Host gene expression response to Toxoplasma gondii infection is not different between RH?ku80 and RH?ku80?rop5 parasites.
ABSTRACT: The normally virulent type-I RH parasite is rendered avirulent when lacking ROP5. The avirulent phenotype is a consequence of interaction with the host innate immune system. We sought to understand if ROP5 alters host gene expression in order to escape host defenses. We saw no gene expression differences between host cells infected with wt (RH?ku80) or RH?ku80?rop5 parasites. We have included uninfected HFF samples that were harvested in parallel with the infected samples. Host gene expression in response to infection with Toxoplasma gondii. Two independent samples per sample type. Three sample types: HFF infected with RH?ku80, HFF infected with RH?ku80?rop5, and uninfected HFF.
Project description:The normally virulent type-I RH parasite is rendered avirulent when lacking ROP5. The avirulent phenotype is a consequence of interaction with the host innate immune system. We sought to understand if ROP5 alters host gene expression in order to escape host defenses. We saw no gene expression differences between host cells infected with wt (RH∆ku80) or RH∆ku80∆rop5 parasites. We have included uninfected HFF samples that were harvested in parallel with the infected samples. Host gene expression in response to infection with Toxoplasma gondii. Two independent samples per sample type. Three sample types: HFF infected with RH∆ku80, HFF infected with RH∆ku80∆rop5, and uninfected HFF.
Project description:Infection of RAW264.7 cells with RH?ku80 parasites or mock-infection for 24 hours To measure changes in gene expression induced in macropahges upon Toxoplasma infection, we infected RAW 264.7 macrophages in cell culture with RH?ku80 parasites or syringe-lysed human foreskin fibroblast monolayers (mock-infected). RNA was harvested 24 hours post infection. Cells were infected with Toxoplasma or mock-infected in vitro, in duplicate
Project description:The in vitro effect of infection with different strains of Toxoplasma gondii was tested 24 hours after infection of Human Foreskin Fibroblasts (HFF) The strains tested include RH, VEG, and transgenic strains of RH overexpressing ROP38 or ROP21 Overall design: Total RNA of Toxoplasma gondii infected HFF cell was compared to uninfected cells
Project description:The in vitro effect of infection with different strains of Toxoplasma gondii was tested 24 hours after infection of Human Foreskin Fibroblasts (HFF) The strains tested include RH, VEG, and transgenic strains of RH overexpressing ROP38 or ROP21 Total RNA of Toxoplasma gondii infected HFF cell was compared to uninfected cells
Project description:The intracellular parasite Toxoplasma gondii infects a wide variety of vertebrate species globally. Infection in most hosts causes a lifelong chronic infection and generates immunological memory responses that protect the host against new infections. In regions where the organism is endemic, multiple exposures to T. gondii likely occur with great frequency, yet little is known about the interaction between a chronically infected host and the parasite strains from these areas. A widely used model to explore secondary infection entails challenge of chronically infected or vaccinated mice with the highly virulent type I RH strain. Here, we show that although vaccinated or chronically infected C57BL/6 mice are protected against the type I RH strain, they are not protected against challenge with most strains prevalent in South America or another type I strain, GT1. Genetic and genomic analyses implicated the parasite-secreted rhoptry effectors ROP5 and ROP18, which antagonize the host's gamma interferon-induced immunity-regulated GTPases (IRGs), as primary requirements for virulence during secondary infection. ROP5 and ROP18 promoted parasite superinfection in the brains of challenged survivors. We hypothesize that superinfection may be an important mechanism to generate T. gondii strain diversity, simply because two parasite strains would be present in a single meal consumed by the feline definitive host. Superinfection may drive the genetic diversity of Toxoplasma strains in South America, where most isolates are IRG resistant, compared to North America, where most strains are IRG susceptible and are derived from a few clonal lineages. In summary, ROP5 and ROP18 promote Toxoplasma virulence during reinfection.Toxoplasma gondii is a widespread parasite of warm-blooded animals and currently infects one-third of the human population. A long-standing assumption in the field is that prior exposure to this parasite protects the host from subsequent reexposure, due to the generation of protective immunological memory. However, this assumption is based on clinical data and mouse models that analyze infections with strains common to Europe infections with strains common to Europe and North America. In contrast, we found that the majority of strains sampled from around the world, in particular those from South America, were able to kill or reinfect the brains of hosts previously exposed to T. gondii. The T. gondii virulence factors ROP5 and ROP18, which inhibit key host effectors that mediate parasite killing, were required for these phenotypes. We speculate that these results underpin clinical observations that pregnant women previously exposed to Toxoplasma can develop congenital infection upon reexposure to South American strains.
Project description:Host resistance to Toxoplasma gondii relies on CD8 T cell IFN? responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFN? responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFN?. T57 IFN? responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFN? responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFN? differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFN? production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFN? responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFN? production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFN? responses to a vacuolar antigen.
Project description:BACKGROUNDS:The prevalence of toxoplasmosis is higher in schizophrenics than in the general population. It has been suggested that certain symptoms of schizophrenia, including changes in olfactory functions, are in fact symptoms of toxoplasmosis that can be easily detected in schizophrenics only due to the increased prevalence of toxoplasmosis in this population. Schizophrenics have impaired identification of odors and lower sensitivity of odor detection, however, no information about these parameters of non-schizophrenic Toxoplasma-infected subjects is available. METHODS:Here we searched for differences in olfactory functions between 62 infected and 61 noninfected non-schizophrenic subjects using the case-controls experimental design. RESULTS:The infected men scored better than the non-infected controls in the standard odor-identification test. The infected women rated all smells as more intensive while the infected men rated nearly all smells as less intensive. Infected women rated the pleasantness of the smell of the cat urine as higher than the non-infected women and the opposite was true for the men-in contrast, higher pleasantness of odor in infected men and lower in infected women were observed and described in the 2011 study. Toxoplasmosis, Rh, and toxoplasmosis-Rh interaction were not associated with the rated pleasantness of the smell of other stimuli. However, our sample contained only 17 Rh negative men and 30 Rh negative women. Therefore, all results concerning the main effects of Rh factor and the interaction with Rh factor must be considered only preliminary. CONCLUSIONS:Our results suggest that latent toxoplasmosis is associated with changes in the olfactory functions in humans; however, the observed changes differ from those observed in schizophrenics.
Project description:Toxoplasma gondii evades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronic Toxoplasma infection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cysts in vivo without affecting the parasite growth rate or the ability to differentiate into cysts in vitro Acute virulence of the Pru?gra2, Pru?gra3, and Pru?gra4 mutants was reduced but not abolished. In contrast, the Pru?gra12 mutant was avirulent in mice and Pru?gra12 parasites failed to establish a chronic infection. High-virulence type I strain RH?gra12 parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-?)-activated macrophages, type I RH?gra12 and type II Pru?gra12 parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RH?ku80 and type II Pru?ku80 strains, respectively. Despite this resistance, ?gra12 PVs ultimately succumbed to IFN-?-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-? and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCE Toxoplasma gondii cysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cysts in vivo A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-?). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.
Project description:Proteomic analysis of ionomycin-treated and untreated mammary epithelial MCF10A cells elucidated differences in Ku80 cleavage. Ku80, a subunit of the Ku protein complex, is an initiator of the non-homologous, end-joining (NHEJ), double-strand breaks (DSBs) repair pathway. The nuclear Ku80 was cleaved in a calcium concentration-dependent manner by m-calpain but not by m-calpain. The cleavage of nuclear Ku80 at its ?/? domain was validated by Western blotting analysis using flag-tagged expression vectors of truncated versions of Ku80 and a flag antibody and was confirmed in m-calpain knock-down cells and in vitro cell-free evaluation with recombinant proteins of calpains, Ku70, and Ku80. In addition, the cleaved Ku80 still formed a Ku heterodimer and promoted DNA DSB repair activity. Taken together, these findings indicate that translocated m-calpain enhances the NHEJ pathway through the cleavage of Ku80. Based on the present study, m-calpain in DNA repair pathways might be a novel anticancer drug target, or its mechanism might be a possible route for resistance acquisition of DNA damage-inducing chemotherapeutics.