Identification of a New Rhoptry Neck Complex RON9/RON10 in the Apicomplexa Parasite Toxoplasma gondii

Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells

La pharmacie en contexte hospitalier : une mission à définir

<div><p>Obligate intracellular pathogens satisfy their nutrient requirements by coupling to host metabolic processes, often modulating these pathways to facilitate access to key metabolites. Such metabolic dependencies represent potential targets for pathogen control, but remain largely uncharacterized for the intracellular protozoan parasite and causative agent of Chagas disease, <i>Trypanosoma cruzi</i>. Perturbations in host central carbon and energy metabolism have been reported in mammalian <i>T</i>. <i>cruzi</i> infection, with no information regarding the impact of host metabolic changes on the intracellular amastigote life stage. Here, we performed cell-based studies to elucidate the interplay between infection with intracellular <i>T</i>. <i>cruzi</i> amastigotes and host cellular energy metabolism. <i>T</i>. <i>cruzi</i> infection of non-phagocytic cells was characterized by increased glucose uptake into infected cells and increased mitochondrial respiration and mitochondrial biogenesis. While intracellular amastigote growth was unaffected by decreased host respiratory capacity, restriction of extracellular glucose impaired amastigote proliferation and sensitized parasites to further growth inhibition by 2-deoxyglucose. These observations led us to consider whether intracellular <i>T</i>. <i>cruzi</i> amastigotes utilize glucose directly as a substrate to fuel metabolism. Consistent with this prediction, isolated <i>T</i>. <i>cruzi</i> amastigotes transport extracellular glucose with kinetics similar to trypomastigotes, with subsequent metabolism as demonstrated in ¹³C-glucose labeling and substrate utilization assays. Metabolic labeling of <i>T</i>. <i>cruzi</i>-infected cells further demonstrated the ability of intracellular parasites to access host hexose pools <i>in situ</i>. These findings are consistent with a model in which intracellular <i>T</i>. <i>cruzi</i> amastigotes capitalize on the host metabolic response to parasite infection, including the increase in glucose uptake, to fuel their own metabolism and replication in the host cytosol. Our findings enrich current views regarding available carbon sources for intracellular <i>T</i>. <i>cruzi</i> amastigotes and underscore the metabolic flexibility of this pathogen, a feature predicted to underlie successful colonization of tissues with distinct metabolic profiles in the mammalian host.</p></div

<i>T</i>. <i>cruzi</i> infection increases host mitochondrial content and respiration.

<p><b>(A)</b> Extracellular lactate measured in culture supernatants of uninfected and <i>T</i>. <i>cruzi</i>-infected NHDF monolayers (48 hpi). Mean ± SD shown for 2 biological replicates each. Student’s t-test was applied. <b>(B)</b> Oxygen consumption rate (OCR) in uninfected and <i>T</i>. <i>cruzi</i>-infected NHDF monolayers (48hpi) before and after injection of oligomycin (O), FCCP (F), and rotenone and antimycin A (R/A). Mean ± SD shown for 4 biological replicates. <b>(C)</b> <i>T</i>. <i>cruzi</i>-infected NHDF monolayers were treated with 1 μM ELQ300 to selectively remove amastigote respiration from the total OCR signal (Infected, <i>±</i>ELQ300). Increased host respiration during <i>T</i>. <i>cruzi</i> infection (Infected, +ELQ300). Mean ± SD shown for 4 biological replicates per condition. Two-way ANOVA with Tukey’s multiple comparisons test was applied (*p<0.05, ***p<0.001, ****p<0.0001). <b>(D)</b> Geometric mean fluorescence intensity of mitochondrial mCherry signal for each condition relative to uninfected controls in NHDF and C2C12 myoblast. Infected cells were discriminated based on <i>T</i>. <i>cruzi</i> GFP expression (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006747#ppat.1006747.s002" target="_blank">S2D Fig</a>), and the geometric mean mCherry fluorescence was determined from each subpopulation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006747#ppat.1006747.s002" target="_blank">S2E Fig</a>). Mean ± SD for 2 independent experiments. Two-way ANOVA with Dunnett’s multiple comparisons test was applied (*p< 0.05, **p< 0.01).</p

Acquisition and metabolism of glucose by intracellular <i>T</i>. <i>cruzi</i> amastigotes.

<p>Isolated <i>T</i>. <i>cruzi</i> amastigotes utilize exogenous substrates as determined by increased <b>(A)</b> oxygen consumption rate (OCR) and <b>(B)</b> extracellular acidification rate (ECAR). After establishing baseline rates, glucose (5 mM), glutamine (5 mM) or buffer were injected as substrates (subs), followed by 100 mM 2-DG to rapidly inhibit glycolysis, and 1 μM rotenone and antimycin A (R/A) to shut down mitochondrial respiration. Mean ± SD of 3 biological replicates. <b>(C)</b> Initial rate (V₀) of [³H]-2-DG uptake by isolated <i>T</i>. <i>cruzi</i> amastigotes or trypomastigotes plotted for a range of substrate concentrations. Mean ± SD of two independent experiments with biological duplicates shown for each lifecycle stage. Inset shows Lineweaver-Burk plot. <b>(D)</b> Intracellular <i>T</i>. <i>cruzi</i> amastigotes access exogenous hexose <i>in situ</i>. <i>T</i>. <i>cruzi-</i>infected monolayers were incubated with 10 μCi [³H]-2-DG in the absence or presence of cytochalasin B (15 μM) for 20 minutes prior to isolation of intracellular amastigotes for scintillation counts. Mean ± SD of two independent experiments. Student’s t-test was applied (**p< 0.01). <b>(E)</b> [³H]-2-DG is internalized by intracellular amastigotes. Following isolation from monolayers pulsed with [³H]-2-DG, treatment of amastigotes with 0.05 mg/mL alamethicin released internalized, non-bound substrate. Mean ± SD of two independent experiments. <b>(F)</b> ATP levels measured in intracellular-derived amastigotes 24 hours after incubation with the indicated carbon substrate relative to initial ATP levels of freshly isolated parasites. Mean ± SD of 3 biological replicates per condition. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the substrate deficient condition (***p< 0.001, ****p< 0.0001).</p

<i>T</i>. <i>cruzi</i> amastigotes incorporate exogenous glucose into multiple metabolic pathways.

<p><i>T</i>. <i>cruzi</i> amastigotes incorporate exogenous glucose into multiple metabolic pathways.</p

Intracellular <i>T</i>. <i>cruzi</i> replication is sensitive to exogenous glucose but not host mitochondrial electron transport chain activity.

<p><b>(A)</b> Proliferation of <i>T</i>. <i>cruzi</i> amastigotes in human dermal fibroblasts with ETC complex III deficiency (CIII mutant) or two independent control fibroblast lines (Normal 1 and 2) derived from flow cytometric data (as detailed in Methods). Data are normalized to represent the percentage of initial amastigotes (18 hpi) that divided the indicated number of times by 48 hpi. Mean ± SD of 2 independent experiments. Dotted lines represent average number of complete amastigote divisions achieved by 48 hpi in each condition. <b>(B)</b> Proliferation of <i>T</i>. <i>cruzi</i> amastigotes in NHDF cultured in medium with varying glucose concentrations. Dotted lines represent average number of amastigote divisions achieved by 48 hpi as determined by flow cytometry of CFSE-labeled parasites. <b>(C)</b> Dose-dependent inhibition of <i>T</i>. <i>cruzi</i> growth in NHDF by 2-deoxyglucose (2-DG) in varying glucose concentrations. Relative number of <i>T</i>. <i>cruzi</i>-ß-galactosidase parasites assessed by Beta-Glo luminescence at 66 hpi shown with nonlinear fit using log(inhibitor) vs. response with variable slope. Mean ± SD of 4 biological replicates per point. <b>(D)</b> Arrest of <i>T</i>. <i>cruzi</i> amastigote proliferation in NHDF in the presence of 2 mM 2-DG under conditions of glucose depletion. Dotted lines represent average number of amastigote divisions achieved by 48 hpi as determined by flow cytometry of CFSE-labeled parasites. <b>(E)</b> Fluorescence micrographs of aldehyde-fixed, DAPI-stained NHDF monolayers corresponding to conditions used in panel D, in which host cell nuclei (large) and parasite DNA (smaller dots) are readily observed. Arrows point to 2 intracellular amastigotes that persist after severe growth restriction caused by glucose withdrawal and 2-DG treatment.</p

<i>T</i>. <i>cruzi</i> infection increases glucose uptake by host cells.

<p><b>(A)</b> Uptake of [³H]-2-deoxyglucose ([³H]-2-DG) into uninfected or <i>T</i>. <i>cruzi</i>-infected NHDF monolayers at 48 hours post infection (48 hpi), in which infection was established with a varying multiplicity of infection (MOI). Mean ± SD shown for 3 biological replicates per MOI. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the uninfected control group (*p<0.05, ****p<0.0001). <b>(B)</b> Cytochalasin B (10 μM) blocks uptake of [³H]-2-DG in uninfected and infected NHDF monolayers (48 hpi). Mean ± SD shown for 3 biological replicates. Two-way ANOVA with Tukey’s multiple comparisons test was applied (****p<. 0001)<b>. (C)</b> [³H]-2-DG uptake by NHDF or <b>(D)</b> C2C12 myoblasts following a 48 h infection with <i>T</i>. <i>cruzi</i> Tulahuén, CL Brener or CL-14 strains. NHDF were infected for 2 hours with MOI 40 for Tulahuén strain and MOI 150 for CL Brener and CL-14 strains. C2C12 were infected for 2 hours with MOI 80 for Tulahuén strain and MOI 150 for CL Brener and CL-14 strains. Mean ± SD for 3 biological replicates. One-way ANOVA with Dunnett’s multiple comparison test was applied for individual comparisons to the uninfected control group (**p< 0.01, ***p< 0.001, ****p< 0.0001).</p

<i>Tg</i>DegP is a rhoptry bulb protein.

<p>(A) Graph representing microarray data of transcripts encoding some known rhoptry proteins (RON2, RON4, ROP5, ROP12, ROP18) along with DegP transcript hourly following thymidine synchronization (from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189556#pone.0189556.ref029" target="_blank">29</a>]). (B) Primary structure of <i>Tg</i>DegP. <i>Tg</i>DegP is a putative serine protease of 956 amino acids belonging to HtrA family. Signal peptide is shaded in black (SP), the catalytic domain is represented in red and H, D and S indicate the positions of the catalytic triad. The two PDZ domains are represented in green. The rat anti-DegP has been made against the complete DegP recombinant protein and the mouse anti-DegP* has been generated against a recombinant protein encompassing residues 426 to 726. (C) Immunofluorescence performed on intracellular parasites using the anti-DegP serum, anti-RON4 and anti-ROP1. On parasites fixed with methanol (upper panel), the rabbit polyclonal anti-RON4 antibodies revealed the neck of the rhoptry. On paraformaldehyde-fixed parasites (lower panel), the DegP protein co-localizes with the bulbous ROP1 protein (labeled with the rabbit polyclonal anti-ROP1) in the bulb of the rhoptry. Parasite boundaries are represented by dashed lines. Scale bar = 1μm. (D) Generation of a knock-out <i>DegP</i> parasites in type I strain RHΔ<i>Ku80</i>. Scheme depicting the strategy used to obtain a KO-<i>DegP</i>^<i>I</i> cell line. The promoter of <i>TgDegP</i> is represented by an arrow. Integration of the plasmid by a single homologous recombination at the <i>DegP</i> locus results in a truncated version of DegP driven by the endogenous <i>DegP</i> promoter while the <i>DegP</i> coding sequence is now promoter less. HXGPRT: hypoxanthine guanine phosphoribosyl transferase selection marker. The solid arrows represent the primers used to verify the integration of the integrative vector and the expected size of the fragment is shown in italics. ML673 hybridized to a sequence specific of the vector, while ML871 is located in the 5’UTR of <i>DegP</i> outside the cloned fragment. (E) PCR verification of the correct integration of the vector at the endogenous <i>DegP</i> locus. The primers ML673 and ML871 are used in this PCR. The recombined locus was detectable only in transgenic parasites KO-<i>DegP</i>^<i>I</i>, as shown by a specific amplification of a 707 bp fragment that is not amplified in the parental strain. (F) Immunofluorescence performed on KO-<i>DegP</i>^<i>I</i> or RHΔ<i>Ku80</i> cell lines with the rat anti-<i>Tg</i>DegP antibodies and rabbit anti-ROP2 antibodies as control for rhoptry staining. The DegP labelling is absent in the KO-<i>DegP</i>^<i>I</i> transgenic parasites. ROP2 staining remains unchanged in the mutant. Scale bar = 1 μm.</p

Deletion of DegP in type II strain affects the <i>in vivo</i> virulence of the parasite.

<p>(A) Confluent monolayers of human fibroblasts were infected with PruΔ<i>Ku80</i> or KO-<i>DegP</i>^<i>II</i> parasites and grown for seven days. Quantification of the size of the lysis plaques is shown in the graph. The size of the lysis plaques is similar between control (PruΔ<i>Ku80</i>) and KO-<i>DegP</i>^<i>II</i> strains. A.U.: arbitrary units. Values represent means ± SEM, n = 3, from a representative experiment out of two independent assays (p = 0.82; unpaired t-test). (B) Intracellular growth rate was assessed by counting the numbers of parasites per vacuole after 20 hours infection of HFF cells with PruΔ<i>Ku80</i> or KO-<i>DegP</i>^<i>II</i> parasites. Data Values represent means ± SEM, n = 3, from a representative experiment out of two independent assays (0.05<*<0.1; unpaired t-test). (C) Host cell invasion efficiencies of RHΔ<i>Ku80</i> or KO-<i>DegP</i>^<i>II</i> strains determined by a two-color staining protocol that distinguishes intracellular from extracellular parasites. Data are mean values ± SEM determined by triplicate assays, performed in two separate experiments (p = 0.48; unpaired t-test). (D) Mouse survival was monitored daily for 40 days after i.p injection of 10⁵ or 10⁶ parasites of PruΔ<i>Ku80</i> or KO-<i>DegP</i>^<i>II</i> parasites. N = 10 mice per group. Representative data out of 2 experiments. The immune response of surviving animals was tested by Western blotting against PruΔ<i>Ku80</i> tachyzoite lysates. In Swiss mice, KO-<i>DegP</i>^<i>II</i> strain is statistically less virulent than RHΔ<i>Ku80</i> (p<0.001, by Logrank test for10⁵ and 10⁶ i.p injections). (E) Mouse survival in immunodeficient NOG mice after i.p. injection of 10⁵ parasites PruΔ<i>Ku80</i> or KO-<i>DegP</i>^<i>II</i> parasites. N = 10 mice per group. Representative data out of 2 experiments. In NOG mice, the virulence of KO-<i>DegP</i>^<i>II</i> and RHΔ<i>Ku80</i> was not statistically different (p = 0.146, by Logrank test).</p

Characterization of <i>Toxoplasma</i> DegP, a rhoptry serine protease crucial for lethal infection in mice

<div><p>During the infection process, Apicomplexa discharge their secretory organelles called micronemes, rhoptries and dense granules to sustain host cell invasion, intracellular replication and to modulate host cell pathways and immune responses. Herein, we describe the <i>Toxoplasma gondii</i> Deg-like serine protein (<i>Tg</i>DegP), a rhoptry protein homologous to High temperature requirement A (HtrA) or Deg-like family of serine proteases. <i>Tg</i>DegP undergoes processing in both types I and II strains as most of the rhoptries proteins. We show that genetic disruption of the <i>degP</i> gene does not impact the parasite lytic cycle <i>in vitro</i> but affects virulence in mice. While in a type I strain DegP^I appears dispensable for the establishment of an infection, removal of DegP^II in a type II strain dramatically impairs the virulence. Finally, we show that KO-<i>DegP</i>^<i>II</i> parasites kill immunodeficient mice as efficiently as the wild-type strain indicating that the protease might be involved in the complex crosstalk that the parasite engaged with the host immune response. Thus, this study unravels a novel rhoptry protein in <i>T</i>. <i>gondii</i> important for the establishment of lethal infection.</p></div