Toxoplasma gondii is an apicomplexan parasite infecting human and animals, causing huge public health concerns and economic losses. Swine alveolar macrophage plays an important role in controlling T. gondii infection. However, the mechanism by which macrophages infected with T. gondii function in the immunity to the infection is unclear, especially for local isolates such as TgHB1 isolated in China. RNA-seq as a valuable tool was applied to simultaneously analyze transcriptional changes of pig alveolar macrophages infected with TgRH (typeI), TgME49 (typeII) or TgHB1 at different time points post infection (6, 12, and 24 h). Paired-end clean reads were aligned to the Sscrofa10.2 pig genome and T. gondii ME49 genome. The differentially expressed genes of macrophages and T. gondii were enriched through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, respectively. Compared to the TgRH and TgME49 infection groups, 307 down-regulated macrophage genes (mainly enriched for development and metabolism) and 419 up-regulated genes (mainly enriched for immune pathways) were uniquely expressed in the TgHB1 infection group. Additionally, 557 down-regulated and 674 up-regulated T. gondii genes (mainly enriched in metabolism and biosynthesis) were uniquely expressed in the TgHB1 infection group. For validation purposes, some of the differentially expressed genes of macrophages involved in immune-related signaling pathways were used for further analysis via real time quantitative reverse-transcription polymerase-chain reaction (qRT-PCR). This work provides important insights into the temporal immune responses of swine alveolar macrophages to infection by the strain TgHB1 isolated from China, and is helpful for better understanding of the T. gondii genotype-associated activation of macrophages during early phase of the infection.
Background: It has been reported that the NF-κB pathway, an important component of host defense system against pathogens infections, can be modulated by GRA15 of type 2 Toxoplasma gondii strains. A newly reported Toxoplasma strain (T.gHB1) belonging to type 1 showed polymorphic phenotypes comparable to the classic type 1 parasite, such as virulence determinant mechanisms. Therefore, it is worth investigating whether the novel type 1 Toxoplasma strain (T.gHB1) has an effect on host NF-κB signaling pathway.Methods: The effects of T.gHB1 on host NF-κB pathway was investigated in HEK293T cells. The GRA15 gene product was analysed by bioinformatics method and its effect on NF-κB was examined by detection of the levels of phosphorylation and nuclear translocation of p65 by Western blotting (WB) and indirect fluorescence assay (IFA). Functional domain of GRA15 was mapped by using the luciferase reporter assay and its effects on p65 nuclear translocation and IκBα phosphorylation were examined by IFA and WB, respectively. Results: We demonstrated that the NF-κB pathway signaling can be activated by the newly identified type 1 T.gHB1 strain of Toxoplasma, while other known type 1 RH parasites exerted an inhibitory effect. T.gHB1 GRA15 possesses only one transmembrane region with an extended C terminal region, which is distinct from other known GRA15 from type 2 parasites. T.gHB1 GRA15 could clearly induce IκBα phosphorylation and p65 nuclear translocation. Dual luciferase assays in HEK293T cells revealed a requirement for 194-518 aa of T.gHB1 GRA15 to effectively activate NF-κB. Conclusions: The overall results indicated that the newly discovered type 1 isolate T.gHB1 and its GRA15 gene product can activate NF-κB through a classical pathway in mammalian cells. These results provide new sights for our understanding of the interaction between Toxoplasma parasites and its host.
Background It has been reported that the NF-κB pathway, an important component of host defense system against pathogens infections, can be differentially modulated by different Toxoplasma gondii strains, depending on the polymorphism of the GRA15 protein. The recently isolated Toxoplasma strain T.gHB1 is a type 1 (ToxoDB#10) strain but shows different virulence determination mechanisms compared to the classic type 1 strains like RH (ToxoDB#10). Therefore, it is worth investigating whether the T.gHB1 strain (ToxoDB#10) affects the host NF-κB signaling pathway. Methods The effects of T.gHB1 (ToxoDB#10) on host NF-κB pathway were investigated in HEK293T cells. The GRA15 gene product was analyzed by bioinformatics, and its effect on NF-κB activation was examined by Western blotting and nuclear translocation of p65. Different truncations of T.gHB1 GRA15 were constructed to map the critical domains for NF-κB activation. Results We demonstrated that the NF-κB pathway signaling pathway could be activated by the newly identified type 1 T.gHB1 strain (ToxoDB#10) of Toxoplasma, while the classic type 1 strain RH (ToxoDB#10) did not. T.gHB1 GRA15 possesses only one transmembrane region with an extended C terminal region, which is distinct from that of classic type 1 (ToxoDB#10) and type 2 (ToxoDB#1) strains. T.gHB1 GRA15 could clearly induce IκBα phosphorylation and p65 nuclear translocation. Dual luciferase assays in HEK293T cells revealed a requirement for 194–518 aa of T.gHB1 GRA15 to effectively activate NF-κB. Conclusions The overall results indicated that the newly isolated type 1 isolate T.gHB1 (ToxoDB#10) had a unique GRA15, which could activate the host NF-κB signaling through inducing IκBα phosphorylation and p65 nuclear translocation. These results provide new insights for our understanding of the interaction between Toxoplasma parasites and its hosts. Graphical Abstract
Background It has been reported that the NF-κB pathway, an important component of host defense system against pathogens infections, can be modulated by GRA15 of type 2 Toxoplasma gondii strains. A newly reported Toxoplasma strain (T.gHB1) belonging to type 1 showed polymorphic phenotypes comparable to the classic type 1 parasite, such as virulence determinant mechanisms. Therefore, it is worth investigating whether the novel type 1 Toxoplasma strain (T.gHB1) has an effect on host NF-κB signaling pathway. Methods The effects of T.gHB1 on host NF-κB pathway was investigated in HEK293T cells. The GRA15 gene product was analysed by bioinformatics method and its effect on NF-κB was examined by detection of the levels of phosphorylation and nuclear translocation of p65 by Western blotting (WB) and indirect fluorescence assay (IFA). Functional domain of GRA15 was mapped by using the luciferase reporter assay and its effects on p65 nuclear translocation and IκBα phosphorylation were examined by IFA and WB, respectively. Results We demonstrated that the NF-κB pathway signaling can be activated by the newly identified type 1 T.gHB1 strain of Toxoplasma, while other known type 1 RH parasites exerted an inhibitory effect. T.gHB1 GRA15 possesses only one transmembrane region with an extended C terminal region, which is distinct from other known GRA15 from type 2 parasites. T.gHB1 GRA15 could clearly induce IκBα phosphorylation and p65 nuclear translocation. Dual luciferase assays in HEK293T cells revealed a requirement for 194–518 aa of T.gHB1 GRA15 to effectively activate NF-κB. Conclusions The overall results indicated that the newly discovered type 1 isolate T.gHB1 and its GRA15 gene product can activate NF-κB through a classical pathway in mammalian cells. These results provide new sights for our understanding of the interaction between Toxoplasma parasites and its host.
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