Influence of Two Major Toxoplasma Gondii Virulence Factors (ROP16 and ROP18) on the Immune Response of Peripheral Blood Mononuclear Cells to Human Toxoplasmosis Infection

Toxoplasma gondii ROP16 and ROP18 proteins have been identified as important virulence factors for this parasite. Here, we describe the effect of ROP16 and ROP18 proteins on peripheral blood mononuclear cells (PBMCs) from individuals with different clinical status of infection. We evaluated IFN-?, IL-10, and IL-1? levels in supernatants from PBMCs cultures infected with tachyzoites of the T. gondii wild-type RH strain or with knock-out mutants of the rop16 and rop18 encoding genes (RH?rop16 and RH?rop18). Cytokine secretion was compared between PBMCs obtained from seronegative individuals (n = 10), with those with chronic asymptomatic (n = 8), or ocular infection (n = 12). We also evaluated if polymorphisms in the genes encoding for IFN-?, IL-10, IL-1?, Toll-like receptor 9 (TLR9), and purinoreceptor P2RX7 influenced the production of the encoded proteins after ex vivo stimulation. In individuals with chronic asymptomatic infection, only a moderate effect on IL-10 levels was observed when PBMCs were infected with RH?rop16, whereas a significant difference in the levels of inflammatory cytokines IFN-? and IL-1? was observed in seronegative individuals, but this was also dependent on the host's cytokine gene polymorphisms. Infection with ROP16-deficient parasites had a significant effect on IFN-? production in previously non-infected individuals, suggesting that ROP16 which is considered as a virulence factor plays a role during the primary infection in humans, but not in the secondary immune response. © Copyright © 2019 Hernández-de-los-Ríos, Murillo-Leon, Mantilla-Muriel, Arenas, Vargas-Montes, Cardona, de-la-Torre, Sepúlveda-Arias and Gómez-Marín

The Rhoptry Proteins ROP18 and ROP5 Mediate Toxoplasma gondii Evasion of the Murine, But Not the Human, Interferon-Gamma Response

The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans

Identificación de sustratos proteicos en celulas humanas para proteínas ROP cinasas de Toxoplasma gondii

Summary Background: Toxoplasma secretes proteins known as ROP kinases; (ROPs) which are important for establishing the survival and growth of the parasite within the host cell. It has been shown that the Toxoplasma ROP5/ROP18 complex is the major effector related to virulence in mice. This complex specifically phosphorylates interferon-induced GTPases (IRGs). There is no evidence whether this complex or other ROP kinases are involved in the development of human toxoplasmosis. Aim: To search human substrates for Toxoplasma gondii ROP11, ROP39 ROP5 and ROP18 in a human cell line. Methodology 1a: We designed an algorithm called MSCA (Multiple Spectral Comparison Alignment) for the search of interactions between proteins (Toxoplasma and Human). 2a: recombinant proteins ROP11, ROP39, and GBP5 were cloned into pGEX4T1 and expressed in E. coli ROSSETTA DE3; with these proteins we performed Pull-down assays either with mass spectrometry (MS) or Western blot (WB). 2b: yeast 2 hybrids (Y2H) protein-protein interaction screening was performed between some ROPs and human GBPs 1-5. 2c: pro-IL1β levels were compared in human PBMCs infected ex-vivo with Toxoplasmas RH or RHΔROP18. Results 1a: MSCA proposed the interactions ROP11-SLC3A2, ROP11-ANXA6, ROP39-SHMT2, ROP39-ROP5, hGBP5-ROP5, and hGBP5-ROP18. 2a the interactions ROP11-SLC3A2, ROP11-ANXA6, ROP39-SHMT2, and ROP39-ROP5 were confirmed by Pull-down MS. 2b: the interactions hGBP5-ROP18 and hGBP5-ROP5 were observed in the yeast 2 hibrid (Y2H) screening interaction. The interactions ROP39-ROP5 and hGBP5-ROP5 were confirmed by Pull-down-WB. 2c: we observed a greater amount of pro-IL1β in human cells infected with RHΔROP18 than RH. Conclusions: ROP39-ROP5 and hGBP5-ROP5 interactions were confirmed by two different techniques. We model that ROP5 interacts with human GBP5 and this interaction could alter the activation of the NLRP3-inflamosome in human PBMCs in a ROP18-dependent manner.RESUMEN Introducción: Toxoplasma secreta unas proteínas conocidas como ROP cinasas; (ROPs) las cuales son importantes para establecer la supervivencia y el crecimiento del parásito en el interior de la célula hospedera. Se ha evidenciado que el complejo ROP5/ROP18 secretado por Toxoplasma, es el mayor efector relacionado con virulencia en ratones; este complejo fosforila específicamente proteínas GTPasas inducidas por interferón (IRGs). No hay evidencia si este complejo u otras ROP cinasas están implicadas en el desarrollo de la toxoplasmosis humana. Objetivo: Evidenciar sustratos humanos para las proteínas cinasas ROP11, ROP39 ROP5 y ROP18 de Toxoplasma gondii en una línea celular humana. Metodología 1a: diseñamos un algoritmo llamado MSCA (Multiple Spectral Comparison Alignment) para la búsqueda de interacciones entre proteínas (Toxoplasma y Humano). 2a: las proteínas recombinantes ROP11, ROP39, y GBP5 se clonaron en pGEX4T1 y se expresaron en E. coli ROSSETTA DE3; con estas proteínas se realizaron ensayos de Pull-down Mass spectrometry (MS) y Pull-down Western blot (WB). 2b: utilizando el sistema de 2 híbridos en levadura (Y2H) realizamos ensayos de interacción proteína-proteína entre ROPs y GBPs humanas (Guanylate binding proteins). 2c: nosotros comparamos los niveles de pro-IL1β en PBMCs humanos infectados ex-vivo con Toxoplasma cepa RH o RHΔROP18. Resultados 1ª: MSCA propone las interacciones ROP11-SLC3A2, ROP11-ANXA6, ROP39-SHMT2, ROP39-ROP5, hGBP5-ROP5 y hGBP5-ROP18-. 2a: las interacciones ROP11-SLC3A2, ROP11-ANXA6, ROP39-SHMT2 y ROP39-ROP5 fueron confirmadas por Pull-down MS. 2b: observamos las interacciones hGBP5-ROP18 y hGBP5-ROP5- por Y2H. Se confirmaron las interacciones ROP39-ROP5 y ROP5-GBP5 por Pull-down-WB. 2c: Observamos mayor cantidad de pro-IL1β en PBMCs infectadas con RHΔROP18. Conclusiones: las interacciones ROP39-ROP5 y hGBP5-ROP5 fueron confirmadas por 2 técnicas diferentes. Sugerimos que ROP5 interacciona con GBP5 humana y esta interacción podría alterar la activación del inflamosoma dependiente de ROP18 en PBMCs humanos.I. Introducción .............................................................................. 8 II. Marco Conceptual ....................................................................10 III. Planteamiento del Problema.................................................... 23 IV. Objetivo.................................................................................... 25 V. Materiales y Métodos................................................................ 25 VI. Resultados............................................................................... 36DoctoradoDoctor en Ciencias Biomédica

The rhoptry proteins ROP18 and ROP5 mediate Toxoplasma gondii evasion of the murine, but not the human, interferon-gamma response.

The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans

Influence of Two Major Toxoplasma Gondii Virulence Factors (ROP16 and ROP18) on the Immune Response of Peripheral Blood Mononuclear Cells to Human Toxoplasmosis Infection

Toxoplasma gondii ROP16 and ROP18 proteins have been identified as important virulence factors for this parasite. Here, we describe the effect of ROP16 and ROP18 proteins on peripheral blood mononuclear cells (PBMCs) from individuals with different clinical status of infection. We evaluated IFN-?, IL-10, and IL-1? levels in supernatants from PBMCs cultures infected with tachyzoites of the T. gondii wild-type RH strain or with knock-out mutants of the rop16 and rop18 encoding genes (RH?rop16 and RH?rop18). Cytokine secretion was compared between PBMCs obtained from seronegative individuals (n = 10), with those with chronic asymptomatic (n = 8), or ocular infection (n = 12). We also evaluated if polymorphisms in the genes encoding for IFN-?, IL-10, IL-1?, Toll-like receptor 9 (TLR9), and purinoreceptor P2RX7 influenced the production of the encoded proteins after ex vivo stimulation. In individuals with chronic asymptomatic infection, only a moderate effect on IL-10 levels was observed when PBMCs were infected with RH?rop16, whereas a significant difference in the levels of inflammatory cytokines IFN-? and IL-1? was observed in seronegative individuals, but this was also dependent on the host's cytokine gene polymorphisms. Infection with ROP16-deficient parasites had a significant effect on IFN-? production in previously non-infected individuals, suggesting that ROP16 which is considered as a virulence factor plays a role during the primary infection in humans, but not in the secondary immune response. © Copyright © 2019 Hernández-de-los-Ríos, Murillo-Leon, Mantilla-Muriel, Arenas, Vargas-Montes, Cardona, de-la-Torre, Sepúlveda-Arias and Gómez-Marín

ROP39 is an Irgb10-specific parasite effector that modulates acute Toxoplasma gondii virulence.

Toxoplasma gondii (T. gondii) is a zoonotic apicomplexan parasite that is an important cause of clinical disability in humans. On a global scale, one third of the human population is infected with T. gondii. Mice and other small rodents are believed to be responsible for transmission of T. gondii to the domestic cat, its definitive host. Interferon-inducible Immunity-Related GTPases (IRG proteins) are important for control of murine T. gondii infections. Virulence differences between T. gondii strains are linked to polymorphic rhoptry proteins (ROPs) that cooperate to inactivate individual IRG family members. In particular, the pseudokinase ROP5 isoform B is critically important in laboratory strains of mice. We identified T. gondii ROP39 in complex with ROP5B and demonstrate its contribution to acute T. gondii virulence. ROP39 directly targets Irgb10 and inhibits homodimer formation of the GTPase leading to an overall reduction of IRG protein loading onto the parasitophorous vacuolar membrane (PVM). Maintenance of PVM integrity rescues the parasite from IRG protein-mediated clearance in vitro and in vivo. This study identifies a novel T. gondii effector that is important for specific inactivation of the IRG resistance system. Our data reveal that yet unknown T. gondii effectors can emerge from identification of direct interaction partners of ROP5B

GRA2, but not ROP16 or GRA15, affects IRG evasion.

<p>WT MEFs stimulated for 24 hours with IFNγ and infected with the indicated strains for 1 hour and fixed for immunofluorescence or allowed to form plaques for 4–7 days. (A,B) Quantification of Irgb6 localization on the parasite containing vacuole and percent plaque loss on stimulated MEFs compared to unstimulated MEFs infected with the indicated strains. Mean + SEM, n>4 experiments. ***p<0.001, Student's t-test.</p

ROP5-A, ROP5-B/C and ROP18 account for strain differences in IRG evasion in non-canonical strains.

<p>(A)Quantification of percentage of PVs with Irgb6 localization and percentage plaque loss on IFNγ-stimulated MEFs compared to unstimulated MEFs infected with the indicated strains. Strains that have greater than 90% mortality in CD-1 outbred mice <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002784#ppat.1002784-Khan3" target="_blank">[11]</a> are indicated in red. The predicted reason for high IRG coating is indicated below the graph, which for CEP, VEG, CASTELLS and P89 is the unexpressed <i>ROP18_III</i> allele, and for Pru, ME49, COUGAR and BOF is divergent or missing <i>ROP5</i> alleles. (B) Phylogenetic tree of <i>ROP5-A</i>, <i>B</i> and<i>C</i> and previously reported major and minor alleles <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002784#ppat.1002784-Behnke1" target="_blank">[34]</a> constructed from full-length coding nucleotide sequences using Neighbor-Joining with 1000 bootstraps. (C) Cumulative behavior, codon by codon, of synonymous (red), nonsynonymous (green) and insertion/deletion (black) mutations in <i>ROP5-A</i> (left) and <i>ROP5-B/C</i> (right). (D) Relative expression of <i>ROP5-A, ROP5-B/C</i> and <i>ROP18</i> determined by RNA-Seq of murine BMDM infected for 24 hours with the indicated strains. Samples were matched for similar levels of <i>Toxoplasma</i> RNA. <i>ROP5-A</i> and <i>B/C</i> copy number estimated by sequencing coverage of the <i>ROP5</i> locus versus the average genome coverage for strains for which the genome has been sequenced is indicated in the table below the graph.</p

Strain differences in survival in IFNγ-stimulated human cells.

<p>Monolayers of HFFs, either previously stimulated for 24 hours with IFNγ or left untreated, were infected with 100–300 parasites. The number of plaques that form after 4–7 days of growth was determined. (A) Percent plaque loss of type I strains RH and GT1, type II strains ME49 and Pru, type III strains CEP and VEG, as well as the non-canonical strains CASTELLS, GUY-MAT, GUY-KOE, RUB, GUY-DOS and VAND. Mean + Std. dev., n≥3 experiments. (B) Percent plaque loss for type III, type III + ROP18_I and type III + ROP18_II. Mean + Std. dev., n≥5 experiments. (C) Percent plaque loss of S22, S22 + LC37, Pru and Pru + LC37. Mean + Std. dev, n≥4 experiments, * p<0.05, Student's t-test.</p

A type III strain expressing type I or type II ROP18 inhibits Irgb6 accumulation and killing.

<p>WT MEFs were stimulated for 24 hours with IFNγ and infected with type I (GT1), type II (Pru), type III (CEP), CEP + ROP18_I or CEP + ROP18_II expressing GFP. (A) Cells were fixed after 1 hour and stained by immunofluorescence for Irgb6 (Red) and with Hoechst (blue). Scale bar represents 5 µm. (B) Quantification of Irgb6 localization on the parasite containing vacuole and percentage plaque loss after 4–7 days on stimulated MEFs compared to unstimulated MEFs. Mean + SEM, n = 5 experiments, ***p<0.001, Student's t test.</p