Identification of Toxoplasma Gondii Tyrosine Hydroxylase (TH) Activity and Molecular Immunoprotection against Toxoplasmosis.

Zhenchao Zhang,Shuai Wang,Zhongshan Ma,Xiangrui Li,Haoran Li,Yuhua Li,Mengyao Tan,Xiaoxiao Song,Shuyue Liu,Haoran Lu,Yi Zhao

doi:10.3390/vaccines8020158

Zhenchao Zhang, Shuai Wang + Show 9 more

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https://doi.org/10.3390/vaccines8020158

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Abstract

The neurotropic parasite Toxoplasma gondii (T. gondii) infection can change the behavior of rodents and cause neuropsychological symptoms in humans, which may be related to the change in neurotransmitter dopamine in the host brain caused by T. gondii infection. T. gondii tyrosine hydroxylase (TgTH) is an important factor in increasing the neurotransmitter dopamine in the host brain. In this study, the enzyme activity of TgTH catalytic substrate for dopamine production and the molecular characteristics of TgTH were identified. In order to amplify the open reading frame (ORF), the designing of the specific primers for polymerase chain reaction (PCR) was on the basis of the TgTH sequence (GenBank Accession No. EU481510.1), which was inserted into pET-32a (+) for the expression of recombined TgTH (rTgTH). The sequence analysis indicated that the gene of TgTH directed the encoding of a 62.4-kDa protein consisting of 565 amino acid residues, which was predicted to have a high antigen index. The enzyme activity test showed that rTgTH and the soluble proteins extracted separately from T. gondii RH strain and PRU strain could catalyze the substrate to produce dopamine in a dose-dependent manner, and the optimum catalytic temperature was 37 °C. The result of the Western Blotting assay revealed that the rTgTH and the native TgTH extracted from somatic of T. gondii RH tachyzoite were successfully detected by the sera of mice infected with T. gondii and the rat serum after rTgTH immune, respectively. Immunofluorescence analysis using antibody against rTgTH demonstrated that the protein was expressed and located on the surface of T. gondii RH tachyzoite. Freund’s adjuvant was used to emulsify the rTgTH, which was subsequently applied to BALB/c mouse immune thrice on week 0, week 2, and week 4, respectively. The result of the animal challenge experiments showed an integral increase in IgG, IgG2a, IgG1, and IFN-γ, IL-4, and IL17 were as well significantly increased, and that the rTgTH vaccinated animals apparently had a prolonged survival time (14.30 ± 2.41) after infection with the RH strain of T. gondii compared with that of the non-vaccinated control animals, which died within 11 days. Additionally, in the rTgTH vaccination group, the number of brain cysts (1275 ± 224) significantly decreased (p < 0.05) compared to the blank control group (2375 ± 883), and the size of the brain cysts in the animals immunized with rTgTH vaccination was remarkably smaller than that of the control mice. All the findings prove that TgTH played an important role in increasing the neurotransmitter dopamine in the host brain and could be used as a vaccine candidate antigen to mediate cell-mediated and humoral immunity.

Highlights

T. gondii, an obligate intracellular apicomplexan parasite, is the causative agent of the anthropozoonotic disease called toxoplasmosis, which can be widely parasitic in a broad range of warm-blooded vertebrates
T. gondii tyrosine hydroxylase (TgTH) played an important role in increasing the neurotransmitter dopamine in the host brain and could be used as a vaccine candidate antigen to mediate cell-mediated and humoral immunity
A 1698 bp open reading frame (ORF) of TgTH, which encode a protein of 565 amino acids with a molecular mass of 62.40 kDa, was found that started from the ATG initiation codon and ended at the stop codon of TAG by gel electrophoresis (Figure 1A) and sequencing

Summary

Introduction

T. gondii, an obligate intracellular apicomplexan parasite, is the causative agent of the anthropozoonotic disease called toxoplasmosis, which can be widely parasitic in a broad range of warm-blooded vertebrates. Toxoplasmosis seriously threatens the health of human and livestock, and it is spread by animals and distributed all over the world [1]. 30% of the world’s population has been infected with T. gondii [2]. The infection can cause high rates of morbidity and mortality for some patients and can result in such patients developing AIDS [3]. The infection of toxoplasma leads to abortion, stillbirth, as well as death of neonates, causing enormous economic losses [4,5]. Infected livestock are the main route of transmission to humans [6]

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