Bioinformatics of excretory/secretory proteins of Toxoplasma gondii strain ME49.

Toxoplasma gondii is an obligate intracellular parasite that is globally distributed and can infect almost all warm-blooded animals, including humans. While vaccines are not available for prophylaxis, there are limited therapeutic options which often do not result in eradication of parasites from patients. ME49 is a cystogenic strain of T. gondii with a potential as a vaccine candidate. Excretory and secretory (ES) proteins are thought to play crucial roles in host-parasite interactions. Hence, we predicted and functionally annotated the ES proteins in T. gondii ME49 using public databases. ES proteins were further examined for the characteristics and possible functions through gene ontology (GO) term enrichment and analyses of metabolic pathways, enzyme code distribution (EC distributing) and protein domains. The potential antigenicity of T. gondii ME49 ES proteins was evaluated for the first time using Abundance of Antigenic Regions (AAR) value to predict the antigenic potential by measuring sequence length and number of antigenic regions. The results showed that the ES proteins have different AAR values at RNA and microarray level. Our studies provide valuable information for future experimental studies, including the identification of novel ES proteins of therapeutic, diagnostic and immunoprophylactic interests.

Inhibition of GSDMD-mediated pyroptosis triggered by Trichinella spiralis intervention contributes to the alleviation of DSS-induced ulcerative colitis in mice.

BACKGROUND: Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is increasing worldwide. Although there is currently no completely curative treatment, helminthic therapy shows certain therapeutic potential for UC. Many studies have found that Trichinella spiralis (T.s) has a protective effect on UC, but the specific mechanism is still unclear. METHODS: Balb/c mice drank dextran sulfate sodium (DSS) to induce acute colitis and then were treated with T.s. In vitro experiments, the LPS combination with ATP was used to induce the pyroptosis model, followed by intervention with crude protein from T.s (T.s cp). Additionally, the pyroptosis agonist of NSC or the pyroptosis inhibitor vx-765 was added to intervene to explore the role of pyroptosis in DSS-induced acute colitis. The degree of pyroptosis was evaluated by western blot, qPCR and IHC, etc., in vivo and in vitro. RESULTS: T.s intervention significantly inhibited NLRP3 inflammasome activation and GSDMD-mediated pyroptosis by downregulating the expression of pyroptosis-related signatures in vitro (cellular inflammatory model) and in vivo (DSS-induced UC mice model). Furthermore, blockade of GSDMD-mediated pyroptosis by the caspase-1 inhibitor vx-765 has a similar therapeutic effect on DSS-induced UC mice with T.s intervention, thus indicating that T.s intervention alleviated DSS-induced UC in mice by inhibiting GSDMD-mediated pyroptosis. CONCLUSION: This study showed that T.s could alleviate the pathological severity UC via GSDMD-mediated pyroptosis, and it provides new insight into the mechanistic study and application of helminths in treating colitis.

Virulence-related gene wx2 of Toxoplasma gondii regulated host immune response via classic pyroptosis pathway.

BACKGROUND: Toxoplasma gondii is known as the most successful parasite, which can regulate the host immune response through a variety of ways to achieve immune escape. We previously reported that a novel gene wx2 of T. gondii may be a virulence-related molecule. The objective of this study was to explore the mechanism of wx2 regulating host immune response. METHODS: The wx2 knockout strain (RHwx2-/- strain) and complementary strain (RHwx2+/+ strain) were constructed by the CRISPR/Cas9 technique, and the virulence of the wx2 gene was detected and changes in pyroptosis-related molecules were observed. RESULTS: Compared with the wild RH and RHwx2+/+ strain groups, the survival time for mice infected with the RHwx2-/- strain was prolonged to a certain extent. The mRNA levels of pyroptosis-related molecules of caspase-1, NLRP3, and GSDMD and et al. in mouse lymphocytes in vivo and RAW267.4 cells in vitro infected with RHwx2-/- strain increased to different degrees, compared with infected with wild RH strain and RHwx2+/+ strain. As with the mRNA level, the protein level of caspase-1, caspase-1 p20, IL-1ß, NLRP3, GSDMD-FL, GSDMD-N, and phosphorylation level of NF-κB (p65) were also significantly increased. These data suggest that wx2 may regulate the host immune response through the pyroptosis pathway. In infected RAW264.7 cells at 48 h post-infection, the levels of Th1-type cytokines of IFN-γ, Th2-type cytokines such as IL-13, Th17-type cytokine of IL-17 in cells infected with RHwx2-/- were significantly higher than those of RH and RHwx2+/+ strains, suggesting that the wx2 may inhibit the host's immune response. CONCLUSION: wx2 is a virulence related gene of T. gondii, and may be involved in host immune regulation by inhibiting the pyroptosis pathway.

Epitope vaccine design for Toxoplasma gondii based on a genome-wide database of membrane proteins.

BACKGROUND: There is presently no effective and safe vaccine for Toxoplasma gondii for humans. The study described here was designed to search for a novel group of optimal B cell and T cell epitopes from Toxoplasma membrane proteins using genome-wide comprehensive screening. METHODS: The amino acid sequences of membrane proteins of T. gondii were obtained from the UniProt database. The ABCPred and BepiPred servers were employed to predict the linear B cell epitopes. The Immune Epitope Database (IEDB) online service was utilized to forecast T cell epitopes within T. gondii membrane proteins that bind to human leukocyte antigen (HLA) class I (HLA-I) or HLA-II molecules. RESULTS: From the 314 membrane proteins of T. gondii, a total of 14 linear B cell epitopes embedded in 12 membrane proteins were identified. Eight epitopes for major histocompatibility complex (MHC) class I (MHC-I) molecules and 18 epitopes for MHC-II molecules were ultimately selected, for which world population coverage percentiles were 71.94% and 99.76%, respectively. The top rated combinations of linear B cell epitopes and T cell epitopes covering both BALB/c mice and a majority of the human population were identified for the development of a protective vaccine. CONCLUSIONS: The ultimate vaccine construct described here, which comprises B cells, MHC-I and MHC-II epitopes, might protect individuals against T. gondii infection by inducing humoral and cellular immune responses.

Corrigendum: A Novel wx2 Gene of Toxoplasma gondii Inhibits the Parasitic Invasion and Proliferation in vitro and Attenuates Virulence in vivo via Immune Response Modulation.

[This corrects the article DOI: 10.3389/fmicb.2020.00399.].

A Novel wx2 Gene of Toxoplasma gondii Inhibits the Parasitic Invasion and Proliferation in vitro and Attenuates Virulence in vivo via Immune Response Modulation.

Toxoplasma gondii (T. gondii) is an obligate intracellular apicomplexan protozoan that can parasitize most warm-blooded animals and cause severe diseases in immunocompromised individuals or fetal abnormalities in pregnant woman. The treatment of toxoplamosis has been limited by effective drugs. Our previous work indicated that the novel gene wx2 of T. gondii may serve as a vaccine antigen candidate. To further investigate the molecular functions of wx2 in highly virulent T. gondii (RH strain), a wx2 gene deletion mutant RH strain (KO-wx2) was established using CRISPR-Cas9. The phynotype of KO-wx2 was analyzed by plaque, invasion, and replication assays in vitro as well as in vivo virulence assays. The results indicated that the targeted deletion of the wx2 gene significantly inhibited in vitro parasite growth and replication in the host cells as well as attenuated parasite virulence in the mouse model. Notably, the percentage of pro-inflammatory factors of interferon gamma (IFN-γ) and interlukin-17A (IL-17A) and anti-inflammatory factor of interlukin-10 (IL-10) in the lymph nodes were upregulated in mice infected with the KO-wx2 strain. Our data suggested that the wx2 gene plays an important role in the process of the parasite's life cycle and virulence in mice. In addition, it also plays an important role in the host's immunity reaction, mainly via Th1 and Th17 cellular immunity, not Th2.