suggesting hijacking of nonvesicular lipid transport pathways. Our fluorescence microscopy observations illustrate the recruitment at the PV of the host ER proteins VAPA and VAPB that both function as mammalian MCS tethering factors that bind to proteins with FFAT motifs. Our EM data show no host ER at PV in VAPA- and VAPB-deficient HeLa cells (DKO VAP, gift from P. De Camilli) infected up to 6h, suggesting Toxoplasma recognition of VAPA/VAPB for ER recruitment.However, 24h p.i. in DKO VAP cells, host ER was observed at the PV (27% coverage) though less than in WT HeLa (43%), suggesting that VAPA/VAPB are not critical for recruitment later in infection. We identified a PVM-localized Toxoplasma protein (we named TgVIP) with two putative FFAT motifs, as a potential host VAP interactor; a knock-out vip mutant exhibits a growth delay. Our EM data show TgVIP KO still recruits host ER, suggesting TgVIP is not involved in host ER-PVM attachment, but it may bind to host VAPs following host ER attachment to the PVM; we are assessing TgVIP and VAP interaction in situ. Previously, we demonstrated that Toxoplasma retrieves nutrients from host organelles (e.g., endolysosomes, lipid droplets, Rab vesicles) trapped in tubules of the intravacuolar network (IVN). The IVN-deficient parasite (∆gra2∆gra6), impaired in intra-PV host organelle sequestration, forms an extensive network of PVM projections (PVMP) extending into the host cell. As this mutant retains virulence, we hypothesize that its survival correlates with increased PVM-host organelle MCS formation. Indeed, host VAPA and a related host ER MCS protein MOSPD2 localize to ∆gra2∆gra6 PVMP. In DKO VAP cells at 24h pi, less host ER is recruited to ∆gra2∆gra6 PV and PV-associated host mitochondria show aberrant morphology. These observations point to a physiological relevance for MCS formation to support parasite infectivity.

The role of the GPI in Toxoplasma gondii pathogenesis and mRNA vaccines

A fully protective vaccine to prevent human parasitic disease has remained elusive. Our investigation of acquired immunity to Toxoplasma gondii that follow either natural infection or vaccination has led us reconsider the role of glycosylphosphatidylinositols (GPIs) in parasite pathogenesis and vaccination. GPIs are highly conserved anchors for eukaryotic cell surface proteins. T. gondii&quot;s plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in T. gondii has remained largely unknown without an understanding of its biosynthesis. We became curious of the gene Tg_207750 as it was one of the most differentially expressed genes between type I strains that either evade immunological memory (GT1) or are controlled by it (RH) and is a predicted glycosyltransferase that adds hexoses to mannose chains. Here we report the identification and disruption of Tg_207750 (&quot;PIGJ&quot;) and Tg_266320 (&quot;PIGE&quot;) GPI sidechain glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. Parasite numbers are not affected by Δpigj early in the infection, suggesting a breakdown of infectious tolerance, which is dependent on host galectin-3 but not TLR2/4 GPI interacting partners. In contrast, humoral recognition of GIPL is entirely dependent on the terminal Glucose as inferred from PIGE mutants. Furthermore, we present evidence that the GPI of type I RH but not GT1 strains lacks sidechains, further implicating this pathway in secondary infection immunity. To directly investigate the role of the GPI in humoral responses, we have tested SAG1 immunization with and without GPI attachment through mRNA vaccination, which revealed a fundamental requirement for the GPI in eliciting humoral responses to GPI-anchored proteins. Ongoing results will be discussed.

A new Toxoplasma rhoptry protein is a major virulence factor that prevents inflammatory host cell death

Programmed-cell death is an antimicrobial mechanism of defense that promotes rapid clearance of intracellular pathogens. Toxoplasma counteracts host immune defenses by secreting effector proteins into host cells; however, how the parasite evades inflammatory cell death and the effectors involved remain poorly characterized. We identified a new Toxoplasma virulence factor that ensures parasite survival by blocking host cell death. RNA-Seq analysis revealed that this rhoptry protein acts as a repressor of host pro-inflammatory responses. THP1 human monocytes infected with a mutant Toxoplasma strain, showed increased nuclear translocation of NF-kB p65, IL-1β and LDH release, compared to infection with wild type or complemented parasites. Moreover, mutant parasites were dramatically impaired in virulence in mice also preventing NF-κB signalling and lytic cell death in BMDM. These findings unravel the role of a major virulence factor of Toxoplasma, supressing inflammatory cell death as a strategy to evade parasite clearance.

A high-throughput screen to elucidate the function of Toxoplasma gondii rhoptry proteins within human host cells

Toxoplasma gondii contain apicomplexan-specific rhoptry organelles whose proteins are essential in mediating invasion of host cells and host-pathogen interactions at early intracellular timepoints. Rhoptry proteins (ROPs) are deposited within the host prior to invasion and localize to various host compartments to rewire the host cell for the parasite&quot;s survival. While some of these proteins have been characterized, the function of the majority of ROPs remains unknown. Here we systematically screen the function of ROPs within host cells utilizing a BioID approach, where ROP baits fused to miniTurboID, are expressed in HEK293 cells and biotinylated proximal prey are identified by mass spectrometry. We have generated a partial dataset containing 23 rhoptry baits and 1472 prey. These data recapitulate known features of rhoptry host biology. Toxofilin prey are significantly enriched in actin cytoskeleton localizing proteins, including the known interactor actin. ROP18 prey are significantly enriched in endoplasmic reticulum proteins, including the known substrate RPN1. Furthermore, ROPs that localize to host compartments do so in this experimental system: ROP47 and PP2C-hn prey are significantly enriched in nucleoplasm proteins and ROP39 prey are significantly enriched in mitochondrial proteins. Novel insights include an enrichment of microtubule proteins in ROP19A prey and peroxisomal proteins in ROP13 prey. Together these results establish the efficacy of this approach and highlight the potential for novel discoveries regarding the function of rhoptry proteins in human host cells.

Rhoptry-associated perforation of the host cell plasma membrane during Toxoplasma gondii invasion

The exocytosis and delivery of rhoptry proteins into the host cell is essential for Toxoplasma gondii invasion and virulence. The rhoptry secretory apparatus (RSA) has become increasingly well-defined and shows conservation among Apicomplexans. While we now know a great deal about both the RSA structure and the individual rhoptry effector proteins that are released, comparatively little is known about how those proteins are delivered into the host cell. Previous electrophysiology experiments revealed that invasion by T. gondii is preceded by a transient increase in host cell plasma membrane conductance. We hypothesize that the conductance transient represents a perforation in the host cell plasma membrane through which rhoptry proteins are delivered into the host cell. To further investigate this event, we have developed a higher throughput assay that utilizes high-speed, multi-wavelength, fluorescence imaging to simultaneously visualize the host cell perforation and invasion of the parasite. Using this assay, we have interrogated a panel of mutant parasites conditionally depleted of key invasion-related proteins. Parasites lacking the rhoptry effector RON2 generate the perforation, suggesting that perforation is upstream of effector injection and moving junction formation. In contrast, parasites lacking components of the rhoptry exocytosis pathway (e.g., RASP2, Nd9) or proteins that regulate rhoptry secretion (e.g., CLAMP), are largely unable to induce the perforation. These data, which have been corroborated by electrophysiology studies, demonstrate that rhoptry exocytosis is required for formation of the perforation. Our results are consistent with a model in which rhoptry exocytosis results in transient perforation of the host cell membrane, enabling the subsequent delivery of rhoptry effector proteins into the host cell.

Session II: Parasite-Host Interactions & Signalling I

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