GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response

Abstract

The obligate intracellular parasite Toxoplasma gondii is able to infect a broad range of hosts and cell types due, in part, to the diverse arsenal of effectors it secretes into the host cell. Here, using genetic crosses between type II and type III Toxoplasma strains and quantitative trait locus (QTL) mapping of the changes they induce in macrophage gene expression, we identify a novel dense granule protein, GRA25. Encoded on chromosome IX, GRA25 is a phosphoprotein that is secreted outside the parasites and is found within the parasitophorous vacuole. In vitro experiments with a type II ⌬gra25 strain showed that macrophages infected with this strain secrete lower levels of CCL2 and CXCL1 than those infected with the wild-type or complemented control parasites. In vivo experiments showed that mice infected with a type II ⌬gra25 strain are able to survive an otherwise lethal dose of Toxoplasma tachyzoites and that complementation of the mutant with an ectopic copy of GRA25 largely rescues this phenotype. Interestingly, the type II and type III versions of GRA25 differ in endogenous expression levels; however, both are able to promote parasite expansion in vivo when expressed in a type II ⌬gra25 strain. These data establish GRA25 as a novel virulence factor and immune modulator.T oxoplasma gondii is an obligate intracellular parasite with the remarkable ability to infect a broad range of mammalian and avian hosts. During infection in the mouse, a natural Toxoplasma host, macrophages are known to play a key role in mounting an immune response to infection via the secretion of cytokines such as interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-␣) (1). The secretion of these cytokines leads to the activation of NK cells and CD4 ϩ and CD8 ϩ T cells (2-4). Although macrophages can themselves be targets of parasite infection, they also can control parasite dissemination through cell-intrinsic, antimicrobial strategies, such as the production of nitric oxide (NO) and reactive oxygen species (ROS) and, in mice, the activation of immunityrelated GTPases (IRGs) (5, 6). Recent studies have shown that inflammatory monocytes, in particular, are recruited to the site of infection by the cytokine CCL2 (monocyte chemotactic protein-1 [MCP-1]) (7, 8). As has been described for infection with several pathogens (9), CCL2 is essential for control of parasite spread and survival of infection in both intraperitoneal (i.p.) and oral Toxoplasma infections (7,8).The strains of Toxoplasma that predominate in Europe and North America, termed types I, II, and III, differ in a wide range of phenotypes, including how they interface with the immune response. The tachyzoite stage of the parasite interacts with the host through secretion of polymorphic proteins from specialized organelles termed rhoptries, which house "ROP" proteins, and dense granules, which contain "GRA" proteins. One such polymorphic protein is the tyrosine kinase ROP16. Upon injection into the host cell, it directly phosphorylates STAT3 and STAT6, and in cells infec...

An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells

“…Consistent with the NF-κB results, GRA15 and GRA25 , which encode two dense granule proteins known to modulate NF-κB activity and chemokine secretion (Ccl2 and Cxcl1), respectively, during infection with tachyzoites [62,78], were expressed in both forms albeit at somewhat higher levels in the infecting sporozoites. Transcripts encoding other well-characterized tachyzoite effectors [51,53], namely ROP16, ROP5, and ROP18 were also all expressed at similar levels in both sporozoites and tachyzoites.…”

Human placental syncytiotrophoblasts restrictToxoplasma gondiivertical transmission at two distinct stages and induce CCL22 in response to infection

“…Given that CCL22 induction in PHT cells required live parasites and was not induced by N. caninum , we reasoned that CCL22 induction was likely the result of a T. gondii -specific parasite effector that would be secreted after host cell invasion. T. gondii MYR1 is a recently identified dense granule protein that is required for the export/secretion of multiple dense granule effectors (including GRA24, GRA25 and GRA16;(15, 20, 21)) and was discovered based on its role in mediating T. gondii -specific activation of the transcription factor c-Myc(14). When we infected PHT cells with T. gondii lacking MYR1 (RH:Δ MYR1 ) and complemented control parasites (RH: Δ MYR1 : MYR1c ), we found that CCL22 production by PHT cells was entirely dependent upon MYR1 ( Figure 4F ), which provides strong evidence that T. gondii CCL22 induction in PHT cells is driven by (a) MYR1-dependent secreted effector(s).…”

The Secreted Acid Phosphatase Domain-Containing GRA44 from Toxoplasma gondii Is Required for c-Myc Induction in Infected Cells

“…Proteomic analysis of proteins that coimmunoprecipitated with GRA44 revealed interactions with known secreted proteins, including numerous GRAs. Among these interactors are GRA9, part of the intravacuolar tubular network (30); GRA16, an effector altering the host cell cycle through the p53 pathway (31); GRA25, a macrophagedependent immune modulator (32); and GRA33, GRA34, GRA45, GRA50, GRA52, and members of the multicopy mitochondrion association factor 1 (MAF1) family. MAF1 is involved in recruiting the host mitochondria to the PV membrane surface (33).…”