Toxoplasma gondii Dense Granule Proteins 7, 14, and 15 Are Involved in Modification and Control of the Immune Response Mediated via NF-κB Pathway

Abstract

Toxoplasma gondii infects almost all warm-blooded animals, including humans, leading to both cellular and humoral immune responses in the host. The virulence of T. gondii is strain specific and is defined by secreted effector proteins that disturb host immunity. Here, we focus on nuclear factor-kappa B (NFκB) signaling, which regulates the induction of T-helper type 1 immunity. A luciferase assay for screening effector proteins, including ROPs and GRAs that have biological activity against an NFκB-dependent reporter plasmid, found that overexpression of GRA7, 14, and 15 of a type II strain resulted in a strong activity. Thus, our study was aimed at understanding the involvement of NFκB in the pathogenesis of toxoplasmosis through a comparative analysis of these three molecules. We found that GRA7 and GRA14 were partially involved in the activation of NFκB, whereas GRA15 was essential for NFκB activation. The deletion of GRA7, GRA14, and GRA15 in the type II Prugniaud (Pru) strain resulted in a defect in the nuclear translocation of RelA. Cells infected with the Pru gra15 parasite showed reduced phosphorylation of inhibitor-κBα. GRA7, GRA14, and GRA15 deficiency decreased the levels of interleukin-6 in RAW246.7 cells, and RNA-seq analysis revealed that GRA7, GRA14, and GRA15 deficiency predominantly resulted in downregulation of gene expression mediated by NFκB. The virulence of all mutant strains increased, but Pru gra14 only showed a slight increase in virulence. However, the intra-footpad injection of the highly-virulent type I RH gra14 parasites in mice resulted in increased virulence. This study shows that GRA7, 14, and 15-induced host immunity via NFκB limits parasite expansion.

Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins

“…TgGRA15 is an interesting TgGRA14-interacting protein since it has also been demonstrated by immunoprecipitation to interact with several ESCRT components [ 60 ]. Although its role in the recruitment of the ESCRT machinery remains to be further elucidated, the known function of TgGRA15 in type II strains is to activate NF-κB signaling [ 61 ], a host immune response that is also moderately stimulated by TgGRA14[ 47 ]. The extent to which TgGRA15 or other TgGRA14-interacting dense granule proteins take part in the uptake of host cytosolic proteins remains unknown.…”

The pathogenicity and virulence of Toxoplasma gondii

“… Active NF-κB pathway and macrophage activation Not studied [ 164 , 171 , 197 , 199 ] GRA12 TGGT1_288650 IVN/PVM Unknown Inhibits IFN-γ mediated parasite killing Active Active Not studied [ 172 ] GRA14 TGGT1_239740 PV/PVM/IVN Unknown Activation of NF-κB pathway and recruitment of macrophages in type II parasites, potentially in other strains too Active Active Not studied [ 173 , 199 ] GRA15 TGGT1_275470 PVM TRAF2/TRAF6 GBP1 In type II parasites, activation of NF-κB pathway via TRAF2/6 interaction leading to IL-12 and IL-1B expression. Also linked to the inhibition of lysosomes fusion and GBP loading to the PVM Truncated (and thus inactive) in some strains Active Active, but less than type II parasites [ 174 , 198 , 199 , 205 , 206 ] GRA16 TGGT1_208830 Nucleus PP2A-B55, HAUSP Modulates the expression of host cell genes involved in the control of cell-cycle progression, p53 signaling, steroids and lipids metabolism Less active? (no virulence phenotype for the KO) Active Not studied [ …”

“…Some PVM-located GRAs also have important pro-survival functions for tachyzoites, like GRA7 that promotes IRG turnover [ 197 ]. Yet, the same protein, along with other PVM-located GRAs like GRA14 and GRA15, can also be an activator of the NF-κB pathway and promote macrophage activation in strains of T. gondii which are less virulent for mice [ 198 , 199 ].…”

“…In type I parasites ROP16 activates the SAT3/STAT6 pathway to favor parasite growth [ 182 ], while type II parasites contain a polymorphic form of ROP16 that is a poor STAT activator [ 204 ]. GRA15, which is more expressed in type II than in type III parasites, can stimulate the NF-κB-dependent production of inflammatory cytokines and activates macrophages [ 198 , 199 ], but is truncated and thus inactive in the type I lab-adapted RH strain. GRA15 also stimulates the recruitment of GBP1 to the PVM through an unknown mechanism [ 205 ].…”

Dense granule protein, GRA64 interacts with host cell ESCRT proteins duringToxoplasma gondiiinfection

“…GRA6 has been shown to influence host cell NFAT activity (27), while a specific GRA15 allele has been shown to induce host cell NF-B activity (28), presumably through host cell exposure of GRA15 at the PVM. Although GRA15 is the main regulator of NF-B, GRA7 and GRA14 also modify nuclear localization of RelA/p65, a member of the NF-B, complex, from the PVM (29). Certain GRA proteins have even been found to operate beyond the PVM interface, entering the host cell cytoplasm and nucleus and influencing host cell function by interacting with specific host cell proteins (30,31).…”