Phosphorylation of Immunity-Related GTPases by a Toxoplasma gondii-Secreted Kinase Promotes Macrophage Survival and Virulence

Abstract

SUMMARY Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.

TheToxoplasma gondiirhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

“…4B). These results clearly indicate that GRA7 is an important component within the T. gondii kinase complex, contributing to phosphorylation of Irga6 by ROP5 and ROP18 (Fentress et al ., 2010; Steinfeldt et al ., 2010; Fleckenstein et al ., 2012). …”

“…4) and its apparent specificity for Irga6 (Fig. 6) stimulated us to investigate whether the overall impact of ROP18 on the IRG resistance system (Fentress et al ., 2010; Steinfeldt et al ., 2010; Behnke et al ., 2012; Fleckenstein et al ., 2012; Niedelman et al ., 2012) is also due only to specific inhibition of Irga6.…”

“…The rhoptry proteins ROP5 and ROP18 have been shown to inhibit the IRG resistance system by phosphorylation of highly conserved threonine residues in the switch I region of the nucleotide binding site (Fentress et al ., 2010; Steinfeldt et al ., 2010; Behnke et al ., 2012; Fleckenstein et al ., 2012; Niedelman et al ., 2012). In this study, we could confirm that the dense granule protein GRA7 is an additional component of the T. gondii ROP5/ROP18 kinase complex (Alaganan et al ., 2014).…”

“…In addition to Irga6, also Irgb6 and Irgb10 have been shown to be phosphorylated upon infection with T. gondii virulent type I strains in cells of laboratory mice (Fentress et al ., 2010; Steinfeldt et al ., 2010; Behnke et al ., 2012; Lilue et al ., 2013), and inactivation by the ROP5/ROP18/GRA7 kinase complex is accompanied by reduced accumulation at the PVM (e.g. Fig.…”

“…Different polymorphic rhoptry proteins were shown to be important for inhibition of the IRG resistance system. The catalytically active kinase ROP18 of T. gondii virulent type I strains phosphorylates IRG proteins at highly conserved threonines in the switch I region of the nucleotide binding site (Fentress et al ., 2010; Steinfeldt et al ., 2010). Phosphorylation of multiple family members could be demonstrated, but the target sites identified in Irga6 most closely match the ROP18 substrate phosphorylation motif (Lim et al ., 2013).…”