ROP16-Mediated Activation of STAT6 Suppresses Host Cell Reactive Oxygen Species Production, Facilitating Type III Toxoplasma gondii Growth and Survival

Abstract

Polymorphic effector proteins determine the susceptibility of Toxoplasma gondii strains to IFN-γ-mediated clearance mechanisms deployed by murine host cells. However, less is known about the influence of these polymorphic effector proteins on IFN-γ-independent clearance mechanisms. Here, we show that deletion of one such polymorphic effector protein, ROP16, from a type III background leads to a defect in parasite growth and survival in unstimulated human fibroblasts and murine macrophages. Rescue of these defects requires a ROP16 with a functional kinase domain and the ability to activate a specific family of host cell transcription factors (STAT3, 5a, and 6). The growth and survival defects correlate with an accumulation of host cell reactive oxygen species (ROS) and are prevented by treatment with an ROS inhibitor. Exogenous activation of STAT3 and 6 suppresses host cell ROS production during infection with ROP16-deficient parasites and depletion of STAT6, but not STAT3 or 5a, causes an accumulation of ROS in cells infected with wild-type parasites. Pharmacological inhibition of NOX2 and mitochondrially derived ROS also rescues growth and survival of ROP16-deficient parasites. Collectively, these findings reveal an IFN-γ-independent mechanism of parasite restriction in human cells that is subverted by injection of ROP16 by type III parasites. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects up to one-third of the world’s population. Control of the parasite is largely accomplished by IFN-γ-dependent mechanisms that stimulate innate and adaptive immune responses. Parasite suppression of IFN-γ-stimulated responses has been linked to proteins that the parasite secretes into its host cell. These secreted proteins vary by T. gondii strain and determine strain-specific lethality in mice. How these strain-specific polymorphic effector proteins affect IFN-γ-independent parasite control mechanisms in human and murine cells is not well known. This study shows that one such secreted protein, ROP16, enables efficient parasite growth and survival by suppressing IFN-γ-independent production of ROS by human and mouse cells.

ROP16-mediated activation of STAT6 facilitates encystment of type IIIToxoplasma gondiiin neurons

“…Having determined that ROP16 III -dependent host cell manipulation is essential for efficient type III encystment, we next sought to determine what domains or functions of ROP16 were required for this phenotype. To accomplish this goal, we used a previously generated panel of IIIΔ rop16 complemented strains 37 . This panel of strains includes ROP16 mutants which are catalytically inactive (ROP16 IIIKD ) or lack a nuclear localization sequence (ROP16 IIIΔNLS ) ( Fig.…”

“…This panel of strains includes ROP16 mutants which are catalytically inactive (ROP16 IIIKD ) or lack a nuclear localization sequence (ROP16 IIIΔNLS ) ( Fig. 3A ) 37 . In addition, as prior work demonstrated that a single polymorphic amino acid on ROP16 determined the strain-specific activation of STAT3/5a/6 34 , this panel also included strains which express a ROP16 III in which the leucine at position 503 was switched to a serine, rendering it “STAT-dead” (ROP16 IIISD ), and a strain that expresses a ROP16 II in which the serine at position 503 was changed to a leucine, rendering it “STAT-active” (ROP16 IISA ) 37 ( Fig.…”

“…As previously described, all parasite strains used in this study were maintained through serial passage in human foreskin fibroblasts (gift of John Boothroyd, Stanford University, Stanford, CA) using DMEM, supplemented with 10% fetal bovine serum, 2 mM glutagro, and 100 IU/ml penicillin and 100 μg/ml streptomycin. In addition to the strains described below, the previously described type III (CEP), IIIΔrop16 strains, and ROP16III parasites expressing mCherry or tdTomato were used [35][36][37]49 .…”

“…ROP16 affected encystment, we infected human foreskin fibroblasts (HFFs) with WTIII, IIIΔrop16, or strains where we re-introduced the type III allele of rop16 (ROP16III) or the type II allele of rop16 (ROP16II) [35][36][37] . We induced encystment by placing the infected cultures under alkaline stress (pH 8.2) in combination with CO2 deprivation (<5%) and tracked differentiation using immunofluorescent staining of T. gondii stage-specific surface antigens 39,40 and Dolichos biflorus agglutinin (DBA), which binds sugar moieties present on the proteins that form the cyst wall 41 .…”

Toxoplasmosis: Current and Emerging Parasite Druggable Targets

“…Moreover, direct phosphorylation of STAT3 by ROP16 mimics the IL-10 activity and downregulates IFN-γ, hence enhancing the virulence of T. gondii [134]. Recently, ROP16-mediated activation of STAT6 proved important for type III T. gondii survival through suppression of host cell reactive oxygen species production [136]. Moreover, ROP16 kinase activity silences the cyclin B1 gene promoter, hijacking the function of the host cell epigenetic machinery [137].…”