Background Alzheimer's disease presents an abnormal cognitive behavior. TgCtwh6 is one of the predominant T. gondii strains prevalent in China. Although T. gondii type II strain infection can cause host cognitive behavioral abnormalities, we do not know whether TgCtwh6 could also cause host cognitive behavioral changes. So, in this study, we will focus on the effect of TgCtwh6 on mouse cognitive behavior and try in vivo and in vitro to explore the underlying mechanism by which TgCtwh6 give rise to mice cognitive behavior changes at the cellular and molecular level. Methods C57BL/6 mice were infected orally with TgCtwh6 cysts. From day 90 post-infection on, all mice were conducted through the open field test and then Morris water maze test to evaluate cognitive behavior. The morphology and number of cells in hippocampus were examined with hematoxylin-eosin (H&E) and Nissl staining; moreover, Aβ protein in hippocampus was determined with immunohistochemistry and thioflavin S plaque staining. Synaptotagmin 1, apoptosis-related proteins, BACE1 and APP proteins and genes from hippocampus were assessed by western blotting or qRT-PCR. Hippocampal neuronal cell line or mouse microglial cell line was challenged with TgCtwh6 tachyzoites and then separately cultured in a well or co-cultured in a transwell device. The target proteins and genes were analyzed by immunofluorescence staining, western blotting and qRT-PCR. In addition, mouse microglial cell line polarization state and hippocampal neuronal cell line apoptosis were estimated using flow cytometry assay. Results The OFT and MWMT indicated that infected mice had cognitive behavioral impairments. The hippocampal tissue assay showed abnormal neuron morphology and a decreased number in infected mice. Moreover, pro-apoptotic proteins, as well as BACE1, APP and Aβ proteins, increased in the infected mouse hippocampus. The experiments in vitro showed that pro-apoptotic proteins and p-NF-κBp65, NF-κBp65, BACE1, APP and Aβ proteins or genes were significantly increased in the infected HT22. In addition, CD80, pro-inflammatory factors, notch, hes1 proteins and genes were enhanced in the infected BV2. Interestingly, not only the APP and pro-apoptotic proteins in HT22, but also the apoptosis rate of HT22 increased after the infected BV2 were co-cultured with the HT22 in a transwell device. Conclusions Neuron apoptosis, Aβ deposition and neuroinflammatory response involved with microglia polarization are the molecular and cellular mechanisms by which TgCtwh6 causes mouse cognitive behavioral abnormalities. Graphical Abstract

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Background The predominant genotype of Toxoplasma in China is the Chinese 1 (ToxoDB#9) lineage. TgCtwh3 and TgCtwh6 are two representative strains of Chinese 1, exhibiting high and low virulence to mice, respectively. Little is known regarding the virulence mechanism of this non-classical genotype. Our previous RNA sequencing data revealed differential mRNA levels of TgMIC1 in TgCtwh3 and TgCtwh6. We aim to further confirm the differential expression of TgMIC1 and its significance in this atypical genotype. Methods Quantitative real-time PCR was used to verify the RNA sequencing data; then, polyclonal antibodies against TgMIC1 were prepared and identified. Moreover, the invasion and proliferation of the parasite in HFF cells were observed after treatment with TgMIC1 polyclonal antibody or not. Results The data showed that the protein level of TgMIC1 was significantly higher in high-virulence strain TgCtwh3 than in low-virulence strain TgCtwh6 and that the invasion and proliferation of TgCtwh3 were inhibited by TgMIC1 polyclonal antibody. Conclusion Differential expression of TgMIC1 in TgCtwh3 and TgCtwh6 may explain, at least partly, the virulence mechanism of this atypical genotype.

AbstractThe 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.

BackgroundChronic Toxoplasma gondii (T. gondii) infection evokes abnormal cognitive behavior of the host. Recent studies suggest that the polarization of microglia to the phenotype of classically activated macrophage (M1) and the deposition of beta-amyloid (Aβ) may be induced in brain of mice chronically infected with T. gondii. However, so far, there is no definite explanation for the relationship mechanism underlying the above between microglia polarization, Aβ deposition and T. gondii. Our previous investigations indicated that in vitro T. gondii type Ⅱ strain dense granule protein 15 (GRA15Ⅱ), one of the genotype-associated effectors of T. gondii Ⅱ strain may induce mouse macrophage to M1. While T. gondii type Ⅰ/Ⅲ strain rhoptry protein 16 (ROP16Ⅰ/Ⅲ) can drive the mouse macrophage to the phenotype of alternatively activated macrophage (M2). Unlike the archetypal strains of types Ⅰ, Ⅱ, and Ⅲ, T. gondii Chinese 1 genotype Wh6 strain (TgCtwh6) possesses both GRA15Ⅱ and ROP16Ⅰ/Ⅲ proteins, indicating the unique pathogenesis of Toxoplasma-related cognitive behavioral abnormalities.MethodsIn this study, we constructed mice model of cognitive behavioral abnormalities through chronic infection of TgCtwh6 via the oral route, and used mouse hippocampal neuronal cell line (HT22) and mouse microglial cell line (BV2) infected with TgCtwh6 in co-culture system to explore the mechanism with which TgCtwh6 infection induced mouse abnormal cognitive behavior. The immunohistochemistry, immunofluorescence, western blotting, cell culture assays, as well as an array of mouse behavior tests were adopted in the research.ResultsIn our research, the infected group showed abnormal cognitive behavior in the water maze and open field experiments in comparison with the control group. Further study showed that the number of synapses and hippocampal neurons decreased and the expression of Aβ increased in brain. In vitro, our research indicated that TgCtwh6 infection could not only directly lead to the HT22 apoptosis but also directly induce BV2 activation to M1 possibly through Notch pathway. Activated BV2 secreted pro-inflammatory factors resulting in HT22 apoptosis indirectly in transwell device. Meanwhile, our reseach demonstrated that TgCtwh6 infection caused a notable expression of β-secretase 1 (BACE1)、amyloid precursor protein (APP) and Aβ in HT22 through NF-κB signaling. Furthmore, BV2 activated by TgCtwh6 infection produced pro-inflammatory factors, such as IL-6, TNF-α and iNOS, which promoted HT22 to express APP in co-culture system. In all, our results suggested that TgCtwh6 gave rise to mouse abnormal cognitive behavior due to hippocampal neuronal apoptosis and Aβ deposition driven by indirect and indirect TgCtwh6 infection to hippocampal neuron. In this pathogenic process, microglia activation played an important role in mediating hippocampal neuronal apoptosis and Aβ deposition.ConclusionsThis study demonstrates TgCtwh6 infection can cause mice to develop AD-like symptoms and give rise to hippocampal neuronal apoptosis and Aβ deposition. Besides, microglia activation played an functional role in the pathological development.