RESUMEN

Lung adenocarcinoma is the most prevalent subtype of lung cancer, which is the leading cause of cancer-related mortality worldwide. Toxoplasma gondii (T.gondii) Rhoptry protein 16 (ROP16) has been shown to quickly enter the nucleus, and through activate host cell signaling pathways by phosphorylation STAT3 and may affect the survival of tumor cells. This study constructed recombinant lentiviral expression vector of T. gondii ROP16 I/II/III and stably transfected them into A549 cells, and the effects of ROP16 on cell proliferation, cell cycle, apoptosis, invasion, and migration of A549 cells were explored by utilizing CCK-8, flow cytometry, qPCR, Western blotting, TUNEL, Transwell assay, and cell scratch assay, and these effects were confirmed in the primary human lung adenocarcinoma cells from postoperative cancer tissues of patients. The type I and III ROP16 activate STAT3 and inhibited A549 cell proliferation, regulated the expression of p21, CDK6, CyclinD1, and induced cell cycle arrest at the G1 phase. ROP16 also regulated the Bax, Bcl-2, p53, cleaved-Caspase3, and Caspase9, inducing cell apoptosis, and reduced the invasion and migration of A549 cells, while type II ROP16 protein had no such effect. Furthermore, in the regulation of ROP16 on primary lung adenocarcinoma cells, type I and III ROP16 showed the same anticancer potential. These findings confirmed the anti-lung adenocarcinoma effect of type I and III ROP16, offering fresh perspectives on the possible application of ROP16 as a target with adjuvant therapy for lung adenocarcinoma and propelling the field of precision therapy research toward parasite treatment of tumors.

Asunto(s)

Adenocarcinoma del Pulmón , Apoptosis , Proliferación Celular , Neoplasias Pulmonares , Proteínas Protozoarias , Toxoplasma , Humanos , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/tratamiento farmacológico , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Toxoplasma/genética , Toxoplasma/metabolismo , Movimiento Celular , Células A549 , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Proteínas Tirosina Quinasas

RESUMEN

cGAS-STING signaling is a major pathway in inducing type Ⅰ IFN, which plays a crucial role in the defense against T. gondii infection. In contrast, T. gondii develops multiple strategies to counteract the host defense, causing serious diseases in a wide range of hosts. Here, we demonstrate that T. gondii rhoptry protein 16 (ROP16) dampens type I interferon signaling via the inhibition of the cGAS (cyclic GMP-AMP synthase) pathway through the polyubiquitination of STING. Mechanistically, ROP16 interacts with STING through the SignalP domain and inhibits the K63-linked ubiquitination of STING in an NLS (nuclear localization signal)-domain-dependent manner. Consequently, knocking out the ROP16 in PRU tachyzoites promotes the STING-mediated production of type I IFNs and limits the replication of T. gondii. Together, these findings describe a distinct pathway where T. gondii exploits the ubiquitination of STING to evade host anti-parasite immunity, revealing new insights into the interaction between the host and parasites.

Asunto(s)

Interferón Tipo I , Toxoplasma , Proteínas de la Membrana/metabolismo , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , Ubiquitinación , Interferón Tipo I/metabolismo

RESUMEN

Introduction: Toxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic "Regulator Of CD8 T cell Response" (ROCTR). Methods: Therefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages. Results: Genetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii. Discussion: Collectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii.

Asunto(s)

Toxoplasma , Animales , Ratones , Sitios de Carácter Cuantitativo , Proteínas Protozoarias/metabolismo , Interferón gamma/metabolismo , Linfocitos T CD8-positivos , Diferenciación Celular

RESUMEN

BACKGROUND: T. gondii infection is characterized by a high global prevalence. Nearly, 16-40% of people have been infected by T. gondii. Although T. gondii often causes subclinical infection, it may cause severe complications in newborns with congenital infection and immunocompromised individuals. Constant attempts of scientists have made valuable findings in the development of T. gondii candidate vaccines. However, an effective vaccine has not been successfully developed yet. In this study, multi-epitope SAG1, MIC4, ROP16, M2AP, GRA12, and multi-epitope ROP8 were injected into BALB/c mice intramuscularly, as cocktailed plasmids or as single-gene plasmids to assess the immune response against chronic and acute Toxoplasma infection. METHODS: BALB/c mice were immunized on days 0, 21, and 42. The immune responses of both vaccinated and control groups were evaluated using cytokine and antibody measurements, lymphocyte proliferation assay, survival time, and average number of cysts in each brain. RESULTS: The results indicated that DNA vaccination using multi-epitope ROP8 and multi-epitope SAG1, ROP16, MIC4, GRA12, M2AP could elicit both cellular and humoral immune responses, and enhanced the survival time in BALB/c mice. Also, the administration of multi-epitope ROP8 plus multi-epitope SAG1, ROP16, MIC4, GRA12, M2AP could enhance the concentrations of IgG antibody, elicit a mixed IgG1/IgG2a reaction with the predominance of the IgG2a, increase the release of IFN-γ cytokine, prolonge the survival time, and reduce the brain cysts. CONCLUSIONS: Here, we report that vaccination using cocktailed plasmids could induce better protective immunity compared to single plasmid for acute and chronic T. gondii infection.

Asunto(s)

Vacunas Antiprotozoos , Toxoplasma , Toxoplasmosis , Vacunas de ADN , Ratones , Animales , Ratones Endogámicos BALB C , Epítopos de Linfocito T , Antígenos de Protozoos/genética , Anticuerpos Antiprotozoarios , Proteínas Protozoarias/genética , Toxoplasma/genética , Inmunoglobulina G , Citocinas , Linfocitos T , ADN

RESUMEN

Polymorphic effector proteins determine the susceptibility of Toxoplasma gondii strains to IFN-γ-mediated clearance mechanisms deployed by murine host cells. However, less is known about the influence of these polymorphic effector proteins on IFN-γ-independent clearance mechanisms. Here, we show that deletion of one such polymorphic effector protein, ROP16, from a type III background leads to a defect in parasite growth and survival in unstimulated human fibroblasts and murine macrophages. Rescue of these defects requires a ROP16 with a functional kinase domain and the ability to activate a specific family of host cell transcription factors (STAT3, 5a, and 6). The growth and survival defects correlate with an accumulation of host cell reactive oxygen species (ROS) and are prevented by treatment with an ROS inhibitor. Exogenous activation of STAT3 and 6 suppresses host cell ROS production during infection with ROP16-deficient parasites and depletion of STAT6, but not STAT3 or 5a, causes an accumulation of ROS in cells infected with wild-type parasites. Pharmacological inhibition of NOX2 and mitochondrially derived ROS also rescues growth and survival of ROP16-deficient parasites. Collectively, these findings reveal an IFN-γ-independent mechanism of parasite restriction in human cells that is subverted by injection of ROP16 by type III parasites.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that infects up to one-third of the world's population. Control of the parasite is largely accomplished by IFN-γ-dependent mechanisms that stimulate innate and adaptive immune responses. Parasite suppression of IFN-γ-stimulated responses has been linked to proteins that the parasite secretes into its host cell. These secreted proteins vary by T. gondii strain and determine strain-specific lethality in mice. How these strain-specific polymorphic effector proteins affect IFN-γ-independent parasite control mechanisms in human and murine cells is not well known. This study shows that one such secreted protein, ROP16, enables efficient parasite growth and survival by suppressing IFN-γ-independent production of ROS by human and mouse cells.

Asunto(s)

Interacciones Huésped-Parásitos , Proteínas Tirosina Quinasas/genética , Proteínas Protozoarias/genética , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Factor de Transcripción STAT6/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasma/genética , Animales , Línea Celular , Fibroblastos/parasitología , Humanos , Inmunidad Innata , Macrófagos , Ratones , Proteínas Tirosina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Factor de Transcripción STAT6/metabolismo , Células THP-1 , Toxoplasma/inmunología , Toxoplasma/fisiología

RESUMEN

Toxoplasmosis, caused by Toxoplasma gondii, is a common parasitic disease, affecting almost one-third of the world's population. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the production of appropriate vaccines against this pathogen is an important goal to avoid toxoplasmosis. considering the role of rhoptry antigens like ROP16 in virulence and satisfactory immunogenicity, they can be used as promising vaccine candidates against T. gondii. In the present study, an in silico approach was used to analyze various aspects of the ROP16 protein, including physicochemical characteristics, the potential epitopes of B and T-cells, the secondary and tertiary structure, the subcellular localization, the transmembrane domain, and other important features of this protein using several bioinformatics tools to design a proper vaccine against T. gondii. The results showed that ROP16 protein includes 93 potential post-translational modification sites. The secondary structure of the ROP16 protein comprises 34.23% alpha-helix, 54.46% random coil, and 11.32% extended strand. Moreover, several potential B- and T-cell epitopes were identified for ROP16. Based on the results of Ramachandran plot, 84.64% of the amino acid residues were located in the favored, 10.34% in allowed, and 5.02% in outlier regions. Furthermore, the results of the antigenicity and allergenicity assessment noted that this protein was immunogenic and non-allergenic. Our findings suggested that structural and functional predictions applied to ROP16 protein using in silico tools can reduce the failure risk of the laboratory studies. This research provided an important basis for further studies and also developed an effective vaccine against acute and chronic toxoplasmosis by various strategies. Further studies are needed on the development of vaccines in vivo using ROP16 alone or in combination with other antigens in the future.

RESUMEN

Toxoplasmosis, caused by the apicomplexan parasite Toxoplasma gondii, is one of the most common infections in the world due to the lifelong persistence of this parasite in a latent stage. This parasite hijacks host signaling pathways through epigenetic mechanisms which converge on key nuclear proteins. Here, we report a new parasite persistence strategy involving T. gondii rhoptry protein ROP16 secreted early during invasion, which targets the transcription factor UHRF1 (ubiquitin-like containing PHD and RING fingers domain 1), and leads to host cell cycle arrest. This is mediated by DNMT activity and chromatin remodeling at the cyclin B1 gene promoter through recruitment of phosphorylated UHRF1 associated with a repressive multienzymatic protein complex. This leads to deacetylation and methylation of histone H3 surrounding the cyclin B1 promoter to epigenetically silence its transcriptional activity. Moreover, T. gondii infection causes DNA hypermethylation in its host cell, by upregulation of DNMTs. ROP16 is already known to activate and phosphorylate protective immunity transcription factors such as STAT 3/6/5 and modulate host signaling pathways in a strain-dependent manner. Like in the case of STAT6, the strain-dependent effects of ROP16 on UHRF1 are dependent on a single amino-acid polymorphism in ROP16. This study demonstrates that Toxoplasma hijacks a new epigenetic initiator, UHRF1, through an early event initiated by the ROP16 parasite kinase.

Asunto(s)

Proteínas Potenciadoras de Unión a CCAAT/genética , Ciclina B1/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/fisiología , Toxoplasmosis/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular , Ciclina B1/metabolismo , Epigénesis Genética , Interacciones Huésped-Parásitos , Humanos , Fosforilación , Regiones Promotoras Genéticas , Toxoplasmosis/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo

RESUMEN

The obligate intracellular protozoan parasite Toxoplasma gondii can infect any nucleated cell from a warm-blooded host. However, its interaction with host macrophages plays a critical role in shaping the immune response during infection. Therefore, assessing Toxoplasma-macrophage interactions at a cellular level is important. In this chapter, we describe assays that can be used to characterize Toxoplasma-macrophage interactions. These assays can also be used to evaluate other host-pathogen interactions. We describe multiplex approaches for measuring arginase activity, indoleamine 2,3 dioxygenase activity, cell death, and parasite growth during Toxoplasma-macrophage interactions. These assays can be used to compare how different Toxoplasma strains differ in their interaction with macrophages, and we describe how to properly assess Toxoplasma strain differences in Toxoplasma-macrophage interactions.

Asunto(s)

Macrófagos/citología , Toxoplasma/citología , Animales , Arginasa/metabolismo , Células Cultivadas , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratas , Toxoplasma/efectos de los fármacos , Toxoplasma/metabolismo

RESUMEN

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn's disease. AIM: To explore the beneficial effect of ToxoROP16I/III-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells. METHODS: RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) (M1 cells) were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro. The expression of ToxoROP16I/III was observed in RAW264.7 macrophages that were transfected with pEGFP-rop16 I/III. The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, transforming growth factor (TGF)-ß1, IL-10, inducible nitric oxide synthase (iNOS), and arginase-1 (Arg-1) was detected. The expression of iNOS, Arg-1, signal transducer and activator of transcription 3 (Stat3), p-Stat3, Stat6, p-Stat6, programmed death ligand-2 (PD-L2), caspase-3, -8, and -9 was analyzed by Western blotting, and Griess assays were performed to detect nitric oxide (NO). TNF-α, IL-1ß, IL-6, TGF-ß1, and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay, and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system. RESULTS: M1 cells exhibited significantly increased production of iNOS, NO, TNF-α, IL-1ß, and IL-6, while ToxoROP16I/III induced macrophage bias to M2 cells in vitro, showing increased expression of Arg-1, IL-10 and TGF-ß1 and elevated production of p-Stat3 and p-Stat6. The mixed M1 and M2 cell culture induced by ToxoROP16I/III exhibited decreased production of NO and iNOS and upregulated expression of Arg-1 and PD-L2. Accordingly, Caco-2 cells became apoptotic, and apoptosis-associated proteins such as caspase-3, -8 and -9 were dampened during co-culture of M1 and M2 cells. Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells, but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis. CONCLUSION: ToxoROP16I/III-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages. This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.

Asunto(s)

Enfermedades Inflamatorias del Intestino/fisiopatología , Enfermedades Inflamatorias del Intestino/terapia , Mucosa Intestinal/fisiopatología , Macrófagos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Animales , Apoptosis , Células CACO-2 , Técnicas de Cocultivo , Citocinas/metabolismo , Regulación hacia Abajo , Homeostasis , Humanos , Inmunoterapia , Inflamación , Lentivirus , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Ratones , Fenotipo , Células RAW 264.7

RESUMEN

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-γ, IL-10, and IL-1ß 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 (RHΔrop16 and RHΔrop18). 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-γ, IL-10, IL-1ß, 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 RHΔrop16, whereas a significant difference in the levels of inflammatory cytokines IFN-γ and IL-1ß 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-γ 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.

Asunto(s)

Leucocitos Mononucleares/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Tirosina Quinasas/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Citocinas/metabolismo , Fibroblastos , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunología , Humanos , Leucocitos Mononucleares/metabolismo , Polimorfismo Genético , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT6/metabolismo , Toxoplasma/patogenicidad , Toxoplasmosis/genética , Virulencia , Factores de Virulencia/inmunología

RESUMEN

Imbalance of Th1 and Th2 response at the maternal-fetal interface is considered as a radical event in the pathogenesis of immunity-related pregnant diseases. It has been demonstrated that the ROP16I, a rhoptry protein of Toxoplasma gondii, and the viable parasite with ROP16I may induce M2 macrophage polarization in host innate immunity and may be involved in the adverse pregnant outcomes. However, the mechanisms by which T. gondii-derived effectors subvert the immune tolerance in the pathology of pregnancy remain unclear. Here, we constructed the RH strain with ROP16I deletion (RHΔrop16) to explore the pathogenesis of abnormal pregnancy. We found that C57BL/6 mice infected with RHΔrop16 exhibited the increased resorption of fetuses and more severe adverse pathology of placentae at the early phase of gestation, as compared to the mice infected with RH wild type (RH WT) parasite. Additionally, RHΔrop16 strain infection significantly promoted M1 macrophage phenotypes of CD80 and CD86, and decreased CD206 expression of M2 macrophages, with upregulation of the iNOS and downregulation of the Arg-1 expression in placental homogenates. Simultaneously, the pro-inflammatory cytokines of IL-12 and TNF-α were elevated whereas the anti-inflammatory cytokine of TGF-ß1 was dampened. Moreover, the p38α mitogen-activated protein kinase (p38α MAPK) was notably phosphorylated in placental macrophages infected with both RHΔrop16 and RH WT strains compared with the control. Taken together, our findings indicated that ROP16I deletion of type I RH strain may cause exacerbated adverse pregnant outcomes, which is attributable to subversion of the maternal immune tolerance due to the increased pro-inflammatory cytokines in the pregnant animals. The results also suggest that ROP16I might be a protective factor and other T. gondii-derived molecules might be involved in the M1-Th1 biased pathological process in aberrant pregnancy at the early phase of gestation.

RESUMEN

Toxoplasma gondii infection evokes a strong Th1-type response with interleukin (IL)-12 and interferon (IFN)-γ secretion. Recent studies suggest that the infection of pregnant mice with T. gondii may lead to adverse pregnancy results caused by subversion of physiological immune tolerance at maternofetal interface rather than direct invasion of the parasite. Genotype-associated dense granule protein GRA15II tends to induce classically activated macrophage (M1) differentiation and subsequently activating NK, Th1, and Th17 cells whereas rhoptry protein ROP16I/III drives macrophages to alternatively activated macrophage (M2) polarization and elicits Th2 immune response. Unlike the archetypal strains of types I, II, and III, type Chinese 1 strains possess both GRA15II and ROP16I/III, suggesting a distinct pathogenesis of Toxoplasma-involved adverse pregnancies. We constructed T. gondii type Chinese 1 strain of WH3Δrop16 based on CRISPR/Cas9 technology to explore the ROP16I/III-deficient/GRA15II-dominant parasites in induction of trophoblast apoptosis in vitro and abnormal pregnant outcomes of mice in vivo. Our study showed that Toxoplasma WH3Δrop16 remarkably induced apoptosis of trophoblasts. C57BL/6 pregnant mice injected with the tachyzoites of WH3Δrop16 presented increased absorptivity of fetuses in comparison with the mice infected with WH3 wild type (WH3 WT) parasites although no remarkable difference of virulence to mice was seen between the two strains. Additionally, the mice inoculated with WH3Δrop16 tachyzoites exhibited a notable expression of both IL-17A and IFN-γ, while the percentage of CD4+CD25+FoxP3 [T regulatory cells (Tregs)] were diminished in splenocytes and placenta tissues compared to those infected with WH3 WT parasites. Accordingly, expressions of IL-4, IL-10, and transforming growth factor beta 1, the pivotal cytokines of Th2 and Tregs response, were significantly dampened whereas IFN-γ and IL-12 expressions were upregulated in WH3Δrop16-infected mice, which gave rise to more prominent outcomes of abnormal pregnancies. Our results indicated that the WH3Δrop16 parasites with gra15II background of T. gondii type Chinese 1 strains may cause miscarriage and stillbirth due to subversion of the maternal immune tolerance and system immunity of the animals and the GRA15II effector contributes to the process of adverse pregnant consequences.

Asunto(s)

Antecedentes Genéticos , Predisposición Genética a la Enfermedad , Complicaciones Infecciosas del Embarazo/parasitología , Resultado del Embarazo , Toxoplasma/clasificación , Toxoplasma/genética , Toxoplasmosis Animal/parasitología , Animales , Apoptosis , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Femenino , Marcación de Gen , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/parasitología , Masculino , Ratones , Placenta , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Bazo/inmunología , Bazo/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Toxoplasmosis Animal/inmunología , Trofoblastos/metabolismo

RESUMEN

The aim of this study is to explore the changes and significance of gene expression profile induced by ROPl6 of Toxoplasma gondii (T.gondii)type Ⅰ,Ⅱ,Ⅲ.The A549 cell line was used as the research model.And,stable transfected ROPl6 of type Ⅰ,Ⅱ,Ⅲ of T.gondii cell lines were established.Then,we used gene expression microarray to identify genes that were differentially expressed.Gene Ontology (GO)functional classification and pathway analysis were performed on screened differentially expressed genes.According to the results of the gene expression profile,ROP16Itransfer group owned 1 270 different genes,with 776 genes up-regulated and 494 genes down-regulated;ROP16Ⅲtransfer group owned 1 362 differ-ent genes,with 842 genes up-regulated and 520 genes down-regulated;ROP16Ⅱtransfer group owned 11 052 different genes, with 5 063 genes up-regulated and 5 989 genes down-regulated.The ROP16Ⅰ/Ⅲgroup has the similar expression spectrum. However,ROP16Ⅱtransfer group was very different,which has distinct molecular profiles.Pathway analysis showed 74 path-way changes of the ROP16Ⅱtransfer group,and 19 pathways was solely dominated by it.In addition,these pathways were re-lated to NF-kappa B,Toll-like receptor,Chemokine signa-ling pathway and inflammatory response,immune response and metabolic process.Above all,ROP1 6 could affect host cell gene expression profiles.And,compared to Toxoplas-ma gondii type Ⅰ,Ⅲ,type Ⅱ has unique gene expression profiles and signaling pathway.Understanding its unique signaling pathway is very important for the prevention of type Ⅱ T.gondii.

RESUMEN

Toxoplasma gondii, as a zoonotic protozoan parasite, develops sophisticated strategies to manipulate hosts for efficient intracellular survival. After successful invasion, T. gondii injects many effector proteins into host cells for various purposes. TgROP16 (T. gondii rhoptry protein 16), which is secreted from rhoptries into host cells, can activate the host STAT (signal transducer and activator of transcription) signaling pathway through phosphorylation of STAT3 and STAT6. However, whether there are other host proteins modulated by TgROP16 is currently unknown. In this study, yeast two-hybrid (Y2H) screen was used to look for additional host proteins interacting with TgROP16. Yeast cells expressing a mouse cDNA library cloned into the prey vector were used to mate with yeasts expressing ROP16 without signal peptide. Two mouse proteins, Dnaja1 (DnaJ heat shock protein family member A1) and Gabra4 (gamma-aminobutyric acid A receptor, subunit alpha 4) were identified to interact with ROP16 from this screen. Further analysis suggested that the Predomain of ROP16 played key roles in mediating interactions with these host proteins, whereas the contribution from the Kinase domain was minor. The interactions between Dnaja1 and different parts of ROP16 were also estimated in vivo by co-immunoprecipitation. The results showed that the Predomain of ROP16 was the major region to interact with Dnaja1, which is consistent with the Y2H results. Based on the gene ontology analysis, Dnaja1 is predicted to participate in stress response while Gabra4 is involved in the system development process. The discovery of new host proteins that interact with ROP16 of T. gondii will help us to further investigate the functions of this effector proteins during T. gondii infection.

RESUMEN

We previously demonstrated that the survival time of BALB/c mice challenged with Toxoplasma gondii RH strain was prolonged by immunising the mice with a eukaryotic vector expressing the protein ROP16 of T. gondii. Building upon previous findings, we are exploring improved vaccination strategies to enhance protection. In this work, a novel recombinant canine adenovirus type 2 expressing ROP16 (CAV-2-ROP16) of T. gondii was constructed and identified to express ROP16 in Madin-Darby canine kidney cells (MDCK) cells by western blot (WB) and indirect immunofluorescence (IFA) assays. Intramuscular immunisation of BALB/c mice with CAV-2-ROP16 was performed to evaluate the humoral and cellular immune responses. This vaccination triggered significant humoral and cellular responses, including ROP16-stimulated lymphoproliferation (P<0.05). Compared to control groups, the CAV-2-ROP16 immunised mice had high production of IFN-γ, IL-2 and IL-12 (P<0.05), with a predominance of IgG2a production, but not IL-10 (P>0.05), revealing that a predominant Th1-type response had developed. The cell-mediated cytotoxic activity with high levels of IFN-γ and TNF-α was significantly increased in both CD4+ and CD8+ T-cell compartments in the mice immunised with CAV-2-ROP16 (P<0.05), compared to three control groups. In addition, when immunised mice were challenged with the RH strain of T. gondii, they showed a significantly increased survival rate (25%) 80days post infection compared with control mice that all died within seven days (P<0.05). The 25% protection rate elicited by the recombinant virus CAV-2-ROP16 has not been achieved in the field of anti-T. gondii vaccination until now. Our work presents the successful use of recombinant virus CAV-2-ROP16 in vaccination protocols to protect against intraperitoneal challenge with the virulent RH strain of T. gondii. This system was shown to be extremely efficient in eliciting humoral and cellular immune responses that led to a significant improvement in survival time in mice.

Asunto(s)

Adenovirus Caninos/genética , Proteínas Tirosina Quinasas/inmunología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Citocinas/sangre , Perros , Femenino , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/administración & dosificación , Vacunas Antiprotozoos/química , Vacunas Antiprotozoos/genética , Bazo/citología , Bazo/inmunología , Toxoplasmosis Animal/parasitología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/química , Vacunas de ADN/genética , Vacunas de ADN/inmunología

RESUMEN

Toxoplasma gondii, the causative agent of toxoplasmosis, encodes two casein kinase 1 (CK1) isoforms, CK1α and CK1ß, with only CK1α having enzyme activity. Here we investigated the biological role of CK1α by construction of a CK1α deletion mutant (Δck1α) based on the type I parasite, and complement the mutant with restored expression of CK1α. Deletion of CK1α resulted in markedly defective parasite replication in vitro. Infected mice with Δck1α parasite caused suppression of IL-12 production, severe liver damage, higher tissue burdens, and short survival time relative to the CK1α-positive parental strain. Western blot analysis revealed that deletion of CK1α led to increased activation of the signal transducer and activator of transcription (STAT)-3 in infected mice and bone marrow-derived microphages. The transcriptome analysis showed that deletion of CK1α may increase expression of rhoptry proteins (ROPs). Western blot showed enhanced expression of ROP16 in the Δck1α parasite as compared with the wild-type and complemented parasites. These findings demonstrated that deletion of CK1α may increase acute virulence of T. gondii in mice by increased expression of ROPs, activation of STAT3, and suppression of IL-12 production, which have important implications for elucidating regulation mechanism of virulence factors for T. gondii.

Asunto(s)

Quinasa de la Caseína I/genética , Toxoplasma/genética , Toxoplasma/patogenicidad , Toxoplasmosis/parasitología , Virulencia/genética , Animales , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Femenino , Regulación de la Expresión Génica/genética , Humanos , Interleucina-12/metabolismo , Hígado/parasitología , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Factor de Transcripción STAT3/genética , Análisis de Supervivencia , Toxoplasma/enzimología , Toxoplasmosis/mortalidad , Toxoplasmosis/patología , Transcriptoma , Células Vero

RESUMEN

The protozoan parasite Toxoplasma gondii is one of the most successful known eukaryotic pathogens on Earth. Virulence of T. gondii strains varies greatly in mice, and mounting evidence suggests that such variations may be relevant to the manifestation of human toxoplasmosis. Polymorphic rhoptry-secreted kinases and pseudokinases (ROP) have been demonstrated to account for murine virulence among the archetypal clonal parasite lineages that dominate the populations of North America and Europe. However, the distribution of virulence gene alleles in natural populations and the broad influence of these allele combinations on T. gondii virulence have not been examined in depth. In the present study, we performed PCR-RFLP genotyping analysis on a diverse array of globally distributed T. gondii strains at four ROP gene loci including ROP18, ROP5, ROP16 and ROP17 that were previously implicated in influencing T. gondii virulence and pathogenesis. We demonstrated through correlation with published virulence data that the combination of ROP18 and ROP5 allele types is highly predictive of T. gondii virulence across a broad range of global T. gondii isolates. These findings indicate that the importance of ROP18 and ROP5 in determining strain virulence is not limited to the North American/European archetypal lineages most commonly used in molecular studies, but also appears to apply to diverse isolates from South/central America and Asia. Restriction fragment length polymorphism analysis of these loci may thus serve as a valuable tool in determining the potential virulence of uncharacterized T. gondii strains in future studies.

Asunto(s)

Genes Protozoarios , Proteínas Serina-Treonina Quinasas/genética , Toxoplasma/genética , Toxoplasma/patogenicidad , Alelos , Animales , Ratones , Proteínas Protozoarias , Especificidad de la Especie , Virulencia/genética

RESUMEN

BACKGROUND: ROP16 is a protein kinase of Toxoplasma gondii identified in the mouse model as a virulent marker, but it is unknown whether this finding is relevant in human toxoplasmosis. METHODS: We obtained the Toxoplasma ROP16 locus DNA sequence in samples from 12 patients with ocular toxoplasmosis, 1 sample from a patient with congenital toxoplasmosis, 22 samples from soldiers operating in the jungle, 2 samples from urban soldiers, and 10 samples from meat for human consumption. An enzyme-linked immunosorbent assay specific for antibodies against the ROP16 mouse-virulent peptide was performed in 46 serum specimens from patients with ocular toxoplasmosis and in 28 serum specimens from patients with chronic asymptomatic infection, of whom 19 had congenital infection and 11 had toxoplasmic lymphadenitis. RESULTS: We found a striking divergence of the ROP16 nucleotide sequences. Ten of 12 sequences (83.3%) from patients with ocular toxoplasmosis clustered with those of mouse-virulent strains, whereas 7 of 7 ROP16 sequences (100%) from meat were clustered with those of mouse-avirulent strains. Only 11 of 104 serum specimens (10.5%) had specific antibodies against the mouse-virulent peptide, and there was no association between clinical forms and positive results of serological assays. CONCLUSIONS: The majority of ROP16 nucleotide sequences from Colombian patients with ocular toxoplasmosis belonged to the group of mouse-virulent strains.

Asunto(s)

Variación Genética , Carne/parasitología , Proteínas Tirosina Quinasas/genética , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/aislamiento & purificación , Toxoplasmosis/parasitología , Niño , Colombia , Femenino , Genotipo , Humanos , Masculino , Embarazo , Análisis de Secuencia de ADN