Host Cell Vimentin Restrains Toxoplasma gondii Invasion and Phosphorylation of Vimentin is Partially Regulated by Interaction with TgROP18

Abstract

The obligate intracellular parasite, Toxoplasma gondii, manipulates the cytoskeleton of its host cells to facilitate infection. A significant rearrangement of host cell vimentin around Toxoplasma parasitophorous vacuoles is observed during the course of infection. ROP18 (TgROP18) is a serine-threonine kinase secreted by T. gondii rhoptry and a major virulence factor; however, the mechanisms by which this kinase modulates host factors remain poorly understood. Different and dynamic patterns of vimentin solubility, phosphorylation, and expression levels were observed in host cells infected with T. gondii strain RH and RH Δrop18 strains, suggesting that TgROP18 contributes to the regulation of these dynamic patterns. Additionally, host cell vimentin was demonstrated to interact with and be phosphorylated by TgROP18. A significant increase in T. gondii infection rate was observed in vimentin knockout human brain microvessel endothelial cells (HBMEC), while vimentin knockout or knock down in host cells had no impact on parasite proliferation and egress. These results indicate that host cell vimentin can inhibit T. gondii invasion. Interestingly, western blotting of different mouse tissues indicated that the lowest vimentin expression level was present in the brain, which may explain the mechanism underlying the nervous system tropism of T. gondii, and the phenomenon of huge cyst burdens developing in the mouse brain during chronic infection.

Toxoplasma gondiiROP18 Inhibits Human Glioblastoma Cell Apoptosis through Mitochondrial Pathway by Targeting Host Cell P2X1

“…pECFP-N1-ROP18¡ and pEYFP-C1-P2X1 were co-transfected for the experimental group. pECFP-N1 and pEYFP-C1 were co-transfected as a negative control and pEYFP-CFP was transfected as a positive control [49]. Forty-eight hours post transfection, the cells were fixed in 4% paraformaldehyde at 37°C for 30 minutes and then washed twice with PBS.…”

TgROP18 Targets IL20RB for Host-Defense-Related-STAT3 Activation During T. gondii Infection

“…To perform FRET and co-immunoprecipitation (Co-IP) assays, IL20RB-HA, and its extracellular and intracellular domain was then cloned into pEYFP-C1 to form pEYFPC1-IL20RB-HA, pEYFPC1-IL20RB-Extr-HA (Extr, extracellular domain of IL20RB), and pEYFPC1-IL20RB-Cyt-HA (Cyt, intracellular domain of IL20RB). The construction of pCFPN1-ROP18-3×FLAG has been described in detail elsewhere (16). All the primers used are shown in Supplementary Data (Table S1).…”

“…A chloramphenicol-resistance gene (chloramphenicol acetyltransferase, CAT) was used as the screening marker. The homologous recombination plasmid (pBlue-5'-3'-ROP18-homo-CAT) was constructed as follows (the procedure is also shown in Figure S1A); A 991 bp fragment and a 965 bp fragment homologous to the upstream and downstream of the gRNA targeted site of the rop18 gene, were respectively subcloned to the 5 -end of the CAT gene cassette on pBlue-script SK II (-) plasmid (16). Subsequently, these two recombinant plasmids were co-transfected into RH-Δrop16 tachyzoites by − a n d 3…”

“…C. HaCaT cell treatment with recombinant GST-ROP18 and GST at different amount: The construction of the prokaryotic expression vector and expression of GST-ROP18 and GST have been described in detail previously (16). The Escherichia coli expressed GST-ROP18 and GST proteins were puri ed using the GST fusion protein puri cation kit (GenScript Inc., China) following the manufacturer's instructions.…”

“…Host cell invasion by T. gondii is a rapid but complicated process (9), including cell membrane attachment, protein secretion, moving junction formation and inclusion (10)(11)(12). Host cell activities, including innate and adaptive immune responses, nutrient metabolism, cytoskeletal rearrangement, and apoptosis, are manipulated during Toxoplasma tachyzoite invasion (13)(14)(15)(16). During invasion, many of the effectors are injected into host cells by T. gondii, including rhoptry proteins (ROPs), dense granule proteins (GRAs), and microneme proteins (MIC) (17,18).…”