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

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          Abstract

          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.

          Author Summary

          Toxoplasma gondii can infect any warm-blooded animal and is transmitted orally by consumption of tissue cysts. To facilitate transmission, the parasite must balance induction and evasion of host immune responses to allow parasite growth and persistence, while avoiding excessive parasite burden, which can kill the host before infectious cysts are formed. Different strains of Toxoplasma have likely evolved specific effector molecules to modulate the immune responses of different hosts. In many mammals, including mice but not humans, the cytokine interferon gamma (IFNγ) induces the immunity-related GTPases (IRGs), which are essential to the murine immune response to Toxoplasma. They function by binding to and disrupting the parasite-containing vacuole. However, some Toxoplasma strains prevent the IRGs from disrupting the parasitophorous vacuole. It was previously shown that the secreted Toxoplasma kinase ROP18 promotes virulence in mice by phosphorylating the IRGs, leading to their inactivation. We report that ROP18 requires another virulence factor, the secreted pseudokinase ROP5, to prevent IRG accumulation, and these two proteins determine the majority of strain differences in IRG evasion, even for divergent strains for which virulence determinants have not been studied. Additionally, we show that ROP18 and ROP5 do not affect Toxoplasma survival in IFNγ-stimulated human cells. Thus, ROP18 and ROP5 are strain- and host-specific determinants of Toxoplasma immune evasion.

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          Most cited references 46

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          MEGA2: molecular evolutionary genetics analysis software.

          We have developed a new software package, Molecular Evolutionary Genetics Analysis version 2 (MEGA2), for exploring and analyzing aligned DNA or protein sequences from an evolutionary perspective. MEGA2 vastly extends the capabilities of MEGA version 1 by: (1) facilitating analyses of large datasets; (2) enabling creation and analyses of groups of sequences; (3) enabling specification of domains and genes; (4) expanding the repertoire of statistical methods for molecular evolutionary studies; and (5) adding new modules for visual representation of input data and output results on the Microsoft Windows platform. http://www.megasoftware.net. s.kumar@asu.edu
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            Sensitive and accurate detection of copy number variants using read depth of coverage.

            Methods for the direct detection of copy number variation (CNV) genome-wide have become effective instruments for identifying genetic risk factors for disease. The application of next-generation sequencing platforms to genetic studies promises to improve sensitivity to detect CNVs as well as inversions, indels, and SNPs. New computational approaches are needed to systematically detect these variants from genome sequence data. Existing sequence-based approaches for CNV detection are primarily based on paired-end read mapping (PEM) as reported previously by Tuzun et al. and Korbel et al. Due to limitations of the PEM approach, some classes of CNVs are difficult to ascertain, including large insertions and variants located within complex genomic regions. To overcome these limitations, we developed a method for CNV detection using read depth of coverage. Event-wise testing (EWT) is a method based on significance testing. In contrast to standard segmentation algorithms that typically operate by performing likelihood evaluation for every point in the genome, EWT works on intervals of data points, rapidly searching for specific classes of events. Overall false-positive rate is controlled by testing the significance of each possible event and adjusting for multiple testing. Deletions and duplications detected in an individual genome by EWT are examined across multiple genomes to identify polymorphism between individuals. We estimated error rates using simulations based on real data, and we applied EWT to the analysis of chromosome 1 from paired-end shotgun sequence data (30x) on five individuals. Our results suggest that analysis of read depth is an effective approach for the detection of CNVs, and it captures structural variants that are refractory to established PEM-based methods.
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              Polymorphic secreted kinases are key virulence factors in toxoplasmosis.

              The majority of known Toxoplasma gondii isolates from Europe and North America belong to three clonal lines that differ dramatically in their virulence, depending on the host. To identify the responsible genes, we mapped virulence in F(1) progeny derived from crosses between type II and type III strains, which we introduced into mice. Five virulence (VIR) loci were thus identified, and for two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule. Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                June 2012
                June 2012
                28 June 2012
                : 8
                : 6
                Affiliations
                [1 ]Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
                [2 ]Department of Cell Biology and Immunology, Wageningen University and Research Centre, Wageningen, The Netherlands
                [3 ]Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
                [4 ]Group of Molecular Parasitology, Universidad del Quindio, Quindio, Colombia
                [5 ]Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America
                University of Geneva, Switzerland
                Author notes

                Conceived and designed the experiments: WN JS DAG JPJS. Performed the experiments: WN JS EER DAG AF MBM DL ES. Analyzed the data: WN JS MBM DAG JPJS. Contributed reagents/materials/analysis tools: DL ES MBY. Wrote the paper: WN JS DAG JPJS.

                Article
                PPATHOGENS-D-11-02761
                10.1371/journal.ppat.1002784
                3386190
                22761577
                Niedelman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 16
                Categories
                Research Article
                Biology
                Genetics
                Immunology
                Microbiology
                Molecular Cell Biology

                Infectious disease & Microbiology

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