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      The Polymorphic Pseudokinase ROP5 Controls Virulence in Toxoplasma gondii by Regulating the Active Kinase ROP18

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          Abstract

          Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δ rop5 and Δ rop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3 −/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.

          Author Summary

          The ability of microorganisms to cause disease in their hosts is often mediated by proteins that are secreted by the pathogen into the host cell as a means of disarming host signaling. Previous studies with the protozoan parasite Toxoplasma gondii have revealed that secretion of parasite protein kinases into the host cell mediates virulence in mouse, a natural host for transmission. Curiously, some of these virulence factors are active protein kinases, while other related pseudokinases lack enzymatic activity; hence, it was unclear how they functioned in promoting virulence. In the present work we demonstrate that ROP5, an inactive member of this protein kinase family, regulates the active protein kinase ROP18, which normally prevents clearance of the parasite in interferon-activated macrophages. Allosteric regulation of enzymes is a common theme in biology, but this is the first example of such a mechanism regulating a pathogen virulence factor. The potential advantage of such a layered process is that it might allow greater temporal or spatial control and perhaps protect the parasite from disabling strategies by the host.

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          Most cited references50

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          The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

          Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration. Copyright 2001 Academic Press.
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            Human IRGM induces autophagy to eliminate intracellular mycobacteria.

            Immunity-related p47 guanosine triphosphatases (IRG) play a role in defense against intracellular pathogens. We found that the murine Irgm1 (LRG-47) guanosine triphosphatase induced autophagy and generated large autolysosomal organelles as a mechanism for the elimination of intracellular Mycobacterium tuberculosis. We also identified a function for a human IRG protein in the control of intracellular pathogens and report that the human Irgm1 ortholog, IRGM, plays a role in autophagy and in the reduction of intracellular bacillary load.
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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
                November 2012
                November 2012
                8 November 2012
                : 8
                : 11
                : e1002992
                Affiliations
                [1 ]Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America
                [2 ]Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America
                [3 ]Departments of Medicine, Molecular Genetics and Microbiology, and Immunology, Division of Geriatrics, and Center for the Study of Aging and Human Development, Duke University Medical Center, Durham, North Carolina, United States of America
                [4 ]Geriatric Research, Education, and Clinical Center, VA Medical Center, Durham, North Carolina, United States of America
                Cornell University, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MSB SJF MM LXL LDS. Performed the experiments: MSB SJF MM LXL. Analyzed the data: MSB SJF MM LXL LDS. Contributed reagents/materials/analysis tools: GAT. Wrote the paper: MSB SJF GAT LDS.

                Article
                PPATHOGENS-D-12-01380
                10.1371/journal.ppat.1002992
                3493473
                23144612
                8ae7fb6e-3fa9-4b8e-aaae-74d49b759517
                Copyright @ 2012

                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.

                History
                : 8 June 2012
                : 10 September 2012
                Page count
                Pages: 15
                Funding
                Supported in part by grants from the National Institutes of Health ( www.nih.gov/) (AI036629, AIAI082423). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Genetics
                Immunology
                Microbiology
                Molecular Cell Biology

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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