Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole

Abstract

The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A–D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

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

“…Strikingly and in contrast to RHΔ rop5 , RHΔ rop18 is fully virulent in Irga6 ko mice (Figs 6D and 7C). These results illustrate the interdependent accumulation of IRG proteins at the T. gondii PVM documented earlier (Khaminets et al ., 2010) and clearly demonstrate a molecular function of GRA7 and ROP18 that is specific for Irga6. The fact that loading of Irgb6 and Irgb10 in Irga6 ko cells infected with RHΔ rop18 is markedly lower than in the same cells infected with the completely avirulent RHΔ rop5 shows again the existence of ROP5‐dependent, ROP18‐independent virulence actions against IRG proteins (Fleckenstein et al ., 2012).…”

“…With T. gondii virulent type I strains, IRG protein accumulation at the PVM is markedly reduced compared with type II or III avirulent strains (Zhao et al ., 2009a; 2009c; Khaminets et al ., 2010). The lack of efficient Irga6 phosphorylation by T. gondii RHΔ gra7 (Fig.…”

“…Increased amounts of Irgb6 and Irgb10 at RHΔ gra7 ‐derived vacuoles in wt MEFs (Fig. 6A) are dependent only on elevated levels of Irga6, a cooperative interaction documented earlier (Khaminets et al ., 2010). …”

“…Activated GTP‐bound effector IRG proteins (the G K S group) accumulate in high densities at the parasitophorous vacuolar membrane (PVM) of avirulent T. gondii type II strains early after invasion, leading to PVM rupture and parasite clearance (Martens et al ., 2005; Ling et al ., 2006; Zhao et al ., 2009b). The loading is cooperative and hierarchical, with two family members serving as pioneers for members loading later (Khaminets et al ., 2010). Premature activation of IRG effectors on cellular endomembranes is prevented by three regulatory IRG proteins, Irgm1–Irgm3 (the G M S group), which keep the effector proteins in a GDP‐bound inactive conformation at endogenous cellular membranes (Hunn et al ., 2008; Haldar et al ., 2013; Maric‐Biresev et al ., in preparation).…”

“…With T. gondii virulent type I strains, the initial loading of the PVM with IRG proteins is markedly reduced (Zhao et al ., 2009c; Khaminets et al ., 2010). In order to preserve the integrity of the PVM, T. gondii has evolved several polymorphic effector proteins (Hunter and Sibley, 2012; Alaganan et al ., 2014; Etheridge et al ., 2014).…”