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ROP18 is a key factor responsible for virulence difference between Toxoplasma gondii and Neospora caninum.

ABSTRACT: Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) are both obligate intracellular protozoan parasites and share many common morphological and biological features. Despite these similarities the two parasites differ dramatically in virulence in mice, but the factors involved in virulence differences between the two parasites remain unknown. A secreted serine-threonine kinase called rhoptry protein 18 (ROP18) was identified to play a crucial role on virulence differences among different T. gondii clonal lineages. Intriguingly, we found that ROP18 in Nc1 strain of N. caninum (NcROP18) is a pseudogene due to several interrupting stop codons in the sequence in our previous studies. We assume that the difference of ROP18 leads to virulence difference between T. gondii and N. caninum. We constructed a transgenic N. caninum Nc1 stain by transfecting the TgROP18 from the T. gondii RH strain. Phenotype and virulence assays showed that the expression of TgROP18 in N. caninum did not affect the motility and cell invasion, but resulted in a significant increase in intracellular parasite proliferation and virulence in mice. Immunity-Related GTPase (IRG) phosphorylation assay showed that the transgenic parasite Nc1-TgROP18 was able to phosphorylate IRGs as T. gondii did. The present study indicated that the ROP18 plays a crucial role in virulence of the closely related parasites T. gondii and N. caninum and it is indeed a key factor responsible for the virulence difference between T. gondii and N. caninum.

SUBMITTER: Lei T

PROVIDER: S-EPMC4057265 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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ROP18 is a key factor responsible for virulence difference between Toxoplasma gondii and Neospora caninum.

Lei Tao T Wang Hui H Liu Jing J Nan Huizhu H Liu Qun Q

PloS one 20140613 6

Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) are both obligate intracellular protozoan parasites and share many common morphological and biological features. Despite these similarities the two parasites differ dramatically in virulence in mice, but the factors involved in virulence differences between the two parasites remain unknown. A secreted serine-threonine kinase called rhoptry protein 18 (ROP18) was identified to play a crucial role on virulence differences among differ ...[more]

PMID: 24927100

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Project description:BackgroundRhoptry protein 18 (ROP18) is a key virulence factor of Toxoplasma gondii. The host's immune responses mediated by immune-related GTPases (IRGs) could be blocked by ROP18's kinase activity. ROP18 also interacts with various substrates, such as activating transcription factor 6 beta (ATF6β) and affects multiple physiological functions within host cells, thereby inducing intense virulence. In this study, competitive inhibitors targeted to ROP18 were subjected to virtual screening based on the principle of structure-based drug design (SBDD).MethodsThe preparation of the ROP18 structure was conducted using the "Structure Prepare" function of Molecular Operating Environment (MOE) software. The ATP-binding pocket was selected as the starting point for virtual screening. Construction of the pharmacophore model used Extended Hückel Theory (EHT) half-quantitative measurement and construction, as well as the characteristics of Type I kinase inhibitors. The pharmacophore model of ROP18 was imported into the Specs database for small molecule similarity screening using EHT pharmacophore measurement. Hit compounds were selected using the functions of London dG and generalized-born volume integral/weighted surface area (GBVI/WSA) scoring. The top 100 hits were analyzed by molecular docking and structure activity relationships (SAR) analysis.ResultsThe final pharmacophore comprised three typical characteristics: three hydrogen bond acceptors/donors, two ring aromatic features occupying the hydrophobic core, and one cation group feature targeted to the terminus of ATP. A total of 1314 hit compounds analogous to ROP18 pharmacophore were passed through the Specs. After two rounds of docking, 25 out of 100 hits were identified as belonging to two main scaffold types: phthalimide ring structure, thiazole ring and styrene structure. Additionally, the screen also identified 13 inhibitors with distinct scaffold types. The docking models and SAR analysis demonstrated that these hits could engage in multiple hydrogen bonds, salt bridges halogen bonds, and hydrophobic interactions with ROP18, and para-position halo substituents on the benzene ring may enhance their affinity scoring.ConclusionsA pharmacophore against the ROP18 ATP-binding pocket was successfully constructed, and 25 representative inhibitors from 15 scaffold clusters were screened using the Specs database. Our results provide useful scaffold types for the chemical inhibition of ROP18 or alternative conformations to develop new anti-toxoplasmosis drug leads.

Project description:BACKGROUND:Knowledge about parasitic infections is crucial information for animal health, particularly of free-ranging species that might come into contact with livestock and humans. METHODS:We investigated the seroprevalence of three tissue-cyst-forming apicomplexan parasites (Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti) in 506 individuals of 12 wildlife species in Namibia using in-house enzyme linked immunosorbent assays (indirect ELISAs applying purified antigens) for screening and immunoblots as confirmatory tests. We included six species of the suborder Feliformia, four species of the suborder Caniformia and two species of the suborder Ruminantia. For the two species for which we had most samples and life-history information, i.e. cheetahs (Acinonyx jubatus, n = 250) and leopards (Panthera pardus, n = 58), we investigated T. gondii seroprevalence in relation to age class, sex, sociality (solitary, mother-offspring group, independent sibling group, coalition group) and site (natural habitat vs farmland). RESULTS:All but one carnivore species (bat-eared fox Otocyon megalotis, n = 4) were seropositive to T. gondii, with a seroprevalence ranging from 52.4% (131/250) in cheetahs to 93.2% (55/59) in African lions (Panthera leo). We also detected antibodies to T. gondii in 10.0% (2/20) of blue wildebeest (Connochaetes taurinus). Adult cheetahs and leopards were more likely to be seropositive to T. gondii than subadult conspecifics, whereas seroprevalence did not vary with sex, sociality and site. Furthermore, we measured antibodies to N. caninum in 15.4% (2/13) of brown hyenas (Hyaena brunnea) and 2.6% (1/39) of black-backed jackals (Canis mesomelas). Antibodies to B. besnoiti were detected in 3.4% (2/59) of African lions and 20.0% (4/20) of blue wildebeest. CONCLUSIONS:Our results demonstrate that Namibian wildlife species were exposed to apicomplexan parasites at different prevalences, depending on parasite and host species. In addition to serological work, molecular work is also needed to better understand the sylvatic cycle and the clear role of wildlife in the epidemiology of these parasites in southern Africa.

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ROP18 is a key factor responsible for virulence difference between Toxoplasma gondii and Neospora caninum.

Publications

ROP18 is a key factor responsible for virulence difference between Toxoplasma gondii and Neospora caninum.

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