Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii.
Ontology highlight
ABSTRACT:
SUBMITTER: Ahn HJ
PROVIDER: S-EPMC2532663 | biostudies-literature | 2006 Sep
REPOSITORIES: biostudies-literature
Publications
Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii.
Ahn Hye-Jin HJ Kim Sehra S Nam Ho-Woo HW
The Korean journal of parasitology 20060901 3
Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3o-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5o-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 a ...[more]
Similar Datasets
Project description:Many intracellular pathogens are separated from the cytosol of their host cells by a vacuole membrane. This membrane serves as a critical interface between the pathogen and the host cell, across which nutrients are imported, wastes are excreted, and communication between the two cells takes place. Very little is known about the vacuole membrane proteins mediating these processes in any host-pathogen interaction. During a screen for monoclonal antibodies against novel surface or secreted proteins of Toxoplasma gondii, we identified ROP4, a previously uncharacterized member of the ROP2 family of proteins. We report here on the sequence, posttranslational processing, and subcellular localization of ROP4, a type I transmembrane protein. Mature, processed ROP4 is localized to the rhoptries, secretory organelles at the apical end of the parasite, and is secreted from the parasite during host cell invasion. Released ROP4 associates with the vacuole membrane and becomes phosphorylated in the infected cell. Similar results are seen with ROP2. Further analysis of ROP4 showed it to be phosphorylated on multiple sites, a subset of which result from the action of either host cell protein kinase(s) or parasite kinase(s) activated by host cell factors. The localization and posttranslational modification of ROP4 and other members of the ROP2 family of proteins within the infected cell make them well situated to play important roles in vacuole membrane function.
| S-EPMC522600 | 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:Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events, such as cell proliferation and differentiation. Toxoplasma gondii is an obligate intracellular protozoan that is both a human and animal pathogen. This Apicomplexan causes significant morbidity and mortality in immune-competent and immune-compromised hosts. In humans, the most common manifestations of T. gondii infections are chorioretinitis in congenital infection and encephalitis in immune-compromised patients, such as patients with advanced AIDS. We have identified a T. gondii homolog of the MAPK family that we have called TgMAPK2. Sequence analyses demonstrated that TgMAPK2 has homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. TgMAPK2 has an open reading frame of 2,037 bp, 678 amino acids, and its molecular weight is 73.1 kDa. It contains the typical 12 subdomains of a MAPK and has a TDY motif in the dual phosphorylation and activation subdomains. This suggests that TgMAPK2 may play an important role in stress response. recombinant TgMAPK2 was catalytically active and was not inhibited by a human ERK2 inhibitor, FR180204. A partial TgMAPK2 lacking the ATP-binding motifs GxGxxGxV was successfully regulated by a ligand-controlled destabilization domain (ddFKBP) expression vector system in T. gondii. Since TgMAPK2 is significantly different from its mammalian counterpart, it may be useful as a drug target. This work establishes a foundation for further study for this unique kinase.
| S-EPMC3266180 | biostudies-literature
Project description:A cDNA of 1.1 kb comprising the gene encoding the peroxiredoxin of Toxoplasma gondii (TgPrx) has been cloned. The open reading frame of 591 bp was translated into a protein of 196 amino acids with a molecular mass of 25 kDa. Conserved 2 cysteine domains of Phe-Val-Cys-Pro and Glu-Val-Cys-Pro indicated TgPrx belonged to 2-Cys Prx families. TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9% followed by Entamoeba histolytica with 39.5% by the amino acid sequence alignment. Polyclonal antibody against recombinant TgPrx detected 25 kDa band in T. gondii without binding to host cell proteins. TgPrx was located in the cytoplasm of T. gondii extracellularly or intracellularly by immunofluorescence assay. The expression of TgPrx was increased as early as 30 min after the treatment with artemisinin in the intracellular stage, while no changes in those of host Prx I and TgSOD. This result implies that TgPrx may function as an antioxidant protecting the cell from the attack of reactive oxygen intermediates. It is also suggested that TgPrx is a possible target of chemotherapy.
| S-EPMC2721090 | biostudies-literature
Project description:Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli, a strong lymphoproliferative response, and significant levels of gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-10 production compared with results for other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with pVAX-ROP16 showed a significantly (P < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice, which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggers a strong humoral and cellular response against T. gondii and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.
| S-EPMC3019779 | biostudies-literature
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:Toxoplasma gondii is an obligate intracellular parasite infecting humans and other warm-blooded animals, resulting in serious public health problems and economic losses worldwide. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In the present study, a DNA vaccine expressing rhoptry protein 13 (ROP13) of T. gondii inserted into eukaryotic expression vector pVAX I was constructed, and the immune protection it induced in Kunming mice was evaluated. Kunming mice were immunized intramuscularly with pVAX-ROP13 and/or with interleukin-18 (IL-18). Then, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged with the virulent T. gondii RH strain (type I) and the cyst-forming PRU strain (type II). The results showed that pVAX-ROP13 alone or with pVAX/IL-18 induced a high level of specific anti-T. gondii antibodies and specific lymphocyte proliferative responses. Coinjection of pVAX/IL-18 significantly increased the production of gamma interferon (IFN-?), IL-2, IL-4, and IL-10. Further, challenge experiments showed that coimmunization of pVAX-ROP13 with pVAX/IL-18 significantly (P < 0.05) increased survival time (32.3 ± 2.7 days) compared with pVAX-ROP13 alone (24.9 ± 2.3 days). Immunized mice challenged with T. gondii cysts (strain PRU) had a significant reduction in the number of brain cysts, suggesting that ROP13 could trigger a strong humoral and cellular response against T. gondii cyst infection and that it is a potential vaccine candidate against toxoplasmosis, which provided the foundation for further development of effective vaccines against T. gondii.
| S-EPMC3535859 | biostudies-literature
Project description:Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.
| S-EPMC5450957 | biostudies-literature
Project description:Toxoplasma gondii utilizes specialized secretory organelles called rhoptries to invade and hijack its host cell. Many rhoptry proteins are proteolytically processed at a highly conserved SΦXE site to remove organellar targeting sequences that may also affect protein activity. We have studied the trafficking and biogenesis of a secreted rhoptry metalloprotease with homology to insulysin that we named toxolysin-1 (TLN1). Through genetic ablation and molecular dissection of TLN1, we have identified the smallest rhoptry targeting domain yet reported and expanded the consensus sequence of the rhoptry pro-domain cleavage site. In addition to removal of its pro-domain, TLN1 undergoes a C-terminal cleavage event that occurs at a processing site not previously seen in Toxoplasma rhoptry proteins. While pro-domain cleavage occurs in the nascent rhoptries, processing of the C-terminal region precedes commitment to rhoptry targeting, suggesting that it is mediated by a different maturase, and we have identified residues critical for proteolysis. We have additionally shown that both pieces of TLN1 associate in a detergent-resistant complex, formation of which is necessary for trafficking of the C-terminal portion to the rhoptries. Together, these studies reveal novel processing and trafficking events that are present in the protein constituents of this unusual secretory organelle.
| S-EPMC3375832 | biostudies-literature
Project description:Ingestion 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 Δrop16 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. Δrop5 and Δrop18 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 Δrop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δrop35 or Δrop38/29/19 strain. Resistance to host IRGs was not affected in the Δrop17, Δrop35, or Δrop38/29/19 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. gondii Reactivation 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