Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular ?-tubulin.
Ontology highlight
ABSTRACT:
SUBMITTER: Takemae H
PROVIDER: S-EPMC3824165 | biostudies-other | 2013
REPOSITORIES: biostudies-other
Publications
Characterization of the interaction between Toxoplasma gondii rhoptry neck protein 4 and host cellular β-tubulin.
Takemae Hitoshi H Sugi Tatsuki T Kobayashi Kyousuke K Gong Haiyan H Ishiwa Akiko A Recuenco Frances C FC Murakoshi Fumi F Iwanaga Tatsuya T Inomata Atsuko A Horimoto Taisuke T Akashi Hiroomi H Kato Kentaro K
Scientific reports 20131112
Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion. TgRON4 is exposed on the cytosolic side of the host cell during invasion, but its molecular interactions remain unclear. Here, we identified host cellular β-tubulin as a binding partner of TgRON4, but not Plasmodium RON4. Coimmunoprecipitation studies in mammalian cells demonstrated that the C-terminal 15-kDa region of β-tubulin was sufficient for bin ...[more]
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Project description:Toxoplasma gondii uses unique secretory organelles called rhoptries to inject an array of effector proteins into the host cytoplasm that hijack host cell functions. We have discovered a novel rhoptry pseudokinase effector, ROP54, which is injected into the host cell upon invasion and traffics to the cytoplasmic face of the parasitophorous vacuole membrane (PVM). Disruption of ROP54 in a type II strain of T. gondii does not affect growth in vitro but results in a 100-fold decrease in virulence in vivo, suggesting that ROP54 modulates some aspect of the host immune response. We show that parasites lacking ROP54 are more susceptible to macrophage-dependent clearance, further suggesting that ROP54 is involved in evasion of innate immunity. To determine how ROP54 modulates parasite virulence, we examined the loading of two known innate immune effectors, immunity-related GTPase b6 (IRGb6) and guanylate binding protein 2 (GBP2), in wild-type and ∆rop54II mutant parasites. While no difference in IRGb6 loading was seen, we observed a substantial increase in GBP2 loading on the parasitophorous vacuole (PV) of ROP54-disrupted parasites. These results demonstrate that ROP54 is a novel rhoptry effector protein that promotes Toxoplasma infections by modulating GBP2 loading onto parasite-containing vacuoles. IMPORTANCE The interactions between intracellular microbes and their host cells can lead to the discovery of novel drug targets. During Toxoplasma infections, host cells express an array of immunity-related GTPases (IRGs) and guanylate binding proteins (GBPs) that load onto the parasite-containing vacuole to clear the parasite. To counter this mechanism, the parasite secretes effector proteins that traffic to the vacuole to disarm the immunity-related loading proteins and evade the immune response. While the interplay between host IRGs and Toxoplasma effector proteins is well understood, little is known about how Toxoplasma neutralizes the GBP response. We describe here a T. gondii pseudokinase effector, ROP54, that localizes to the vacuole upon invasion and is critical for parasite virulence. Toxoplasma vacuoles lacking ROP54 display an increased loading of the host immune factor GBP2, but not IRGb6, indicating that ROP54 plays a distinct role in immune evasion.
| S-EPMC4863586 | biostudies-other
Project description:Toxoplasmosis is a cosmopolitan zoonotic infection, caused by a unicellular protozoan parasite known as Toxoplasma gondii that belongs to the phylum Apicomplexa. It is estimated that over one-third of the world's population has been exposed and are latently infected with the parasite. In humans, toxoplasmosis is predominantly asymptomatic in immunocompetent persons, while among immunocompromised individuals may be cause severe and progressive complications with poor prognosis. Moreover, seronegative pregnant mothers are other risk groups for acquiring the infection. The life cycle of T. gondii is very complex, indicating the presence of a plurality of antigenic epitopes. Despite of great advances, recognize and construct novel vaccines for prevent and control of toxoplasmosis in both humans and animals is still remains a great challenge for researchers to select potential protein sequences as the ideal antigens. Notably, in several past years, constant efforts of researchers have made considerable advances to elucidate the different aspects of the cell and molecular biology of T. gondii mainly on microneme antigens, dense granule antigens, surface antigens, and rhoptry proteins (ROP). These attempts thereby provided great impetus to the present focus on vaccine development, according to the defined subcellular components of the parasite. Although, currently there is no commercial vaccine for use in humans. Among the main identified T. gondii antigens, ROPs appear as a putative vaccine candidate that are vital for invasion procedure as well as survival within host cells. Overall, it is estimated that they occupy about 1%-30% of the total parasite cell volume. In this review, we have summarized the recent progress of ROP-based vaccine development through various strategies from DNA vaccines, epitope or multi epitope-based vaccines, recombinant protein vaccines to vaccines based on live-attenuated vectors and prime-boost strategies in different mouse models.
| S-EPMC6369123 | biostudies-literature
Project description:One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.
| S-EPMC2642630 | biostudies-literature
Project description:Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells.
| S-EPMC3299665 | biostudies-literature
Project description:The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.
| S-EPMC3946946 | biostudies-literature
Project description:Toxoplasma gondii is an obligatory intracellular apicomplexan protozoan which can infect any warm-blooded animal and causes severe diseases in immunocompromised individuals or infants infected in utero. The survival and success of this parasite require that it colonizes the host cell, avoids host immune defenses, replicates within an appropriate niche, and exits the infected host cell to spread to neighboring non-infected cells. All of these processes depend on the parasite ability to synthesis and export secreted proteins. Amongst the secreted proteins, rhoptry organelle proteins (ROPs) are essential for the parasite invasion and host cell manipulation. Even though the functions of most ROPs have been elucidated in the less virulent T. gondii (type II), the roles of ROPs in the highly virulent type I strain remain largely un-characterized. Herein, we investigated the contributions of 15 ROPs (ROP10, ROP11, ROP15, ROP20, ROP23, ROP31, ROP32, ROP33, ROP34, ROP35, ROP36, ROP40, ROP41, ROP46, and ROP47) to the infectivity of the high virulent type I T. gondii (RH strain). Using CRISPR-Cas9, these 15 ROPs genes were successfully disrupted and the effects of gene knockout on the parasite's ability to infect cells in vitro and BALB/c mice in vivo were investigated. These results showed that deletions of these ROPs did not interfere with the parasite ability to grow in cultured human foreskin fibroblast cells and did not significantly alter parasite pathogenicity for BALB/c mice. Although these ROPs did not seem to be essential for the acute infectious stage of type I T. gondii in the mouse model, they might have different functions in other intermediate hosts or play different roles in other life cycle forms of this parasite due to the different expression patterns; this warrants further investigations.
| S-EPMC5258691 | biostudies-literature
Project description:Toxoplasma gondii is an obligate intracellular parasite belonging to the phylum Apicomplexa that infects all warm-blooded animals, including humans. T. gondii can replicate in every nucleated host cell by orchestrating metabolic interactions to derive crucial nutrients. In this review, we summarize the current status of known metabolic interactions of T. gondii with its host cell and discuss open questions and promising experimental approaches that will allow further dissection of the host-parasite interface and discovery of ways to efficiently target both tachyzoite and bradyzoite forms of T. gondii, which are associated with acute and chronic infection, respectively.
| S-EPMC6208699 | biostudies-literature
Project description:UnlabelledIngestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the ?rop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5, ROP17, ROP18, ROP35, or ROP38/29/19 (ROP38, ROP29, and ROP19) severely reduced cyst burdens. ?rop5 and ?rop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The ?rop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the ?rop35 or ?rop38/29/19 strain. Resistance to host IRGs was not affected in the ?rop17, ?rop35, or ?rop38/29/19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondiiImportanceReactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections.
| S-EPMC4959664 | biostudies-literature
Project description:Toxoplasma gondii secretes rhoptry (ROP) and dense granule (GRA) effector proteins to evade host immune clearance mediated by interferon gamma (IFN-γ), immunity-related GTPase (IRG) effectors, and CD8+ T cells. Here, we investigated the role of parasite-secreted effectors in regulating host access to parasitophorous vacuole (PV) localized parasite antigens and their presentation to CD8+ T cells by the major histocompatibility class I (MHC-I) pathway. Antigen presentation of PV localized parasite antigens by MHC-I was significantly increased in macrophages and/or dendritic cells infected with mutant parasites that lacked expression of secreted GRA (GRA2, GRA3, GRA4, GRA5, GRA7, GRA12) or ROP (ROP5, ROP18) effectors. The ability of various secreted GRA or ROP effectors to suppress antigen presentation by MHC-I was dependent on cell type, expression of IFN-γ, or host IRG effectors. The suppression of antigen presentation by ROP5, ROP18, and GRA7 correlated with a role for these molecules in preventing PV disruption by IFN-γ-activated host IRG effectors. However, GRA2 mediated suppression of antigen presentation was not correlated with PV disruption. In addition, the GRA2 antigen presentation phenotypes were strictly co-dependent on the expression of the GRA6 protein. These results show that MHC-I antigen presentation of PV localized parasite antigens was controlled by mechanisms that were dependent or independent of IRG effector mediated PV disruption. Our findings suggest that the GRA6 protein underpins an important mechanism that enhances CD8+ T cell recognition of parasite-infected cells with damaged or ruptured PV membranes. However, in intact PVs, parasite secreted effector proteins that associate with the PV membrane or the intravacuolar network membranes play important roles to actively suppress antigen presentation by MHC-I to reduce CD8+ T cell recognition and clearance of Toxoplasma gondii infected host cells.
| S-EPMC6742963 | biostudies-literature
Project description:Toxoplasma gondii, a representative model organism belonging to the phylum Apicomplexa, can infect almost all warm-blooded organisms, including humans. The invasion of host cells via host-parasite interaction is the key step for T. gondii to complete its life cycle. Herein we performed tandem mass tag analysis to investigate global proteomic changes in host cells (human foreskin fibroblasts, HFFs) [HFFs infected with T. gondii (HT) vs. HFFs (H)] and T. gondii [HT vs. T. gondii (T)] during intracellular infection. Overall, 3477 and 1434 proteins were quantified, of which 375 and 1099 proteins were differentially expressed (adjusted p-value < 0.05 and >1.5 or <0.67-fold change) in host cells and T. gondii, respectively. T. gondii invasion relies on the secretion of numerous secretory proteins, which originate from three secretory organelles: micronemes, rhoptries, and dense granules. In the HT vs. T group, few secretory proteins were upregulated, such as microneme proteins (MICs: MIC6, MIC10), rhoptry bulb proteins (ROPs: ROP5, ROP17), and dense granule proteins (GRAs: GRA4, GRA5, GRA12). In contrast, dozens of known secretory proteins were significantly downregulated in T. gondii-infected HFFs. In HFFs, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed a large number of differentially expressed proteins (DEPs) enriched in metabolic processes and immune-associated signaling pathways, such as NF-κB, cAMP, and Rap1 signaling pathways. Further, in case of T. gondii, DEPs were involved in ribosome biogenesis, citrate cycle, and galactose metabolism, indicating that cell biosynthesis and metabolism of T. gondii were altered after host cell invasion. These findings reveal novel modifications in the proteome of host cells as well as T. gondii, helping us better understand the mechanisms underlying host-parasite interaction.
| S-EPMC8158437 | biostudies-literature