Avirulent strains of Toxoplasma gondii infect macrophages by active invasion from the phagosome.
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ABSTRACT:
SUBMITTER: Zhao Y
PROVIDER: S-EPMC4035997 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Research exploring the proteome of Toxoplasma gondii oocysts has gained momentum over the past few years. However, little is known about the oocyst's protein repertoires that contribute to differential virulence among T. gondii strains. Here, we used isobaric tag for relative and absolute quantitation-based proteomic analysis of oocysts of two T. gondii strains exhibiting the virulent PYS (ToxoDB#9) phenotype versus the less virulent PRU (Type II, ToxoDB#1) phenotype. Our aim was to determine protein expression patterns that contribute to the virulence of a particular phenotype. A total of 2,551 proteins were identified, of which 374 were differentially expressed proteins (DEPs) (|log2 fold change| ≥ 0.58 and P < 0.05). DEPs included 192 increased and 182 decreased proteins. Gene Ontology and KEGG pathway analyses revealed a large number of DEPs enriched in various metabolic processes. Protein interaction network analysis using STRING identified inosine monophosphate dehydrogenase (IMPDH), Bifunctional GMP synthase/glutamine amidotransferase protein, Glucose-6-phosphate 1-dehydrogenase, and Citrate synthase as the top four hubs. Of the 22 virulence proteins commonly expressed in the oocysts of the two strains, 13 and 2 proteins were increased in PYS strain and PRU strain, respectively. Also, 10 and 3 of the 22 identified oocyst wall proteins showed higher expression in oocysts of PRU strain and PYS strain, respectively. These findings revealed new proteomic differences in the oocysts of T. gondii strains of different genotypic backgrounds.
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Project description:The phylum Apicomplexa includes parasites responsible for global scourges such as malaria, cryptosporidiosis, and toxoplasmosis. Parasites in this phylum reproduce inside the cells of their hosts, making invasion of host cells an essential step of their life cycle. Characterizing the stages of host-cell invasion, has traditionally involved tedious microscopic observations of individual parasites over time. As an alternative, we introduce the use of compartment models for interpreting data collected from snapshots of synchronized populations of invading parasites. Parameters of the model are estimated via a maximum negative log-likelihood principle. Estimated parameter values and their 95% confidence intervals (95% CI), are consistent with reported observations of individual parasites. For RH strain parasites, our model yields that: (1) penetration of the host-cell plasma membrane takes 26s (95% CI: 22-30s); (2) parasites that ultimately invade, remain attached three times longer than parasites that eventually detach from the host cells, and (3) 25% (95% CI: 19-33%) of parasites invade while 75% (95% CI: 67-81%) eventually detach from their host cells without progressing to invasion. A key feature of the model is the incorporation of invasion stages that cannot be directly observed. This allows us to characterize the phenomenon of parasite detachment from host cells. The properties of this phenomenon would be difficult to quantify without a mathematical model. We conclude that mathematical modeling provides a powerful new tool for characterizing the stages of host-cell invasion by intracellular parasites.
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Project description:Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals, including humans. Its closest extant relative, Hammondia hammondi, has never been found to infect humans and, in contrast to T. gondii, is highly attenuated in mice. To better understand the genetic bases for these phenotypic differences, we sequenced the genome of a H. hammondi isolate (HhCatGer041) and found the genomic synteny between H. hammondi and T. gondii to be >95%. We used this genome to determine the H. hammondi primary sequence of two major T. gondii mouse virulence genes, TgROP5 and TgROP18. When we expressed these genes in T. gondii, we found that H. hammondi orthologs of TgROP5 and TgROP18 were functional. Similar to T. gondii, the HhROP5 locus is expanded, and two distinct HhROP5 paralogs increased the virulence of a T. gondii TgROP5 knockout strain. We also identified a 107 base pair promoter region, absent only in type III TgROP18, which is necessary for TgROP18 expression. This result indicates that the ROP18 promoter was active in the most recent common ancestor of these two species and that it was subsequently inactivated in progenitors of the type III lineage. Overall, these data suggest that the virulence differences between these species are not solely due to the functionality of these key virulence factors. This study provides evidence that other mechanisms, such as differences in gene expression or the lack of currently uncharacterized virulence factors, may underlie the phenotypic differences between these species.
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Project description:Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using cps, an avirulent, nonreplicating uracil auxotroph strain of the parasite Toxoplasma gondii (T. gondii), which preferentially invades immunosuppressive CD11c(+) antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c(+) cells invaded by cps were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to cps treatment of the immunosuppressive ovarian tumor environment, CD11c(+) cells regained the ability to efficiently cross-present antigen and prime CD8(+) T-cell responses. Correspondingly, cps treatment markedly increased tumor antigen-specific responses by CD8(+) T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal cps treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of cps treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with T. gondii. Taken together, our results establish that cps preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8(+) T-cell responses. Immunochemotherapeutic applications of cps might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors.
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Project description:A multiplex PCR assay was designed for multilocus strain typing of Toxoplasma gondii based on length polymorphism of five microsatellite markers. Eight T. gondii strains already sequenced at these five markers were used as control isolates. This method is simple, rapid, reproducible, and adapted to a large set of isolates.
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Project description:Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.
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Project description:We established a conditional site-specific recombination system based on dimerizable Cre recombinase-mediated recombination in the apicomplexan parasite Toxoplasma gondii. Using a new single-vector strategy that allows ligand-dependent, efficient removal of a gene of interest, we generated three knockouts of apicomplexan genes considered essential for host-cell invasion. Our findings uncovered the existence of an alternative invasion pathway in apicomplexan parasites.
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