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.

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.

Identification of drought resistant miRNA in Macleaya cordata by high-throughput sequencing.

Drought is one of the most serious factors affecting crop yields in the world. Macleaya cordata (Willd.) is a draught-tolerant medicinal plant that has been proposed as a pioneer crop to be cultivated in arid areas. However, the exact molecular mechanisms through which M. cordata responds to draught stress remain elusive. In recent years, microRNA (miRNAs) in plants have been associated with stress response. Based on these findings, the current study aimed to shed light on the potential regulatory roles of miRNAs in the draught tolerance of M. cordata by employing high-throughput RNA sequencing and degradation sequencing. Six M. cordata plants were randomly divided into two equal experiment groups, including one draught group and one control group. High-throughput sequencing of the M. cordata samples led to the identification of 895 miRNAs, of which 18 showed significantly different expression levels between the two groups. PsRobot analysis and degradation sequencing predicted the differential miRNAs to target 59 and 36 genes, respectively. Functional analysis showed that 38 of the predicted genes could be implicated in the modulation of stress response. Four miRNAs and eight target genes were selected for quantitative real-time polymerase chain reaction (qRT-PCR) validation. The expression trend of each miRNA analyzed by qRT-PCR was consistent with that determined by sequencing, and was negatively correlated with those of its target genes. The results of our current study supported the involvement of miRNAs in the draught tolerance of M. cordata and could pave the way for further investigation into the related regulatory mechanisms.

Treg expression of CIS suppresses allergic airway inflammation through antagonizing an autonomous TH2 program.

Maintenance of regulatory T (Treg) cells is crucial for the regulatory function of Treg cells in immune homeostasis and self-tolerance; however, the detailed underlying mechanisms remain elusive. In the current study, we found that the cytokine suppressor CIS (cytokine induced SH-2 protein) is required for maintenance of Treg cell identity. Mice with Treg-specific Cis-deficiency displayed aggravated experimental allergic asthma, and in adulthood, developed splenomegaly, lymphadenopathy and spontaneous eosinophilic airway inflammation, accompanied by accumulation of effector memory helper T (TH) cells. Cis-deficiency led to the loss of Foxp3 expression and the decrease in suppressive function of Treg cells. Cis-deficient Treg cells expressed TH2 cell signature genes, Gata3, Irf4 and Il4, and excessive interleukin-4-signal transducer and activator of transcription 6 (IL-4-STAT6) signals resulted in repressive chromatin modification in the Foxp3 locus and permissive modification in the Il4 loci. In vitro, blockade of IL-4 restored the expression of Foxp3 and the suppressive function of inducible Treg (iTreg) cells. Thus, we identified a novel feedback loop in stabilization of Treg cells and suppression of TH2-type inflammation in a Treg-intrinsic manner.

Modulation of benzylisoquinoline alkaloid biosynthesis by overexpression berberine bridge enzyme in Macleaya cordata.

Macleaya cordata produces a variety of benzylisoquinoline alkaloids (BIAs), such as sanguinarine, protopine, and berberine, which are potential anticancer drugs and natural growth promoters. The genes encoding the berberine bridge enzyme (BBE) were isolated from M. cordata and Papaver somniferum, and then the two genes were overexpressed in M. cordata. Through liquid chromatography with triple-quadrupole mass spectrometry analysis, it was determined that McBBE-OX caused higher levels of (S)-norcoclaurine, (S)-coclaurine, (S)-N-cis-methylcoclaurine, (S)-reticuline, (S)-tetrahydrocolumbamine, (S)-tetrahydroberberine, (S)-cheilanthifoline, and (S)-scoulerine than PsBBE-OX, empty vector or control treatments. qRT-PCR analysis demonstrated that the introduced genes in the transgenic lines were all highly expressed. However, the levels of sanguinarine (SAN) and chelerythrine (CHE) in all the transgenic lines were slightly lower than those in the wild-type lines, possibly because the overexpression of McBBE causes feedback-inhibition. This is the first report on the overexpression of potential key genes in M. cordata, and the findings are important for the design of metabolic engineering strategies that target BIAs biosynthesis.

Hairy root induction and benzylisoquinoline alkaloid production in Macleaya cordata.

Sanguinarine is currently widely used to replace antibiotic growth promoters in animal feeding and has demonstrated useful anticancer activity. Currently, the main source of sanguinarine is from an important medicinal plant, Macleaya cordata. To obtain a new source of sanguinarine production, we established hairy root cultures of M. cordata by co-cultivating leaf and stem explants with Agrobacterium rhizogenes. Except the co-cultivation medium, all growth media contained 200 mg/L timentin to eliminate A. rhizogenes. Through comparing the metabolic profiles and gene expression of hairy roots and wild-type roots sampled at five time points, we found that the sanguinarine and dihydrosanguinarine contents of hairy roots were far higher than those of wild-type roots, and we revealed the molecular mechanism that causes these metabolites to increase. Consequently, this study demonstrated that the hairy root system has further potential for bioengineering and sustainable production of sanguinarine on a commercial scale. To the best of our knowledge, this is the first efficient protocol reported for the establishment of hairy root cultures in M. cordata using A. rhizogenes.

Seroprevalence and associated risk factors of Toxoplasma gondii among Manchu pregnant women in northeastern China.

Toxoplasma gondii is an intracellular protozoan parasite infecting human and almost all warm-blooded animals. It may cause several severe symptoms if pregnant women infection with T. gondii, including misbirth. A cross-sectional study was conducted containing 313 Manchu pregnant women attending antenatal care from 2016 to 2017 in Jilin province, northeastern China, and were assessed by enzyme-link immunosorbent assay and the study utilized univariate analysis to identify the potential risk factors for T. gondii infection. Of the 313 investigated Manchu pregnant women, 51 (16.29%, 95% CI 12.2-20.4) were tested as T. gondii-seropositive, with 47 (15.02%) seropositive for T. gondii IgG antibodies and 8 (2.56%) IgM positive. The seroprevalence of T. gondii in different age groups varied from 13.50% (8.7-18.3) to 23.90% (13.9-41.9). Pregnant raising cat at home has a significantly higher seroprevalence of T. gondii than no cat at home. Pregnant consuming raw/undercooked meat has a significantly higher T. gondii seroprevalence than individuals did not consuming raw/undercooked meat. This is the first study of T. gondii infection seroprevalence in Manchu pregnant women. Risk factors analysis suggested that seroprevalence of T. gondii in investigated Manchu pregnant women was mainly related to consumption of raw/undercooked meat and raising cat at home. The findings will provide key and baseline data for prevention and control of toxoplasmosis among Manchu pregnant women and other people.

Enterocytozoon bieneusi in Minks (Neovison vison) in Northern China: A Public Health Concern.

Enterocytozoon bieneusi is the most important causative agent of microsporidiosis and can infect almost all vertebrate and invertebrate hosts, including minks (Neovison vison). In the present study, a total of 298 feces samples (including 79 from Heilongjiang province, 31 from Hebei province, 67 from Jilin province, 90 from Liaoning province, and 31 from Shandong province, Northern China) were examined by nested PCR amplification of the internal transcribed spacer (ITS) region of the rRNA gene. The overall prevalence of E. bieneusi in minks was 10.1%, with 10.5% in Jilin province, 32.3% in Hebei province, 8.9% in Liaoning province, 0% in Shandong province, and 6.3% in Heilongjiang province. Furthermore, multiple logistic regression analysis revealed that region was only risk factors associated with E. bieneusi infection in the investigated minks. Five E. bieneusi ITS genotypes (three known genotypes, namely D, Peru11, and EbpC; two novel genotypes, namely, NCM-1 and NCM-2) were found in the current study. Importantly, genotypes D, Peru11 and EbpC, previously identified in humans, were also found in minks, which suggested that minks are the potential sources of human microsporidiosis. To the best of our knowledge, this is the first report of E. bieneusi infection in minks worldwide. The results of the present survey have implications for the controlling E. bieneusi infection in minks, other animals and humans.

Molecular detection and genotyping of Toxoplasma gondii in free-ranging pigs in Northeastern China.

Pig is the well-known intermediate host of T. gondii, a ubiquitous and obligate intracellular zoonotic pathogen. However, information about prevalence and genotypes of T. gondii infection in free-ranging pigs is scarce. Therefore a total of 186 hilar lymph nodes specimens were collected from free-ranging pigs from rural regions from Jilin (n = 119) and Liaoning (n = 67) provinces, northeastern China, and were investigated from T. gondii infection by semi-nested PCR of the B1 gene. Positive testing samples were genotyped by using polymerase chain reaction followed by restriction-fragment length polymorphism technology (PCR-RFLP) applied to 11 loci. The overall prevalence of T. gondii in investigated free-ranging pigs was 18.3% (34/186), with 16.4% (11/67) in Liaoning Province and 19.3% (23/119) in Jilin Province. Moreover, two genotypes, namely ToxoDB #9 and ToxoDB #10, were detected in investigated pigs. The present study showed a high T. gondii prevalence in free-ranging compared to farmed pigs as reported in previous studies by others, raising a major public health concern. It is essential to establish efficient strategies to prevent and control T. gondii infection in free-ranging pigs, other animals and humans in investigated regions.

Toxoplasma gondii Infection in Dustmen in Northeastern China: A Case-Control Seroprevalence Study.

BACKGROUND: Toxoplasmosis is caused by an intracellular parasite Toxoplasma gondii, which can infect many hosts including humans. METHODS: In order to estimate whether dustmen are more susceptible to T. gondii, a case-control study was conducted containing 332 dustmen from Jilin and Heilongjiang in Northeastern China, as well as 332 general populations from the same regions as control subjects. Serum samples were tested IgG and IgM antibodies to T. gondii using the enzyme-linked immunosorbent assay (ELISA). RESULTS: The overall anti-T. gondii IgG was 15.06% (50/332) in dustmen compared with 9.64% (32/332) in the controls (P = 0.0337). Also, 5 (1.51%) dustmen had anti-T. gondii IgM antibodies compared with 2 (0.60%) control individuals (P = 0.2543). A significant association was only found between dustmen and level of T. gondii IgG in comparison with the control subjects. Seroprevalence of T. gondii IgG antibodies in male dustmen was significant higher than male control subjects (P = 0.0399). Dustmen from Jilin had the significant higher T. gondii IgG rate (P = 0.0143), in comparison with the control subjects from Jilin. Moreover, dustmen raising cat at home had the significant higher T. gondii IgG rate (P = 0.0097), in comparison with the control subjects. Risk factor analysis suggested that raising cat at home and not having habits of washing hand before eating were mainly related to the T. gondii infection in dustmen. CONCLUSIONS: This is the first record of seroprevalence of T. gondii infection in dustmen in Jilin and Heilongjiang provinces in Northeastern China. These findings also suggest that the government departments should pay close attention to the toxoplasmosis in dustmen in Northeastern China.

Isocyanide-Based Multicomponent Bicyclization with Substituted Allenoate and Isatin: Synthesis of Unusual Spirooxindole Containing [5.5]-Fused Heterocycle.

A three-component bicyclization reaction of isocyanide, substituted allenoate, and isatin has been disclosed. This protocol is proposed to proceed through Michael addition, double cyclization, and [1,5]-hydride shift sequence, thus leading to the formation of two new rings and five new chemical bonds, including C-C, C-O, and C-N bonds.