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

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 Δrop5 and Δrop18 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.

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

“…1). Stimulation of ROP18 kinase activity was suggested earlier to be an important molecular function of ROP5 (Behnke et al ., 2012). We analysed ROP5 immunoprecipitations from detergent tachyzoite lysates of different T. gondii strains for binding of ROP18.…”

“…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.…”

“…This conformation is facilitated by direct binding of the pseudokinase ROP5 to IRG proteins (Fleckenstein et al ., 2012; Niedelman et al ., 2012). Although not apparent for an IRG‐specific function of ROP5 in our own experiments (Fleckenstein et al ., 2012), an interaction of ROP5 with ROP18 was reported to control T. gondii virulence by up‐regulation of ROP18 activity (Behnke et al ., 2012). The rop5 locus is represented by a cluster of tandemly repeated genes encoding three different ROP5 isoforms, ROP5A, ROP5B and ROP5C, each containing multiple copies, and all isoforms are predicted to be catalytically inactive (El Hajj et al ., 2006; Reese and Boothroyd, 2011).…”