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Production and characterization of monoclonal antibodies against Toxoplasma gondii ROP18 with strain-specific reactivity.

ABSTRACT: The rhoptry kinase 18 of Toxoplasma gondii (TgROP18) has been identified as a key virulence factor that allows the parasite to escape from host immune defences and promotes its proliferation in host cells. Although much research is focused on the interaction between host cells and TgROP18, the development of monoclonal antibodies (mAbs) against TgROP18 has not been reported till date. To produce mAbs targeting TgROP18, two hybridomas secreting mAbs against TgROP18, designated as A1 and T2, were generated using cell fusion technology. The subtypes of the A1 and T2 mAbs were identified as IgG3 λ and IgM κ, and peptide scanning revealed that the core sequences of the antigenic epitopes were 180LRAQRRRSELVFE192 and 351NYFLLMMRAEADM363, respectively. The T2 mAb specifically reacted with both T. gondii type I and Chinese I, but not with T. gondii type II, Plasmodium falciparum or Schistosoma japonicum. Finally, the sequences of heavy chain and light chain complementarity-determining regions of T2 were amplified, cloned and characterized, making the modification of the mAb feasible in the future. The development of mAbs against TgROP18 would aid the investigation of the molecular mechanisms underlying the modulation of host cellular functions by TgROP18, and in the development of strategies to diagnose and treat Toxoplasmosis.

SUBMITTER: Zhang F

PROVIDER: S-EPMC10317636 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Production and characterization of monoclonal antibodies against <i>Toxoplasma gondii</i> ROP18 with strain-specific reactivity.

Zhang Famin F Su Rui R Han Chengjian C Wang Yang Y Li Jingyang J Zhu Jinjin J Luo Qingli Q Chen Lingzhi L Zhang Junling J Ding Xiaojuan X Du Jian J Chu Deyong D Cai Yihong Y Shen Jilong J Yu Li L

Parasitology 20200212 9

The rhoptry kinase 18 of Toxoplasma gondii (TgROP18) has been identified as a key virulence factor that allows the parasite to escape from host immune defences and promotes its proliferation in host cells. Although much research is focused on the interaction between host cells and TgROP18, the development of monoclonal antibodies (mAbs) against TgROP18 has not been reported till date. To produce mAbs targeting TgROP18, two hybridomas secreting mAbs against TgROP18, designated as A1 and T2, were ...[more]

PMID: 32046796

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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:Toxoplasma gondii is a widespread obligatory parasitic protozoon that infects nearly all warm-blooded animals and causes toxoplasmosis. However, the current treatments for toxoplasmosis are limited by severe side effects. Myrislignan is a natural product from Myristica fragrans Houtt with wide pharmacological activities. In the current study, we tested the anti-T. gondii activity of myrislignan both in vitro and in vivo and explored its potential mechanism of action. The cytotoxicity of myrislignan in African green monkey kidney (Vero) cells was assessed using Cell Counting Kit-8 (CCK-8) assays. The in vitro effects of myrislignan on T. gondii were determined by quantitative PCR and Giemsa staining. An in vivo murine model of T. gondii infection was used to determine the efficacy of myrislignan. The changes in tachyzoites after myrislignan exposure were examined by electron microscopy. The impact of myrislignan on mitochondrial function in tachyzoites was assessed by MitoTracker Red CMXRos staining and an ATP detection kit. In vitro, myrislignan inhibited T. gondii tachyzoite proliferation with a 50% effective concentration of 32.41 μg/ml, and reduced the invasion of cells by tachyzoites (14.63 and 1.92% invasion rates for control and 70 μg/ml myrislignan, respectively). Importantly, myrislignan had no significant cytotoxicity against Vero cells at concentrations less than 132 μg/ml. In addition, surface shrinkage and mitochondrial damage were observed in tachyzoites after myrislignan exposure. The reduced ΔΨm and ATP levels in tachyzoites treated with myrislignan further confirmed mitochondrial damage. In the in vivo murine model, myrislignan treatment significantly reduced the parasite burden in tissues compared to no treatment. In conclusion, myrislignan had potent anti-T. gondii activities both in vitro and in vivo, and these activities might involve the interruption of mitochondrial function. These data suggest that myrislignan might be a useful compound for the treatment of toxoplasmosis.

Dataset Information

Production and characterization of monoclonal antibodies against Toxoplasma gondii ROP18 with strain-specific reactivity.

Publications

Production and characterization of monoclonal antibodies against <i>Toxoplasma gondii</i> ROP18 with strain-specific reactivity.

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