224 results on '"Pengtao GONG"'
Search Results
2. Development of an LFD-RPA Assay for Rapid Detection of Pentatrichomonas hominis Infection in Dogs
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Yao Rong, Xichen Zhang, Xuejiao Chen, Jianhua Li, Pengtao Gong, Xiaocen Wang, Xin Li, Xu Zhang, Taotao Yue, Hongbo Zhang, Xiaofei Zhou, and Nan Zhang
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Pentatrichomonas hominis ,zoonotic pathogen ,LFD-RPA ,dogs ,rapid detection ,Biology (General) ,QH301-705.5 - Abstract
Pentatrichomonas hominis is a trichomonad protozoan that infects the cecum and colon of humans and other mammals. It is a zoonotic pathogen that causes diarrhea in both animals and humans. As companion animals, dogs infected with P. hominis pose a risk of transmitting it to humans. Current methods, such as direct smears and polymerase chain reaction (PCR), used for P. hominis detection have limitations, including low detection rates and the need for specialized equipment. Therefore, there is an urgent need to develop rapid, sensitive, and simple detection methods for clinical application. Recombinase polymerase amplification (RPA) has emerged as a technology for rapid pathogen detection. In this study, we developed a lateral flow dipstick (LFD)-RPA method based on the highly conserved SPO11-1 gene for detecting P. hominis infection by optimizing the primers, probes, and reaction conditions, and evaluating cross-reactivity with genomes of Giardia duodenalis and other parasites. The LFD-RPA method was then used to test 128 dog fecal samples collected from Changchun. The results confirmed the high specificity of the method with no cross-reactivity with the five other parasites. The lowest detection limit of the method was 102 copies/µL, and its sensitivity was 100 times higher than that of the conventional PCR method. Consistent with the positivity rate observed using nested PCR, 12 samples (out of 128) tested positive using this method (positivity rate, 9.38%). In conclusion, the LFD-RPA method developed in this study represents a simple and sensitive assay that allows for the rapid detection of P. hominis infection in dogs, especially in this field.
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- 2023
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3. Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis
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Min Sun, Zhiteng Zhao, Ying Li, Lili Cao, Jianhua Li, Xichen Zhang, Xin Li, Nan Zhang, Shuqin Cheng, Xiaocen Wang, and Pengtao Gong
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Giardia intestinalis ,VSPAS7 ,NLRP3 ,Pyroptosis ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background The unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported. Methods To examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1β secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. Results VSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1β secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated. Conclusions Overall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity. Graphical abstract
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- 2023
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4. A potential role for Giardia chaperone protein GdDnaJ in regulating Giardia proliferation and Giardiavirus replication
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Hongbo Zhang, Chunyan Zhao, Xichen Zhang, Jianhua Li, Pengtao Gong, Xiaocen Wang, Xin Li, Xin Wang, Xu Zhang, Shuqin Cheng, Taotao Yue, and Nan Zhang
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Giardia duodenalis ,Giardiavirus ,RNA-dependent RNA polymerase ,Chaperone protein ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Giardia duodenalis (referred to as Giardia) is a flagellated binucleate protozoan parasite, which causes one of the most common diarrheal diseases, giardiasis, worldwide. Giardia can be infected by Giardiavirus (GLV), a small endosymbiotic dsRNA virus belongs to the Totiviridae family. However, the regulation of GLV and a positive correlation between GLV and Giardia virulence is yet to be elucidated. Methods To identify potential regulators of GLV, we performed a yeast two-hybrid (Y2H) screen to search for interacting proteins of RdRp. GST pull-down, co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assay were used to verify the direct physical interaction between GLV RdRp and its new binding partner. In addition, their in vivo interaction and colocalization in Giardia trophozoites were examined by using Duolink proximal ligation assay (Duolink PLA). Results From Y2H screen, the Giardia chaperone protein, Giardia DnaJ (GdDnaJ), was identified as a new binding partner for GLV RdRp. The direct interaction between GdDnaJ and GLV RdRp was verified via GST pull-down, co-immunoprecipitation and BiFC. In addition, colocalization and in vivo interaction between GdDnaJ and RdRp in Giardia trophozoites were confirmed by Duolink PLA. Further analysis revealed that KNK437, the inhibitor of GdDnaJ, can significantly reduce the replication of GLVs and the proliferation of Giardia. Conclusion Taken together, our results suggested a potential role of GdDnaJ in regulating Giardia proliferation and GLV replication through interaction with GLV RdRp. Graphical Abstract
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- 2023
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5. The NLRP3 inflammasome recognizes alpha-2 and alpha-7.3 giardins and decreases the pathogenicity of Giardia duodenalis in mice
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Panpan Zhao, Jianhua Li, Xin Li, Jingquan Dong, Xiaocen Wang, Nan Zhang, Shan Li, Min Sun, Xichen Zhang, Zhibang Wang, Min Liang, Ying Li, Lili Cao, and Pengtao Gong
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Giardia duodenalis ,Alpha-2 giardin ,Alpha-7.3 giardin ,NLRP3 inflammasome ,Pathogenicity ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Giardia duodenalis is a parasitic organism that can cause giardiasis, an intestinal infection, particularly prevalent in young children, with clinical symptoms of diarrhea. We previously reported that extracellular G. duodenalis triggers intracellular nucleotide-binding oligomerization-like receptor 3 (NLRP3) inflammasome activation and regulates the host inflammatory response by secreting extracellular vesicles (EVs). However, the exact pathogen-associated molecular patterns in G. duodenalis EVs (GEVs) involved in this process and the role of the NLRP3 inflammasome in giardiasis remain to be elucidated. Methods Recombinant eukaryotic expression plasmids of pcDNA3.1(+)-alpha-2 and alpha-7.3 giardins in GEVs were constructed, transfected into primary mouse peritoneal macrophages and screened by measuring the expression levels of the inflammasome target molecule caspase-1 p20. The preliminary identification of G. duodenalis alpha-2 and alpha-7.3 giardins was further verified by measuring the protein expression levels of key molecules of the NLRP3 inflammasome (NLRP3, pro-interleukin-1 beta [IL-1β], pro-caspase-1, and caspase-1 p20), the secretion levels of IL-1β, the level of apoptosis speck-like protein (ASC) oligomerization and the immunofluorescence localization of NLRP3 and ASC. The roles of the NLRP3 inflammasome in G. duodenalis pathogenicity were then evaluated using mice in which NLRP3 activation was blocked (NLRP3-blocked mice), and body weight, parasite burden in the duodenum and histopathological changes in the duodenum were monitored. In addition, we explored whether alpha-2 and alpha-7.3 giardins triggered IL-1β secretion in vivo through the NLRP3 inflammasome and determined the roles of these molecules in G. duodenalis pathogenicity in mice. Results Alpha-2 and alpha-7.3 giardins triggered NLRP3 inflammasome activation in vitro. This led to caspase-1 p20 activation, upregulation of the protein expression levels of NLRP3, pro-IL-1β and pro-caspase-1, significant enhancement of IL-1β secretion, ASC speck formation in the cytoplasm and also induction of ASC oligomerization. Deletion of the NLRP3 inflammasome aggravated G. duodenalis pathogenicity in mice. Compared to wild-type mice gavaged with cysts, mice gavaged with cysts in NLRP3-blocked mice displayed increased trophozoite loads and severe duodenal villus damage, characterized by necrotic crypts with atrophy and branching. In vivo assays revealed that alpha-2 and alpha-7.3 giardins could induce IL-1β secretion through the NLRP3 inflammasome and that immunization with alpha-2 and alpha-7.3 giardins decreased G. duodenalis pathogenicity in mice. Conclusions Overall, the results of the present study revealed that alpha-2 and alpha-7.3 giardins trigger host NLRP3 inflammasome activation and decrease G. duodenalis infection ability in mice, which are promising targets for the prevention of giardiasis. Graphical Abstract
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- 2023
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6. The Detection of Circulating Antigen Glutathione S-Transferase in Sheep Infected with Fasciola hepatica with Double-Antibody Sandwich Signal Amplification Enzyme-Linked Immunosorbent Assay
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Jiahui Duan, Nan Zhang, Shaoxiong Liu, Jianhua Li, Pengtao Gong, Xiaocen Wang, Xin Li, Xu Zhang, Bo Tang, and Xichen Zhang
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F. hepatica ,circulating antigen ,double-antibody sandwich SA-ELISA ,glutathione s-transferase ,Veterinary medicine ,SF600-1100 ,Zoology ,QL1-991 - Abstract
Fasciolosis is a global zoonotic parasitic disease caused by F. hepatica infection that is particularly harmful to cattle and sheep. A biotin–streptavidin signal amplification ELISA (streptavidin-ELISA/SA-ELISA) based on circulating antigens can allow for the early detection of F. hepatica-infected animals and is suitable for batch detection. It is considered to be a better means of detecting F. hepatica infection than traditional detection methods. In this study, using the serum of sheep artificially infected with F. hepatica, the cDNA expression library of F. hepatica was screened, 17 immunodominant antigen genes of F. hepatica were obtained, and glutathione s-transferase (GST) was selected as the candidate detection antigen. Firstly, the GST cDNA sequence was amplified from F. hepatica, followed by the preparation of recombinant protein GST (rFhGST). Then, monoclonal and polyclonal antibodies against rFhGST were prepared using the GST protein. Afterward, the immunolocalization of the target protein in the worm was observed via confocal microscopy, and it was found that the GST protein was localized in the uterus, intestinal tract, and body surface of F. hepatica. Finally, a double-antibody sandwich SA-ELISA based on the detection of circulating antigens was established. There was no cross-reaction with positive sera infected with Dicrocoelium lanceatum (D. lanceatum), Haemonchus contortus (H. contortus), Neospora caninum (N. caninum), or Schistosoma japonicum (S. japonicum). Forty serum and fecal samples from the same batch of sheep in Nong’an County, Changchun City, Jilin Province, China were analyzed using the established detection method and fecal detection method. The positive rate of the SA-ELISA was 17.5%, and the positive rate of the fecal detection method was 15%. The detection results of this method were 100% consistent with commercial ELISA kits. A total of 152 sheep serum samples were tested in Nong’an County, Changchun City, Jilin Province, and the positive rate was 5.92%. This study laid the foundation for the development of serological detection preparations for F. hepatica infection based on the detection of circulating antigens.
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- 2024
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7. Cryptosporidium parvum regulates HCT-8 cell autophagy to facilitate survival via inhibiting miR-26a and promoting miR-30a expression
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Heng Jiang, Xu Zhang, Xin Li, Xiaocen Wang, Nan Zhang, Pengtao Gong, Xichen Zhang, Yanhui Yu, and Jianhua Li
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Cryptosporidium parvum ,microRNAs ,MAPK signaling ,Autophagy ,Parasite proliferation ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Cryptosporidium parvum is an important zoonotic parasite, which not only causes economic losses in animal husbandry but also harms human health. Due to the lack of effective measures for prevention and treatment, it is important to understand the pathogenesis and survival mechanism of C. parvum. Autophagy is an important mechanism of host cells against parasite infection through key regulatory factors such as microRNAs and MAPK pathways. However, the regulatory effect of C. parvum on autophagy has not been reported. Here, we demonstrated that C. parvum manipulated autophagy through host cellular miR-26a, miR-30a, ERK signaling and P38 signaling for parasite survival. Methods The expression of Beclin1, p62, LC3, ERK and P38 was detected using western blotting in HCT-8 cells infected with C. parvum as well as treated with miR-26a-mimic, miR-30a-mimic, miR-26a-mimic or miR-30a-inhibitor post C. parvum infection. The qPCR was used to detect the expression of miR-26a and miR-30a and the number of C. parvum in HCT-8 cells. Besides, the accumulation of autophagosomes was examined using immunofluorescence. Results The expression of Beclin1 and p62 was increased, whereas LC3 expression was increased initially at 0–8 h but decreased at 12 h and then increased again in C. parvum-infected cells. C. parvum inhibited miR-26a-mimic-induced miR-26a but promoted miR-30a-mimic-induced miR-30a expression. Suppressing miR-30a resulted in increased expression of LC3 and Beclin1. However, upregulation of miR-26a reduced ERK/P38 phosphorylation, and inhibiting ERK/P38 signaling promoted Beclin1 and LC3 while reducing p62 expression. Treatment with miR-26a-mimic, autophagy inducer or ERK/P38 signaling inhibitors reduced but treatment with autophagy inhibitor or miR-30a-mimic increased parasite number. Conclusions The study found that C. parvum could regulate autophagy by inhibiting miR-26a and promoting miR-30a expression to facilitate the proliferation of parasites. These results revealed a new mechanism for the interaction of C. parvum with host cells. Graphical Abstract
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- 2022
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8. Establishment and application of a CRISPR-Cas12a-based RPA-LFS and fluorescence for the detection of Trichomonas vaginalis
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Shan Li, Xiaocen Wang, Yanhui Yu, Songgao Cao, Juan Liu, Panpan Zhao, Jianhua Li, Xichen Zhang, Xin Li, Nan Zhang, Min Sun, Lili Cao, and Pengtao Gong
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Trichomonas vaginalis ,CRISPR-Cas12a ,RPA ,Visualization detection ,On-site testing ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Infection with Trichomonas vaginalis can lead to cervicitis, urethritis, pelvic inflammatory disease, prostatitis and perinatal complications and increased risk of HIV transmission. Here, we used an RPA-based CRISPR-Cas12a assay system in combination with a lateral flow strip (LFS) (referred to as RPA-CRISPR-Cas12a) to establish a highly sensitive and field-ready assay and evaluated its ability to detect clinical samples. Methods We developed a one-pot CRISPR-Cas12a combined with RPA-based field detection technology for T. vaginalis, chose actin as the target gene to design crRNA and designed RPA primers based on the crRNA binding site. The specificity of the method was demonstrated by detecting genomes from nine pathogens. To improve the usability and visualize the RPA-CRISPR-Cas12a assay results, both fluorescence detection and LFS readouts were devised. Results The RPA-CRISPR-Cas12a assay platform was completed within 60 min and had a maximum detection limit of 1 copy/µl and no cross-reactivity with Candida albicans, Mycoplasma hominis, Neisseria gonorrhoeae, Escherichia coli, Cryptosporidium parvum, G. duodenalis or Toxoplasma gondii after specificity validation. Thirty human vaginal secretions were tested by RPA-CRISPR-Cas12a assays, and the results were read by a fluorescent reporter and LFS biosensors and then compared to the results from nested PCR detection of these samples. Both RPA-CRISPR-Cas12a assays showed 26.7% (8/30) T. vaginalis-positive samples and a consistency of 100% (8/8). The RPA-CRISPR-Cas12a assays had a higher sensitivity than nested PCR (only seven T. vaginalis-positive samples were detected). Conclusions The T. vaginalis RPA-CRISPR-Cas12a assay platform in this study can be used for large-scale field testing and on-site tests without the need for trained technicians or costly ancillary equipment. Graphical abstract
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- 2022
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9. Prevalence of fish-borne zoonotic trematode infection in Jilin Province, China
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Yuru Wang, Xiaocen Wang, Pengtao Gong, Yanhui Yu, Nan Zhang, Yanyan Ren, Yeting Ma, Zhiteng Zhao, Xichen Zhang, Xin Li, and Jianhua Li
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Fish-borne zoonotic trematodes ,Prevalence ,Clonorchis sinensis ,Metorchis orientalis ,Echinochasmus japonicus ,Zoology ,QL1-991 - Abstract
Fish-borne zoonotic trematodes (FZTs) are the most serious food-borne parasites in Asia and have become a burden to public health and a new challenge in food safety. In Jilin Province, China, the prevalence of FZTs in intermediate and definitive hosts has not been extensively explored. In the present study, we investigated the prevalence of FZTs in Jilin Province, China. From July to November 2020, a total of 132 freshwater snails (the first intermediate host of FZTs), 4122 wild freshwater fishes (the second intermediate host of FZTs) and 143 fecal samples from canines, ducks and swine (the definitive host of FZTs) were collected from the Yitong River basin of Jilin Province. FZT species were identified by morphological observation combined with internal transcribed spacer (ITS) sequence analysis. The prevalence of FZTs was then calculated. The results showed that the prevailing species of FZTs in Jilin Province, China, were Clonorchis sinensis, Metorchis orientalis and Echinochasmus japonicus. The total prevalence of FZTs was 29.74% (1226/4122) in fish, the total infection rates were 2.27% (3/132) in snails, 75.00% (21/28) in canines and 37.18% (29/78) in ducks. The coinfection rates of the two trematodes were 13.39% (552/4122) in fish, 35.71% (10/28) in canines and 7.69% (6/78) in ducks. The coinfection rate of the three flukes was 2.60% (107/4122) in fish. Nine of the 12 fish species examined were infected with FZT metacercariae.
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- 2022
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10. TLR3 activation by Clonorchis sinensis infection alleviates the fluke-induced liver fibrosis.
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Yuru Wang, Pengtao Gong, Xuancheng Zhang, Xiaocen Wang, Xu Zhang, Nan Zhang, Yanhui Yu, Yeting Ma, Haoyang Zhang, Xichen Zhang, Xin Li, and Jianhua Li
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Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Abstract
Clonorchis sinensis is a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. The role of toll-like receptors (TLRs) in C. sinensis infection has not yet been fully elucidated. Here, the TLR3 signaling pathway, cytokine expression and liver fibrosis were examined in C. sinensis-infected wildtype (WT) and TLR3-/- mice. Polyinosinic-polycytidylic acid (Poly (I:C)) was used to treat C. sinensis infections. The results showed that TLR3 deficiency caused severe clonorchiasis with increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-β1/Smad2/3 pathway and myofibroblast activation, exacerbated liver fibrosis (compared to WT mice). Poly (I:C) intervention increased the body weight, decreased mouse mortality and parasite burden, reduced liver inflammation, and alleviated C. sinensis-induced liver fibrosis. Furthermore, C. sinensis extracellular vesicles (CsEVs) promote the production of IL-6, TNF in WT biliary epithelial cells (BECs) via p38/ERK pathway, compared with control group, while TLR3 deletion induced much higher levels of IL-6 and TNF in TLR3-/- BECs than that in WT BECs. Taken together, TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathway, a phenomenon that resulted in the alleviation of C. sinensis-induced liver fibrosis. Poly (I:C) is a potential treatment for clonorchiasis.
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- 2023
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11. Development of Nested Polymerase Chain Reaction with Novel Specific Primers for Detection of Tritrichomonas muris Infection in Laboratory Mice
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Hongbo Zhang, Nan Zhang, Jianhua Li, Panpan Zhao, Xin Li, Xiaocen Wang, Xu Zhang, Bao Yuan, Fei Gao, Pengtao Gong, and Xichen Zhang
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Tritrichomonas muris ,SSU rRNA ,primer ,nested PCR ,laboratory mice ,Veterinary medicine ,SF600-1100 ,Zoology ,QL1-991 - Abstract
A variety of rodent ceca are parasitized by Tritrichomonas muris (T. muris), a flagellated protozoan. To date, there are no ideal methods for the detection of T. muris infections in laboratory mice; thus, new molecular methodologies for its specific detection need to be developed. In this study, using staining and SEM, it was observed that T. muris has a pear-shaped body and contains three anterior flagella. A nested PCR system with novel specific primers was designed based on the conserved regions of the SSU rRNA gene of T. muris. The nested PCR system for T. muris showed good specificity and high sensitivity for at least 100 T. muris trophozoites/mL and 0.1 ng/μL of fecal genomic DNA, which means that 176 trophozoites per gram of mouse feces could be detected. When using this nested PCR system, the detection rate was 18.96% (58/306), which was higher than the detection rate of 14.05% (43/306) detected via smear microscopy in fecal samples from five mouse strains. The sensitivity and specificity of nested PCR in detecting T. muris was found to be 100%, and it demonstrated a 26% increase in diagnostic sensitivity compared to the smear microscopy method in the present study. In conclusion, the nested PCR developed with novel primers based on the SSU rRNA gene of T. muris has good accuracy, specificity, and sensitivity for the detection of T. muris infections in laboratory mice.
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- 2023
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12. Multiple Regulations of Parasitic Protozoan Viruses: A Double-Edged Sword for Protozoa
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Zhiteng Zhao, Xin Li, Nan Zhang, Jianhua Li, Na Zhao, Mengyao Gao, Xichen Zhang, Xiaocen Wang, Panpan Zhao, Lu Li, Min Sun, Lili Cao, and Pengtao Gong
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parasitic protozoan viruses ,PPVs ,morphology ,pathogenicity ,viral infection ,virus-parasite relationship ,Microbiology ,QR1-502 - Abstract
ABSTRACT Parasite infections affect human and animal health significantly and contribute to a major burden on the global economy. Parasitic protozoan viruses (PPVs) affect the protozoan parasites’ morphology, phenotypes, pathogenicity, and growth rates. This discovery provides an opportunity to develop a novel preventive and therapeutic strategy for parasitic protozoan diseases (PPDs). Currently, there is greater awareness regarding PPVs; however, knowledge of viruses and their associations with host diseases remains limited. Parasite-host interactions become more complex owing to PPVs; however, few studies have investigated underlying viral regulatory mechanisms in parasites. In this study, we reviewed relevant studies to identify studies that investigated PPV development and life cycles, the triangular association between viruses, parasites, and hosts, and the effects of viruses on protozoan pathogenicity. This study highlights that viruses can alter parasite biology, and viral infection of parasites may exacerbate the adverse effects of virus-containing parasites on hosts or reduce parasite virulence. PPVs should be considered in the prevention of parasitic epidemics and outbreaks, although their effects on the host and the complexity of the triangular association between PPVs, protozoans, and hosts remain unclear. IMPORTANCE PPVs-based regulation of parasitic protozoa can provide a theoretical basis and direction for PPD prevention and control, although PPVs and PPV regulatory mechanisms remain unclear. In this review, we investigated the differences between PPVs and the unique properties of each virus regarding virus discovery, structures, and life cycles, focused on the Trichomonas vaginalis virus, Giardia lamblia virus, Leishmania RNA virus, and the Cryptosporidium parvum virus 1. The triangular association between PPVs, parasitic protozoa, and hosts reveals the “double-edged sword” property of PPVs, which maintains a balance between parasitic protozoa and hosts in both positive and negative respects. These studies discuss the complexity of parasitic protozoa and their co-existence with hosts and suggest novel pathways for using PPVs as tools to gain a deeper understanding of protozoal infection and treatment.
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- 2023
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13. Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways.
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Yuru Wang, Xu Zhang, Xiaocen Wang, Nan Zhang, Yanhui Yu, Pengtao Gong, Xichen Zhang, Yeting Ma, Xin Li, and Jianhua Li
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Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Abstract
Clonorchis sinensis is an important food-borne zoonotic parasite which has been linked to biliary fibrosis and cholangiocarcinoma. However, the details of the pathogenesis of C. sinensis were unclear. To explore the role and regulatory mechanism of toll-like receptor 2 (TLR2) in C. sinensis-induced biliary fibrosis, we established the C. sinensis-infected C57BL/6 mouse model with TLR2-/- and wild type (WT) mice. The mortality rate, liver lesions, TLR2 and TGF-β1 expression, phosphorylation of Smad2/3, AKT, p38, ERK and p65, and cytokine productions were analyzed. Furthermore, similar parameters were examined in mouse biliary epithelial cells (BECs) co-cultured with C. sinensis excretory/secretory proteins (ESPs). The results showed that TLR2 expression was enhanced significantly in C. sinensis-infected WT mice and mouse BECs. C. sinensis-infected TLR2-/- mice exhibited an increased weight and a decreased mortality rate; significantly alleviated liver lesions and biliary fibrosis, reduced numbers of myofibroblasts; decreased expression of TGF-β1 and phosphorylation level of AKT, p38 and Smad2/3; significantly decreased production of IL-6, TNF-α and IL-4, while increased production of IFN-γ compared with C. sinensis-infected WT mice. Furthermore, C. sinensis ESPs could activate TLR2-mediated AKT and p38 pathways to increase the production of IL-6 in mouse BECs. In conclusion, these data indicate that C. sinensis infection activated TGF-β1-Smad2/3 through TLR2-mediated AKT and p38 pathways to promote IL-6 production, which resulted in myofibroblast activation and aggravating biliary fibrosis in mice.
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- 2023
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14. Trypanosoma evansi evades host innate immunity by releasing extracellular vesicles to activate TLR2-AKT signaling pathway
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Ran Wei, Xin Li, Xiaocen Wang, Nan Zhang, Yuru Wang, Xichen Zhang, Pengtao Gong, and Jianhua Li
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trypanosoma evansi ,immune evasion ,extracellular vesicles ,tlr2 ,akt ,kmp-11 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Surra, one of the most important animal diseases with economic consequences in Asia and South America, is caused by Trypanosoma evansi. However, the mechanism of immune evasion by T. evansi has not been extensively studied. In the present study, T. evansi extracellular vesicles (TeEVs) were characterized and the role of TeEVs in T. evansi infection were examined. The results showed that T. evansi and TeEVs could activate TLR2-AKT pathway to inhibit the secretions of IL-12p40, IL-6, and TNF-α in mouse BMDMs. TLR2−/- mice and mice with a blocked AKT pathway were more resistant to T. evansi infection than wild type (WT) mice, with a significantly lower infection rate, longer survival time and less parasite load, as well as an increased secretion level of IL-12p40 and IFN-γ. Kinetoplastid membrane protein-11 (KMP-11) of TeEVs could activate AKT pathway and inhibit the productions of IL-12p40, TNF-α, and IL-6 in vitro. TeEVs and KMP-11 could inhibit the productions of IL-12p40 and IFN-γ, promote T. evansi proliferation and shorten the survival time of infected mice in vivo. In conclusion, T. evansi could escape host immune response through inhibiting the productions of inflammatory cytokines via secreting TeEVs to activate TLR2-AKT pathway. KMP-11 in TeEVs was involved in promoting T. evansi infection. Extracellular vesicles (EVs) secreted by Trypanosoma evansi (T. evansi) activate the TLR2-AKT signaling pathway to inhibit the production of inflammatory cytokines, thereby escaping the host’s immune response. Kinetoplastid membrane protein-11 (KMP-11) in EVs is related to the promotion of T.evansi infection via AKT pathway.
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- 2021
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15. Giardia duodenalis extracellular vesicles regulate the proinflammatory immune response in mouse macrophages in vitro via the MAPK, AKT and NF-κB pathways
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Panpan Zhao, Lili Cao, Xiaocen Wang, Jianhua Li, Jingquan Dong, Nan Zhang, Xin Li, Shan Li, Min Sun, Xichen Zhang, Min Liang, Xudong Pu, and Pengtao Gong
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Giardia duodenalis ,Extracellular vesicles ,Immune response ,MAPK ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Giardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals. The presentation of giardiasis ranges from asymptomatic to severe diarrhea, and the World Health Organization lists it in the Neglected Diseases Initiative. Extracellular vesicles (EVs) are a key mediator of intracellular communication. Although previous studies have shown that G. intestinalis can regulate a host’s innate immune response, the role of G. intestinalis EVs (GEVs) in triggering a G. intestinalis-induced innate immune response remains to be further explored. Methods In this study, GEVs, G. intestinalis and GEVs + G. intestinalis were inoculated into macrophages, respectively. The transcription and secretion levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α), were measured using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs). The phosphorylation levels of the MAPK, AKT and NF-κB signaling pathways in GEV-stimulated mouse macrophages were examined using western blotting and immunofluorescence methods. The roles of activated pathways in the GEV-triggered inflammatory response were determined using inhibition assays, western blotting and ELISAs. Results The results showed that pretreatment with GEVs enhanced with G. intestinalis (GEVs + G. intestinalis) induced IL-1β, IL-6 and TNF-α transcription and secretion from mouse macrophages compared to stimulation with either GEVs or G. intestinalis alone. Inoculation of mouse macrophages with GEVs upregulated the phosphorylation levels of the p38 MAPK, p44/42 MAPK (Erk1/2), AKT and NF-κB signaling pathways and led to the nuclear translocation of NF-κB p65. Blocking the activated p38, Erk and NF-κB signaling pathways significantly downregulated the secretion of proinflammatory cytokines, and blocking the activated AKT signaling pathway demonstrated reverse effects. Conclusions The results of this study reveal that GEVs can enhance G. intestinalis-induced inflammatory response levels in mouse macrophages through activation of the p38, ERK and NF-κB signaling pathways. The role of GEVs in regulating host cell immune responses may provide insights into exploring the underlying mechanisms in G. intestinalis–host interactions. Graphical abstract
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- 2021
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16. Changes of gut microbiota in colorectal cancer patients with Pentatrichomonas hominis infection
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Hongbo Zhang, Yanhui Yu, Jianhua Li, Pengtao Gong, Xiaocen Wang, Xin Li, Yidan Cheng, Xiuyan Yu, Nan Zhang, and Xichen Zhang
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colon cancer ,Pentatrichomonas hominis ,gut microbiota ,16S rRNA ,high-throughput sequencing ,Microbiology ,QR1-502 - Abstract
Pentatrichomonas hominis is a parasitic trichomonads protozoa that parasitizes in the colon and cecum of humans and other animals. Our previous studies have demonstrated that infection with P. hominis is associated with the incidence of colon cancer (37.93%). However, the mechanism by which P. hominis infections increase the incidence of colon cancer remains unclear. Previous studies have suggested that certain parasites promote colon cancer by regulating gut microbiota. This study aimed to elucidate whether the association between P. hominis infections and the increased incidence of colon cancer is related to changes in gut microbiota. Therefore, the gut microbiota patients with colon cancer who were infected with P. hominis and uninfected patients with colon cancer were analyzed by 16S rRNA high-throughput sequencing. The results demonstrated that patients with colon cancer who were not infected with P. hominis showed increased gut bacterial diversity, a higher relative abundance of Alcaligenes sp., Leucobacter sp., Paraprevotella sp., Ruminococcaceae UCG-002, and a significant reduction in the abundance of Veillonella sp., compared to individuals without colon cancer. Additionally, the relative abundance of the Ruminococcaceae UCG-002 and the Eubacterium eligens groups was reduced, while the relative abundance of bacteria associated with colon cancer, including Flavonifractor sp., Lachnoclostridium sp., and the Ruminococcus gnavus group, increased significantly in patients with colon cancer who were infected with P. hominis, compared to those of uninfected patients with colon cancer. In conclusion, these results suggested that P. hominis infections may aggravate the development of colon cancer and the findings provide new insights for subsequent in-depth studies on the pathogenesis, diagnosis, and prevention of colon cancer.
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- 2022
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17. First case report of Metorchis orientalis from Black Swan
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Yuru Wang, Xin Li, Qingsong Sun, Pengtao Gong, Nan Zhang, Xichen Zhang, Xiaocen Wang, Guojiang Li, and Jianhua Li
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Metorchis orientalis ,Black Swan ,Intermediate hosts ,Definitive hosts ,ITS sequence Analysis ,Zoology ,QL1-991 - Abstract
Metorchis orientalis belongs to the genus Metorchis of Opisthorchiidae, which mainly parasitizes in liver and bile ducts of waterfowl, causing liver dysfunction of the host. It has been reported that M. orientalis also infects humans. As a natural species in Australia and a popular ornamental animal, Black Swan (Cygnus atratus) has been imported into many countries. At present there has been no report of M. orientalis infection in Black Swan. In the present study M. orientalis infection in Black Swan was identified by a combination of different techniques, including morphological observation and molecular analysis. M. orientalis adults were found in the gallbladder and bile duct of a three-year-old female Black Swan, which was further confirmed by internal transcribed spacer (ITS) sequence analysis. In addition, the intermediate and definitive hosts of M. orientalis from the ‘Qing’ lake (a man-made lake in Changchun, China) that Black Swan lived were investigated and the infection route was preliminarily determined. Parafossarulus striatulus functioned as the first intermediate host which contained M. orientalis DNA, and fishes such as Pseudorasbora parva and Rhodeinae served as the second intermediate hosts with M. orientalis metacercariae in the fish flesh. M. orientalis eggs were found in the feces of three other Swans and six ducks that lived in the ‘Qing’ lake. This was the first reported case about M. orientalis infection of Black Swan. Our study described the course of the infection and provided new information about potential carriers and disseminators of M. orientalis.
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- 2020
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18. Neospora caninum Evades Immunity via Inducing Host Cell Mitophagy to Inhibit Production of Proinflammatory Cytokines in a ROS-Dependent Manner
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Xu Zhang, Yuru Wang, Pengtao Gong, Xiaocen Wang, Nan Zhang, Mengge Chen, Ran Wei, Xichen Zhang, Xin Li, and Jianhua Li
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N. caninum ,mitophagy ,proinflammatory cytokines ,ROS ,immune escape ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Neospora caninum is an intracellular protozoan that mainly infects cattle to cause abortion and significant economic losses worldwide. A better understanding of the immune evasion mechanisms of N. caninum could help to search for an effective approach to prevent and treat neosporosis. Mitophagy is used by some viruses to evade host immune surveillance. However, host cell mitophagy and its effect on N. caninum infection is unclear. In the present study, N. caninum-induced host cell mitophagy and its role in parasite infection were investigated in vitro and in vivo. Furthermore, the regulation of N. caninum-induced host cell mitophagy on the production of Reactive Oxygen Species (ROS), the secretions of proinflammatory cytokines, and the signals of p38, ERK, and Nlrp3 inflammasome were explored. Our results showed that autophagosomes and co-localization of LC3 with mitochondria were observed in N. caninum-infected macrophages. The mtDNA/nDNA ratio and the levels of mitochondrial marker proteins (Hsp60 and Tim23) were decreased with the increase of N. caninum numbers or infection time. N. caninum could induce mitophagy in brain and peritoneal lavage fluid cells of mice. Promoting mitophagy via mitophagy inducers (CCCP) could shorten survival time, decrease body weight, increase parasite load, and attenuate secretion of cytokines in N. caninum infected mice. CCCP treatment decreased the production of cytokines and Reactive Oxygen Species (ROS), and increased parasite burden in N. caninum-infected macrophages. Furthermore, CCCP or NAC (ROS inhibitor) treatment could inhibit ERK signal, Nlrp3 inflammasome, and cytokine production, while promote p38 signal in N. caninum-infected macrophages. The opposite results were obtained when using a mitophagy inhibitor (Mdivi1). Taken together, N. caninum-induced mitophagy could regulate the activations of p38, ERK, Nlrp3 inflammasome to inhibit the production of inflammatory cytokines in a ROS-dependent manner to escape host immune surveillance.
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- 2022
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19. The Protective Role of TLR2 Mediates Impaired Autophagic Flux by Activating the mTOR Pathway During Neospora caninum Infection in Mice
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Jielin Wang, Xiaocen Wang, Pengtao Gong, Fu Ren, Xin Li, Nan Zhang, Xu Zhang, Xichen Zhang, and Jianhua Li
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Neospora caninum ,autophagy ,mTOR ,TLR2 ,anti-infection ,innate immune ,Microbiology ,QR1-502 - Abstract
Autophagy has been shown to play an essential role in defending against intracellular bacteria, viruses, and parasites. Mounting evidence suggests that autophagy plays different roles in the infection process of different pathogens. Until now, there has been no conclusive evidence regarding whether host autophagy is involved in Neospora caninum infection. In the current study, we first monitored the activation of autophagy by N. caninum, which occurred mainly in the early stages of infection, and examined the role of host autophagy in N. caninum infection. Here, we presented evidence that N. caninum induced an increase in autophagic vesicles with double-membrane structures in macrophages at the early stage of infection. LC3-II expression peaked and decreased as infection continued. However, the expression of P62/SQSTM1 showed significant accumulation within 12 h of infection, indicating that autophagic flux was blocked. A tandem fluorescence protein mCherry-GFP-LC3 construct was used to corroborate the impaired autophagic flux. Subsequently, we found that N. caninum infection induced the activation of the TLR2–AKT–mTOR pathways. Further investigation revealed that TLR2–mTOR, accompanied by the blockade of autophagic flux, was responsible for impaired autophagy but was not associated with AKT. In vitro and in vivo, N. caninum replication was strongly blocked by the kinase inhibitor 3-methyladenine (3-MA, autophagy inhibitor). In contrast, rapamycin (Rapa, an autophagy inducer) was able to promote intracellular proliferation and reduce the survival rate of N. caninum-infected mice. On the other hand, the accumulation of autophagosomes facilitated the proliferation of N. caninum. Collectively, our findings suggest that activation of host autophagy facilitates N. caninum replication and may counteract the innate immune response of the host. In short, inhibition of the early stages of autophagy could potentially be a strategy for neosporosis control.
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- 2021
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20. Correction: Establishment and application of a CRISPR-Cas12a-based RPA-LFS and fluorescence for the detection of Trichomonas vaginalis
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Shan Li, Xiaocen Wang, Yanhui Yu, Songgao Cao, Juan Liu, Panpan Zhao, Jianhua Li, Xichen Zhang, Xin Li, Nan Zhang, Min Sun, Lili Cao, and Pengtao Gong
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Infectious and parasitic diseases ,RC109-216 - Published
- 2022
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21. Evaluation of Protective Immune Responses Induced in BALB/c Mice and Goats by the Neospora caninum Surface SRS Proteins and Interleukin-18
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Pu Wang, Xiaocen Wang, Weirong Wang, Pengtao Gong, Nan Zhang, Renzhe Zhang, Huan Zeng, Qian Sun, Wanqing Li, Xin Li, Shuqin Cheng, Xu Zhang, Xinyi Huang, Chenyang Gao, Yadong Zheng, Jianhua Li, and Xichen Zhang
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Neospora caninum ,IL-18 ,SRS17 ,SRS2 ,SRS52 ,immunization ,Veterinary medicine ,SF600-1100 ,Zoology ,QL1-991 - Abstract
Neosporosis is caused by Neospora caninum (N. caninum), which mainly infects cattle and goats and severely threatens the animal industry. In this study, the inhibitory effects of polyclonal antiserum anti-NcSRS17, NcSRS2 and NcSRS52 were explored. Cytokines in mice or goat serum were detected after immunization. After infection, the survival of mice was recorded. The pathological changes and parasite loads were observed and detected in tissues. The results showed that anti-NcSRS2, NcSRS17 and NcSRS52 antibodies all inhibit the invasion and proliferation of N. caninum. The IFN-γ level in the NcSRS17 group was higher than that in the NcSRS2 and NcSRS52 groups, and higher in the NcSRS2-mIL-18 group than in the NcSRS2 group. The survival rates of mice were 16% in the positive control group, 67% in the SRS52 group, 83% in the SRS2 and mIL-18 groups and 100% in the SRS17 and SRS2-mIL-18 groups. Goats immunized with NcSRS17-gIL-18 developed high levels of IL-4, IL-12 and IFN-γ compared with those immunized with NcSRS-17. Parasite loads in the brains of animals in the NcSRS17 and NcSRS17-gIL-18 groups were significantly reduced, and were significantly lower in the NcSRS17-gIL-18 group (p ≤ 0.01). This study indicates that SRS17 may be an antigen candidate for vaccine development against neosporosis, and IL-18 can enhance the immune protective efficiency of antigen candidates.
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- 2022
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22. Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways
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Xudong Pu, Xin Li, Lili Cao, Kaiming Yue, Panpan Zhao, Xiaocen Wang, Jianhua Li, Xichen Zhang, Nan Zhang, Zhiteng Zhao, Min Liang, and Pengtao Gong
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Giardia duodenalis ,TLR9 ,p38 ,ERK ,cytokines ,Biology (General) ,QH301-705.5 - Abstract
Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, is an important opportunistic, pathogenic, zoonotic, protozoan parasite that infects the small intestines of humans and animals, causing giardiasis. Several studies have demonstrated that innate immunity-associated Toll-like receptors (TLRs) are critical for the elimination of G. duodenalis; however, whether TLR9 has a role in innate immune responses against Giardia infection remains unknown. In the present study, various methods, including reverse transcriptase–quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, inhibitor assays, and small-interfering RNA interference, were utilized to probe the role of TLR9 in mouse macrophage-mediated defenses against G. lamblia virus (GLV)–free or GLV-containing Giardia trophozoites. The results revealed that in G. duodenalis–stimulated mouse macrophages, the secretion of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, was enhanced, concomitant with the significant activation of TLR9, whereas silencing TLR9 attenuated the host inflammatory response. Notably, the presence of GLV exacerbated the secretion of host proinflammatory cytokines. Moreover, G. duodenalis stimulation activated multiple signaling pathways, including the nuclear factor κB p65 (NF-κB p65), p38, ERK, and AKT pathways, the latter three in a TLR9-dependent manner. Additionally, inhibiting the p38 or ERK pathway downregulated the G. duodenalis–induced inflammatory response, whereas AKT inhibition aggravated this process. Taken together, these results indicated that G. duodenalis may induce the secretion of proinflammatory cytokines by activating the p38 and ERK signaling pathways in a TLR9-dependent manner in mouse macrophages. Our in vitro findings on the mechanism underlying the TLR9-mediated host inflammatory response may help establish the foundation for an in-depth investigation of the role of TLR9 in the pathogenicity of G. duodenalis.
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- 2021
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23. Extracellular vesicles secreted by Giardia duodenalis regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways.
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Panpan Zhao, Lili Cao, Xiaocen Wang, Jingquan Dong, Nan Zhang, Xin Li, Jianhua Li, Xichen Zhang, and Pengtao Gong
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Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Abstract
Giardia duodenalis, also known as G. intestinalis or G. lamblia, is the major cause of giardiasis leading to diarrheal disease with 280 million people infections annually worldwide. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism participating in cells communications. The aim of this study is to explore the roles of G. duodenalis EVs (GEVs) in host-pathogen interactions using primary mouse peritoneal macrophages as a model. Multiple methods of electron microscopy, nanoparticle tracking analysis, proteomic assays, flow cytometry, immunofluorescence, qPCR, western blot, ELISA, inhibition assays, were used to characterize GEVs, and explore its effects on the host cell innate immunity as well as the underlying mechanism using primary mouse peritoneal macrophages. Results showed that GEVs displayed typical cup-shaped structure with 150 nm in diameter. GEVs could be captured by macrophages and triggered immune response by increasing the production of inflammatory cytokines Il1β, Il6, Il10, Il12, Il17, Ifng, Tnf, Il18, Ccl20 and Cxcl2. Furthermore, activation of TLR2 and NLRP3 inflammasome signaling pathways involved in this process. In addition, CA-074 methyl ester (an inhibitor of cathepsin B) or zVAD-fmk (an inhibitor of pan-caspase) pretreatment entirely diminished these effects triggered by GEVs exposure. Taken together, these findings demonstrated that GEVs could be internalized into mouse peritoneal macrophages and regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways.
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- 2021
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24. High prevalence of Pentatrichomonas hominis infection in gastrointestinal cancer patients
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Nan Zhang, Hongbo Zhang, Yanhui Yu, Pengtao Gong, Jianhua Li, Ziyi Li, Ting Li, Zhanjie Cong, Chunying Tian, Xiaofeng Liu, Xiuyan Yu, and Xichen Zhang
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Pentatrichomonas hominis ,Gastrointestinal cancer ,Colorectal cancer ,Stomach cancer ,Epidemiology ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Pentatrichomonas hominis is a flagellated protozoan that inhabits the large intestine of humans. Although several protozoans have been proposed to have a role in cancer progression, little is known about the epidemiology of P. hominis infection in cancer patients. Methods To determine the prevalence of P. hominis in patients with digestive system malignancies, we collected 195 and 142 fecal samples from gastrointestinal cancer patients and residents without any complaints related to the digestive system, respectively. Each sample was detected for the presence of P. hominis by nested PCR amplifying the internal transcribed spacer (ITS) region and partial 18S rRNA gene. Results A significantly higher prevalence of P. hominis was found in cancer patients than that in the control population (41.54 vs 9.15%, χ 2 = 42.84, df = 1, P
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- 2019
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25. Protective Immunity Against Neospora caninum Infection Induced by 14-3-3 Protein in Mice
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Shan Li, Nan Zhang, Shaoxiong Liu, Jianhua Li, Li Liu, Xiaocen Wang, Xin Li, Pengtao Gong, and Xichen Zhang
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Neospora caninum ,14-3-3 ,extracellular vesicles ,cytokines ,immunity ,Veterinary medicine ,SF600-1100 - Abstract
Neospora caninum is an apicomplexan parasite that infects many mammals and remains a threatening disease worldwide because of the lack of effective drugs and vaccines. Our previous studies demonstrated that N. caninum 14-3-3 protein (Nc14-3-3), which is included in N. caninum extracellular vesicles (NEVs), can induce effective immune responses and stimulate cytokine expression in mouse peritoneal macrophages. However, whether Nc14-3-3 has a protective effect and its mechanisms are poorly understood. Here, we evaluated the immune responses and protective effects of Nc14-3-3 against exposure to 2 × 107 Nc-1 tachyzoites. Antibody (IgG, IgGl, and IgG2a) levels and Th1-type (IFN-γ and IL-12) and Th2-type (IL-4 and IL-10) cytokines in mouse serum, survival rates, survival times, and parasite burdens were detected. In the present study, the immunostimulatory effect of Nc14-3-3 was confirmed, as it triggered Th1-type cytokine (IFN-γ and IL-12) production in mouse serum 2 weeks after the final immunization. Moreover, the immunization of C57BL/6 mice with Nc14-3-3 induced high IgG antibody levels and significant increases in CD8+ T lymphocytes in the spleens of mice, indicating that the cellular immune response was significantly stimulated. Mouse survival rates and times were significantly prolonged after immunization; the survival rates were 40% for Nc14-3-3 immunization and 60% for NEV immunization, while mice that received GST, PBS, or blank control all died at 13, 9, or 8 days, respectively, after intraperitoneal N. caninum challenge. In addition, qPCR analysis indicated that there was a reduced parasite burden and diminished pathological changes in the mice immunized with Nc14-3-3. Our data demonstrate that vaccination of mice with Nc14-3-3 elicits both cellular and humoral immune responses and provides partial protection against acute neosporosis. Thus, Nc14-3-3 could be an effective antigen candidate for vaccine development for neosporosis.
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- 2021
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26. Disruption of Dense Granular Protein 2 (GRA2) Decreases the Virulence of Neospora caninum
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Jingquan Dong, Nan Zhang, Panpan Zhao, Jianhua Li, Lili Cao, Xiaocen Wang, Xin Li, Ju Yang, Xichen Zhang, and Pengtao Gong
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Neospora caninum ,CRISPR ,dense granules protein 2 ,intravacuolar network ,immunofluorescence ,Veterinary medicine ,SF600-1100 - Abstract
Neospora caninum causes abortions in cattle and nervous system dysfunction in dogs. Dense granular proteins (GRAs) play important roles in virulence; however, studies on NcGRA functions are limited. In the present study, multiple methods, including site-directed mutagenesis; CRISPR/Cas9 gene editing; Western blotting; quantitative polymerase chain reaction; confocal microscopy; plaque, invasion, egress, and replication assays; animal assays of survival rate and parasite burden; and hematoxylin–eosin staining, were used to characterize the NcGRA2 protein, construct an NcGRA2 gene disruption (ΔNcGRA2) strain, and explore its virulence in vivo and vitro. The results showed that NcGRA2 shared 31.31% homology with TgGRA2 and was colocalized with NcGRA6 at the posterior end of tachyzoites and the intravacuolar network of parasitophorous vacuoles (PVs). Cell fractionation analysis showed that NcGRA2 behaved as a transmembrane and membrane-coupled protein. The ΔNcGRA2 strain was constructed by coelectroporation of the NcGRA2-targeting CRISPR plasmid (pNc-SAG1-Cas9:U6-SgGRA2) and DHFR-TS DNA donor and verified at the protein, genome, and transcriptional levels and by immunofluorescence localization analysis. The in vitro virulence results showed that the ΔNcGRA2 strain displayed smaller plaques, similar invasion and egress abilities, and slower intracellular growth. The in vivo virulence results showed a prolonged survival time, lower parasite burden, and mild histopathological changes. Overall, the present study indicates that NcGRA2, as a dense granular protein, forms the intravacuolar network structure of PVs and weakens N. caninum virulence by slowing proliferation. These data highlight the roles of NcGRA2 and provide a foundation for research on other protein functions in N. caninum.
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- 2021
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27. A Novel MicroRNA From the Translated Region of the Giardiavirus rdrp Gene Governs Virus Copy Number in Giardia duodenalis
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Pengtao Gong, Xianhe Li, Wei Wu, Lili Cao, Panpan Zhao, Xin Li, Baoyan Ren, Jianhua Li, and Xichen Zhang
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Giardia duodenalis ,Giardiavirus ,microRNA ,coding region ,rdrp gene ,Microbiology ,QR1-502 - Abstract
Giardia duodenalis is an important zoonotic parasite that can cause human and animal diarrhea. Giardiavirus (GLV) is a double-stranded RNA virus in Totiviridae family, which specifically infects trophozoites of the primitive protozoan parasite G. duodenalis. However, the GLV infectious and the pathogenicity of the G. duodenalis still remain to be confirmed. The GLV genome is 6,277 bp, which encodes two proteins (Gag and Gag-Pol). The expression of Gag-Pol protein is regulated by a-1 ribosomal frameshift. In this report, we identified a novel microRNA (GLV miRNA1) from the GLV. Split ligation northern results showed that GLV miRNA1 is a special expression product of GLV, and the precursor was also identified by primer extension. Antisense sequence of the GLV miRNA1 could increase the copy number of virus in G. duodenalis. It suggests that GLV miRNA1 governs the copy number of Giardiavirus in G. duodenalis. Most importantly, the GLV miRNA1 lies at the translated region of the rdrp gene, which is the first case that microRNA locates in the translated region of a known protein. It may be implying a novel phenomenon for miRNA biogenesis.
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- 2020
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28. Effects of Dense Granular Protein 6 (GRA6) Disruption on Neospora caninum Virulence
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Panpan Zhao, Nan Zhang, Jingquan Dong, Jianhua Li, Xiaocen Wang, Xin Li, Xiangrui Li, Ju Yang, Pengtao Gong, and Xichen Zhang
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Neospora caninum ,dense granules protein 6 ,intravacuolar network ,CRISPR ,virulence factor ,Veterinary medicine ,SF600-1100 - Abstract
Neospora caninum (N. caninum) is a major cause of abortions in cattle. During its invasion of host cells, a parasitophorous vacuole (PV) is formed, accompanied by an intravacuolar network (IVN). The IVN takes part in parasite ingesting of nutrients from hosts. The dense granular proteins of N. caninum (NcGRAs) play a key role in forming the PV and the IVN, which may influence virulence during N. caninum invasion. The present study aimed to explore the biological function of NcGRA6 in N. caninum by disrupting the NcGRA6 gene in the Nc-1 strain. We successfully constructed an NcGRA6-targeting CRISPR plasmid (pNc-SAG1-Cas9:U6-SgGRA6) and amplified the DHFR-TS DNA donor. The NcGRA6 knockout mutation (ΔNcGRA6) was generated by co-electroporation of the pNc-SAG1::CAS9-U6::sgGRA6 plasmid and the DHFR-TS DNA donor into the Nc-1 strain, which was then cultured under pyrimethamine selection pressure. The ΔNcGRA6 mutation was further verified by identification of NcGRA6 gene disruption using PCR, measurement of NcGRA6 gene transcription levels using qPCR, assessment of NcGRA6 protein expression levels using western blotting, and observation of NcGRA6 protein location using immunofluorescence and immunoelectron microscopy. The results of in vitro virulence assays, including plaque, invasion, egress, and replication assays, showed that the ΔNcGRA6 strain had smaller plaques, similar invasion and egress ability, and slower intracellular replication ability than the Nc-1 strain. The results of in vivo virulence assays showed that the ΔNcGRA6 strain exhibited reduced virulence and improved survival ability in mice compared with the Nc-1 strain. The parasite burden in ΔNcGRA6 strain-infected mouse tissues, including the heart, brain, liver, spleen, lung, and kidney, was significantly reduced compared with that in mice infected with the Nc-1 strain. These data suggest that we successfully constructed a ΔNcGRA6 strain and verify that NcGRA6 is a critical virulence factor. NcGRA6 gene disruption can slow down N. caninum proliferation and lower the pathogenicity to hosts. Our findings provide a foundation for future research on other targeted N. caninum protein functions and may help in exploring the interaction mechanisms between parasites and hosts.
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- 2020
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29. An OTU deubiquitinating enzyme in Eimeria tenella interacts with Eimeria tenella virus RDRP
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Pu Wang, Jianhua Li, Pengtao Gong, Weirong Wang, Yongxing Ai, and Xichen Zhang
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Eimeria tenella ,Etv-RDRP ,Et-OTU ,Interaction ,Deubiquitinase ,Mutation ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Chicken coccidiosis, a disease caused by seven species of Eimeria (Apicomplexa: Coccidia), inflicts severe economic losses on the poultry industry. Eimeria tenella is the one of the most virulent species pathogenic to chickens. Many parasitic protozoans are parasitised by double-stranded (ds) RNA viruses, and the influence of protozoan viruses on parasitic protozoans has been extensively reported. E. tenella RNA virus 1 (Etv) was identified in E. tenella, and the complete genome sequence of Etv was analysed. Here, we screened Etv-RNA-dependent RNA polymerase (RDRP)-interacting host protein E. tenella ovarian tumour (OTU) protein-like cysteine protease (Et-OTU) using a yeast two-hybrid system with pGBKT7-RDRP plasmid serving as bait. A previous study demonstrated that Et-OTU could regulate the telomerase activity of E. tenella, indicating that Et-OTU affects E. tenella proliferation. However, whether Etv-RDRP affects the molecular biological characteristics of E. tenella by interacting with OTU remains unclear. Results We obtained seven positive clones from the initial screen, and six of the seven preys were identified as false-positives. Finally, we identified an RDRP-associated protein predicted to be an E. tenella OTU protein. A α-galactosidase assay showed that the bait vector did not activate the GAL4 reporter gene, indicating no autoactivation activity from the RDRP bait fusion. Pull-down and co-immunoprecipitation assays verified the interaction between Et-OTU and Etv-RDRP both intracellularly and extracellularly. Additionally, Et-OTU was able to deconjugate K48- and K6-linked di-ubiquitin (di-Ub) chains in vitro but not K63-, K11-, K29-, or K33-linked di-Ub chains. The C239A and H351A mutations eliminated the deubiquitinase (DUB) activity of Et-OTU, whereas the D236A mutation did not. Additionally, when combined with RDRP, the DUB activity of Et-OTU towards K48- and K6-linked chains was significantly enhanced. Conclusion Etv-RDRP interacts with Et-OTU both intracellularly and extracellularly. Etv-RDRP enhances the hydrolysis of Et-OTU to K6- or K48-linked ubiquitin chains. This study lays the foundation for further research on the relationship between E. tenella and Etv.
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- 2018
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30. Analysis of Codon Usage Patterns in Giardia duodenalis Based on Transcriptome Data from GiardiaDB
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Xin Li, Xiaocen Wang, Pengtao Gong, Nan Zhang, Xichen Zhang, and Jianhua Li
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Giardia duodenalis ,codon usage bias ,transcriptome ,optimal codon ,evolution ,Genetics ,QH426-470 - Abstract
Giardia duodenalis, a flagellated parasitic protozoan, the most common cause of parasite-induced diarrheal diseases worldwide. Codon usage bias (CUB) is an important evolutionary character in most species. However, G. duodenalis CUB remains unclear. Thus, this study analyzes codon usage patterns to assess the restriction factors and obtain useful information in shaping G. duodenalis CUB. The neutrality analysis result indicates that G. duodenalis has a wide GC3 distribution, which significantly correlates with GC12. ENC-plot result—suggesting that most genes were close to the expected curve with only a few strayed away points. This indicates that mutational pressure and natural selection played an important role in the development of CUB. The Parity Rule 2 plot (PR2) result demonstrates that the usage of GC and AT was out of proportion. Interestingly, we identified 26 optimal codons in the G. duodenalis genome, ending with G or C. In addition, GC content, gene expression, and protein size also influence G. duodenalis CUB formation. This study systematically analyzes G. duodenalis codon usage pattern and clarifies the mechanisms of G. duodenalis CUB. These results will be very useful to identify new genes, molecular genetic manipulation, and study of G. duodenalis evolution.
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- 2021
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31. NLRP3 inflammasome activation in murine macrophages caused by Neospora caninum infection
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Xiaocen Wang, Pengtao Gong, Xu Zhang, Jielin Wang, Lixin Tai, Xu Wang, Zhengkai Wei, Yongjun Yang, Zhengtao Yang, Jianhua Li, and Xichen Zhang
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Neospora caninum ,Macrophages ,NLRP3 inflammasome ,Caspase-1 ,IL-1β ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Neospora caninum is an intracellular parasite that causes significant economic losses in cattle industry. Understanding the host resistance mechanisms in the innate immune response to neosporosis could facilitate the exploration of approaches for controlling N. caninum infection. The NLR inflammasome is a multiprotein platform in the cell cytoplasm and plays critical roles in the host response against microbes. Methods Neospora caninum-infected wild-type (WT) macrophages and Nlrp3 −/− macrophages, and inhibitory approaches were used to investigate inflammasome activation and its role in N. caninum infection. Inflammasome RT Profiler PCR Arrays were used to identify the primary genes involved in N. caninum infection. The expression of the sensor protein NLRP3, processing of caspase-1, secretion of IL-1β and cell death were detected. Neospora caninum replication in macrophages was also assessed. Results Many NLR molecules participated in the recognition of N. caninum, especially the sensor protein NLRP3, and further study revealed that the NLRP3 distribution became punctate in the cell cytoplasm, which facilitated inflammasome activation. Inflammasome activation-mediated caspase-1 processing and IL-1β cleavage in response to N. caninum infection were observed and were correlated with the time of infection and number of infecting parasites. LDH-related cell death was also observed, and this death was regarded as beneficial for the clearance of N. caninum. Treatment of N. caninum-infected macrophages with caspase-1, pan-caspase and NLRP3 inhibitors led to the impaired release of active IL-1β and a failure to restrict parasite replication. And Neospora caninum infected peritoneal macrophages from Nlrp3-deficient mice displayed greatly decreased release of active IL-1β and the failure of caspase-1 cleavage. Conclusions The NLRP3 inflammasome can be activated in N. caninum-infected macrophages, and plays a protective role in the host response to control N. caninum.
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- 2017
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32. 14-3-3 Protein of Neospora caninum Modulates Host Cell Innate Immunity Through the Activation of MAPK and NF-κB Pathways
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Shan Li, Pengtao Gong, Nan Zhang, Xin Li, Lixin Tai, Xu Wang, Zhengtao Yang, Ju Yang, Xingquan Zhu, Xichen Zhang, and Jianhua Li
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Neospora caninum ,14-3-3 ,immune protection ,MAPK ,AKT ,cytokines ,Microbiology ,QR1-502 - Abstract
Neospora caninum is an obligate intracellular apicomplexan parasite, the etiologic agent of neosporosis, and a major cause of reproductive loss in cattle. There is still a lack of effective prevention and treatment measures. The 14-3-3 protein is a widely expressed acidic protein that spontaneously forms dimers within apicomplexan parasites. This protein has been isolated and sequenced in many parasites; however, there are few reports about the N. caninum 14-3-3 protein. Here, we successfully expressed and purified a recombinant fusion protein of Nc14-3-3 (rNc14-3-3) and prepared a polyclonal antibody. Immunofluorescence and immunogold electron microscopy studies of tachyzoites or N. caninum-infected cells suggested that 14-3-3 was localized in the cytosol and the membrane. Western blotting analysis indicated that rNc14-3-3 could be recognized by N. caninum-infected mouse sera, suggesting that 14-3-3 may be an infection-associated antigen that is involved in the host immune response. We demonstrated that rNc14-3-3 induced cytokine expression by activating the MAPK and AKT signaling pathways, and inhibitors of p38, ERK, JNK, and AKT could significantly decrease the production of IL-6, IL-12p40, and TNF-α. In addition, phosphorylated nuclear factor-κB (NF-κB/p65) was observed in wild-type peritoneal macrophages (PMs) treated with rNc14-3-3, and the protein level of NF-κB/p65 was reduced in the cytoplasm but increased correspondingly in the nucleus after 2 h of treatment. These results were also observed in deficient in TLR2-/- PMs. Taken together, our results indicated that the N. caninum 14-3-3 protein can induce effective immune responses and stimulate cytokine expression by activating the MAPK, AKT, and NF-κB signaling pathways but did not dependent TLR2, suggesting that Nc14-3-3 is a novel vaccine candidate against neosporosis.
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- 2019
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33. Extracellular Vesicles Secreted by Neospora caninum Are Recognized by Toll-Like Receptor 2 and Modulate Host Cell Innate Immunity Through the MAPK Signaling Pathway
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Shan Li, Pengtao Gong, Lixin Tai, Xin Li, Xiaocen Wang, Chunyan Zhao, Xu Zhang, Zhengtao Yang, Ju Yang, Jianhua Li, and Xichen Zhang
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Neospora caninum ,extracellular vesicles ,innate immunity ,toll-like receptor 2 ,MAPK ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Neospora caninum is an obligate intracellular parasite, which causes significant economic losses in the cattle industry. However, the immune mechanism of the parasite–host interaction is not yet fully understood. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism by which almost all cells, especially immune and tumor cells, participate in intercellular communications. Although studies have indicated that EVs secreted by Toxoplasma gondii or Trypanosoma brucei promote exchanges of biological molecules important for the host–parasite interplay, however, EVs and their biological activities in N. caninum is not clear. Here, we used multiple methods, including electron microscopy, nanoparticle tracking analysis, RT-PCR, immunofluorescence, western blot, proteomics, and cytokine analyses, to examine the properties of N. caninum EVs. We found that N. caninum produced EVs that are similar to mammalian exosomes, which generally range from 30 to 150 nm in diameter. It was shown that N. caninum EVs could remarkably increase the production of pro-inflammatory cytokines IL-12p40, TNF-α, IL-1β, IL-6, and IFN-γ by wild-type (WT) mouse bone marrow-derived macrophages (BMDMs) whereas the secretion of IL-12p40, TNF-α, and IFN-γ was very strongly downregulated in TLR2−/− mouse BMDMs. The levels of IL-6 were not affected, but the secretion of IL-10 was upregulated. We found that the phosphorylation levels of P38, ERK, and JNK were significantly reduced in the TLR2−/− cells compared with those in WT mouse BMDMs and that treatment with chemical inhibiters of P38, ERK, and JNK resulted in upregulation of IL-6, IL-12p40, and IL-10 production. Together, these results demonstrated that N. caninum EVs could be rapidly internalized to deliver proteins to the host cells and modulate the host cell immune responses through MAPK signaling pathway in a TLR2-dependent manner. Our study is the first to reveal potential roles for N. caninum EVs in host communication and immune response in parasite–host interactions.
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- 2018
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34. NLRP3 Inflammasome Participates in Host Response to Neospora caninum Infection
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Xiaocen Wang, Pengtao Gong, Xu Zhang, Shan Li, Xiangyun Lu, Chunyan Zhao, Qile Yu, Zhengkai Wei, Yongjun Yang, Qun Liu, Zhengtao Yang, Jianhua Li, and Xichen Zhang
- Subjects
Neospora caninum ,NLRP3 inflammasome ,IL-18 ,IFN-γ ,host defense ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii that mainly infects canids as the definitive host and cattle as the intermediate host, resulting in abortion in cattle and leading to financial losses worldwide. Commercial vaccines or drugs are not available for the prevention and treatment of bovine neosporosis. Knowledge about the hallmarks of the immune response to this infection could form the basis of important prevention strategies. The innate immune system first responds to invading parasite and subsequently initiates the appropriate adaptive immune response against this parasite. Upon infection, activation of host pattern-recognition receptors expressed by immune cells triggers the innate immune response. Toll-like receptors, NOD-like receptors, and C-type lectin receptors play key roles in recognizing protozoan parasite. Therefore, we aimed to explore the role of the NLRP3 inflammasome during the acute period of N. caninum infection. In vitro results showed that N. caninum infection of murine bone marrow-derived macrophages activated the NLRP3 inflammasome, accompanied by the release of IL-1β and IL-18, cleavage of caspase-1, and induction of cell death. K+ efflux induced by N. caninum infection participated in the activation of the inflammasome. Infection of mice deficient in NLRP3, ASC, and caspase-1/11 resulted in decreased production of IL-18 and reduced IFN-γ in serum. Elevated numbers of monocytes/macrophages and neutrophils were found at the initial infection site, but they failed to limit N. caninum replication. These findings suggest that the NLRP3 inflammasome is involved in the host response to N. caninum infection at the acute stage and plays an important role in limiting parasite growth, and it may enhance Th1 response by inducing production of IFN-γ. These findings may help devise protocols for controlling neosporosis.
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- 2018
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35. Trichomonas vaginalis Induces Production of Proinflammatory Cytokines in Mouse Macrophages Through Activation of MAPK and NF-κB Pathways Partially Mediated by TLR2
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Ling Li, Xin Li, Pengtao Gong, Xichen Zhang, Zhengtao Yang, Ju Yang, and Jianhua Li
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Trichomonas vaginalis ,TLR2 ,TLR2-/- ,MAPK ,NF-κB ,cytokines ,Microbiology ,QR1-502 - Abstract
Trichomoniasis, caused by Trichomonas vaginalis infection, is the most prevalent sexually transmitted disease in female and male globally. However, the mechanisms by innate immunity against T. vaginalis infection have not been fully elucidated. Toll-like receptor2 (TLR2) has been shown to be involved in pathogen recognition, innate immunity activation, and inflammatory response to the pathogens. Nonetheless, the function of TLR2 against T. vaginalis remains unclear. In the present study, we investigated the role of TLR2 in mouse macrophages against T. vaginalis. RT-qPCR analysis revealed that T. vaginalis stimulation increased the gene expression of TLR2 in wild-type (WT) mouse macrophages. T. vaginalis also induced the secretion of IL-6, TNF-α, and IFN-γ in WT mouse macrophages, and the expression of these cytokines significantly decreased in TLR2-/- mouse macrophages and in WT mouse macrophages pretreated with MAPK inhibitors SB203580 (p38) and PD98059 (ERK). Western blot analysis demonstrated that T. vaginalis stimulation induced the activation of p38, ERK, and p65 NF-κB signal pathways in WT mouse macrophages, and the phosphorylation of p38, ERK, and p65 NF-κB significantly decreased in TLR2-/- mouse macrophages. Taken together, our data suggested that T. vaginalis may regulates proinflammatory cytokines production by activation of p38, ERK, and NF-κB p65 signal pathways via TLR2 in mouse macrophages. TLR2 might be involved in the defense and elimination of T. vaginalis infection.
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- 2018
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36. Caprine Monocytes Release Extracellular Traps against Neospora caninum In Vitro
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Zhengtao Yang, Zhengkai Wei, Carlos Hermosilla, Anja Taubert, Xuexiu He, Xiaocen Wang, Pengtao Gong, Jianhua Li, and Xichen Zhang
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Neospora caninum ,caprine ,monocytes ,extracellular traps ,apicomplexa ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Neospora caninum is an obligate intracellular apicomplexan parasite that causes reproductive loss and severe economic losses in dairy and goat industry. In the present study, we aim to investigate the effects of N. caninum tachyzoites on the release of extracellular traps (ETs) in caprine monocytes and furthermore elucidated parts of its molecular mechanisms. N. caninum tachyzoite-induced monocytes-derived ETs formation was detected by scanning electron microscopy. H3 and myeloperoxidase (MPO) within monocyte-ETs structures were examined using laser scanning confocal microscopy analyses. The results showed that N. caninum tachyzoites were not only able to trigger ETs formation in caprine monocytes, but also that monocyte-released ETs were capable of entrapping viable tachyzoites. Histones and MPO were found to be decorating the DNA within the monocytes derived-ETs structures thus proving the classical components of ETs. Furthermore, inhibitors of NADPH oxidase-, MPO-, ERK 1/2-, or p38 MAPK-signaling pathway significantly decreased N. caninum tachyzoite-triggered caprine monocyte-derived ETosis. This is the first report of ETs release extruded from caprine monocytes after N. caninum exposure and thus showing that this early innate immune effector mechanism might be relevant during the acute phase of caprine neosporosis.
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- 2018
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37. Activation of a Neospora caninum EGFR-Like Kinase Facilitates Intracellular Parasite Proliferation
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Xiaoxia Jin, Guojiang Li, Xichen Zhang, Pengtao Gong, Yanhui Yu, and Jianhua Li
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N. caninum ,EGFR ,AG1478 ,PKC activity ,parasite proliferation ,Microbiology ,QR1-502 - Abstract
The Apicomplexan parasite Neospora caninum, an obligate intracellular protozoan, causes serious diseases in a number of mammalian species, especially in cattle. Infection with N. caninum is associated with abortions in both dairy and beef cattle worldwide which have a major economic impact on the cattle industry. However, the mechanism by which N. caninum proliferates within host cells is poorly understood. Epidermal growth factor receptor (EGFR) is a protein kinase ubiquitously expressed, present on cell surfaces in numerous species, which has been confirmed to be essential in signal transduction involved in cell growth, proliferation, survival, and many other intracellular processes. However, the presence of EGFR in N. caninum and its role in N. caninum proliferation remain unclear. In the present study, we identified a putative EGFR-like kinase in N. caninum, which could be activated in tachyzoites by infection or treatment with rNcMIC3 [containing four epidermal growth factor (EGF) domains] or human EGF. Blockade of EGFR-like in tachyzoites by AG1478 significantly reduced parasite proliferation in host cells. Our data suggested that the activation of tachyzoite EGFR-like might facilitate the intracellular proliferation of N. caninum.
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- 2017
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38. TLR2−/− Mice Display Decreased Severity of Giardiasis via Enhanced Proinflammatory Cytokines Production Dependent on AKT Signal Pathway
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Xin Li, Xichen Zhang, Pengtao Gong, Feifei Xia, Ling Li, Zhengtao Yang, and Jianhua Li
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Giardia ,giardiasis ,TLR2 ,TLR2−/− ,AKT ,cytokines ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Giardia infection is one of the most common causes of waterborne diarrheal disease in a wide array of mammalian hosts, including humans globally. Although numerous studies have indicated that adaptive immune responses are important for Giardia defense, however, whether the host innate immune system such as TLRs recognizes Giardia remains poorly understood. TLR2 plays a crucial role in pathogen recognition, innate immunity activation, and the eventual pathogen elimination. In this study, we investigated the role of TLR2 as a non-protective inflammatory response on controlling the severity of giardiasis. RT-PCR analysis suggested that TLR2 expression was increased in vitro. We demonstrated that Giardia lamblia-induced cytokines expression by the activation of p38 and ERK pathways via TLR2. Interestingly, the expression of IL-12 p40, TNF-α, and IL-6, but not IFN-γ, was enhanced in TLR2-blocked and TLR2−/− mouse macrophages exposed to G. lamblia trophozoites compared with wild-type (WT) mouse macrophages. Further analysis demonstrated that G. lamblia trophozoites reduced cytokines secretion by activating AKT pathway in WT mouse macrophages. Immunohistochemical staining in G. lamblia cysts infected TLR2−/− and WT mice showed that TLR2 was highly expressed in duodenum in infected WT mice. Also, infected TLR2−/− and AKT-blocked mice showed an increased production of IL-12 p40 and IFN-γ compared with infected WT mice at the early stage during infection. Interestingly, infected TLR2−/− and AKT-blocked mice displayed a decreased parasite burden, an increased weight gain rate, and short parasite persistence. Histological morphometry showed shortened villus length, hyperplastic crypt and decreased ratio of villus height/crypt depth in infected WT mice compared with in infected TLR2−/− and AKT-blocked mice. Together, our results suggested that TLR2 deficiency leads to alleviation of giardiasis and reduction of parasite burden through the promotion of proinflammatory cytokines production. For the first time, our results demonstrated that TLR2 played a negative role in host defense against Giardia.
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- 2017
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39. Activation of ERK Signaling via TLR11 Induces IL-12p40 Production in Peritoneal Macrophages Challenged by Neospora caninum
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Xiaoxia Jin, Pengtao Gong, Xichen Zhang, Guojiang Li, Tao Zhu, Mengge Zhang, and Jianhua Li
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N. caninum ,cytokine ,IL-12p40 ,ERK signaling ,TLR11 ,Microbiology ,QR1-502 - Abstract
Neospora caninum, an obligate intracellular protozoan parasite, can infect a large variety of vertebrate hosts including the most economically important cattle. Infection with N. caninum is a main cause of abortion in both dairy and beef cattle, which causes great economic losses worldwide. However, the mechanism of host cell infection by N. caninum has not been fully elucidated, especially in terms of inflammatory responses. In this study, the effect of TLR-ERK signaling pathway on the synthesis of pro-inflammatory interleukin-12p40 in mouse peritoneal macrophages (PMϕ) challenged by N. caninum was investigated. Our results suggested that N. caninum infection quickly activated MEK-ERK signaling via TLR11 in PMϕ. In addition, N. caninum infection also caused upregulated production of IL-12p40 by PMϕ, which was significantly reduced with the blockade of TLR11/MEK/ERK pathway, suggesting that this upregulation of IL-12 p40 was TLR11 and MEK-ERK-activation dependent.
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- 2017
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40. Canine neutrophil extracellular traps release induced by the apicomplexan parasite Neospora Caninum in vitro
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Zhengkai Wei, Carlos Hermosilla, Anja Taubert, Xuexiu He, Xiaocen Wang, Pengtao Gong, Jianhua Li, zhengtao yang, and Xichen Zhang
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DNA ,Neutrophils ,canine ,NETs ,Neospora caninum ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Neosporosis is considered as one of the main causes of abortion and severe economic losses in dairy industry. The Canis genus serving as one of the confirmed definitive hosts of the apicomplexan parasite Neospora caninum (N. caninum) plays a critical role in its life cycle. However, the effects of N. caninum on its definitive hosts of neutrophils extracellular traps (NETs) formation remain unclear. In the present study, N. caninum tachyzoite-induced canine NETs formation was observed by scanning electron microscopy (SEM). Visualization of DNA decorated with H3, NE and MPO within N. caninum tachyzoite-induced NETs were examined using fluorescence confocal microscopy analyses. Furthermore, the formation of canine NETs was quantified using Sytox Green staining, and the LDH levels in supernatants were examined by an LDH Cytotoxicity Assay® kit. The results clearly showed that NETs-like structures were induced by N. caninum tachyzoites, and the major components within these structures induced by N. caninum tachyzoite were further confirmed by fluorescence confocal microscopy visualization. These results suggest that N. caninum tachyzoites strongly induced NETs formation in canine PMN. In functional inhibition assays, the blockings of NADPH oxidase, NE, MPO, SOCE, ERK 1/2 and p38 MAPK signaling pathways significantly inhibited N. caninum tachyzoite-induced NETs formation, which suggests that N. caninum tachyzoite-induced NETs formation is a NADPH oxidase-, NE-, MPO-, SOCE-, ERK 1/2- and p38 MAPK-dependent cell death process. To our knowledge, this study is the first to report the formation of NETs in canine PMN against N. caninum infection.
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- 2016
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41. Cryptosporidium parvum maintains intracellular survival by activating the host cellular EGFR-PI3K/Akt signaling pathway
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Heng, Yang, Mengge, Zhang, Xiaocen, Wang, Pengtao, Gong, Nan, Zhang, Xichen, Zhang, Xin, Li, and Jianhua, Li
- Subjects
Immunology ,Molecular Biology - Abstract
Autophagy is a critical cellular mechanism in helping infected cells remove intracellular pathogens and is countered by pathogens maintaining intracellular survival by regulating autophagy through the manipulation of the host cellular signal transduction pathway. Cryptosporidium parvum is a zoonotic intracellular but extracytoplasmic protozoon that causes diarrhea in infants and young children worldwide. However, it is still unclear how Cryptosporidium adapts to intracellular survival. In the present study, we demonstrated that C. parvum could activate the EGFR-PI3K/Akt signaling pathway to promote intracellular survival in HCT-8 cells. The western blot results showed that C. parvum induced EGFR and Akt phosphorylation in HCT-8 cells. The EGFR inhibitor AG1478 decreased EGFR and Akt phosphorylation, and the PI3K inhibitor LY294002 impaired Akt phosphorylation induced by C. parvum in HCT-8 cells. Inhibition of EGFR or Akt decreased the number of intracellular parasites. Second, low-dose infection of C. parvum triggered complete autophagy and enhanced autophagic flux in HCT-8 cells. The expressions of mTOR and p62 were decreased, and the expressions of LC3 and Beclin1 were increased in C. parvum-infected HCT-8 cells. Transfection with siBeclin1 or siATG7 reduced LC3 accumulation, while lysosome inhibitor E64d+pepA increased LC3 accumulation induced by C. parvum in HCT-8 cells. Intracellular parasite proliferation was decreased when treated with autophagy inducer rapamycin, whereas autophagy inhibitor 3-MA, E64d+pep A, siBeclin1 or siATG7 increased intracellular parasites. Third, C. parvum inhibited autophagy killing to promote its own intracellular survival by activating EGFR-Akt signaling pathway. The EGFR inhibitor AG1478 enhanced autophagic flux, and Akt inhibitor IV increased LC3 accumulation and inhibited C. parvum proliferation in HCT-8 cells. Akt inhibitor IV-inhibited C. parvum proliferation was attenuated by E64d+pepA. In summary, C. parvum could maintain intracellular survival by inhibiting autophagy via EGFR-PI3K/Akt pathway. These results revealed a new mechanism for the interaction of C. parvum with host cells.
- Published
- 2023
42. Trichomonas vaginalis triggers the release of THP-1 extracellular traps
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Fei, Li, Zhengkai, Wei, Weina, Jiang, Lili, Cao, Yuhang, Gao, Zhengtao, Yang, Jianhua, Li, Biao, Yu, Xichen, Zhang, and Pengtao, Gong
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- 2019
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43. Immunol detection of cathepsin L from Fasciola hepatica infection in sheep by monoclonal antibody-based colloidal gold test strip assay.
- Author
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Shaoxiong Liu, Nan Zhang, Qinlei Yu, Jianhua Li, Xiaocen Wang, Xin Li, Xu Zhang, Shuqin Cheng, Taotao Yue, Hongbo Zhang, Pengtao Gong, and Xichen Zhang
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- 2023
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44. Giardia lamblia regulates the production of proinflammatory cytokines through activating the NOD2–Rip2–ROS signaling pathway in mouse macrophages
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Xu, Zhang, Xin, Li, Yanhui, Yu, Xichen, Zhang, Xiaocen, Wang, Nan, Zhang, Mengge, Chen, Pengtao, Gong, and Jianhua, Li
- Subjects
Mice ,Macrophages ,Immunology ,Nod2 Signaling Adaptor Protein ,Animals ,Cytokines ,Immunology and Allergy ,Cell Biology ,Giardia lamblia ,Reactive Oxygen Species ,Signal Transduction - Abstract
Giardia lamblia is a zoonotic protozoan that causes the diarrheal illness giardiasis, with the highest prevalence reported in the tropics and subtropics. Giardia is currently the most frequently identified pathogen in waterborne outbreaks in the United States. Nucleotide oligomerization domain (NOD) 1 and NOD2, intracellular NOD-like receptors, recognize pathogens to induce proinflammatory and antimicrobial responses. However, the roles of NOD1 and NOD2 signaling in Giardia infection have not yet been investigated. In the present study, the activation of NOD1 and NOD2 signaling pathways and the production of proinflammatory cytokines, reactive oxygen species (ROS) and nitric oxide in mouse macrophages stimulated with G. lamblia or parasite excretory-secretory products (ESPs) were examined. The results showed that G. lamblia and ESPs activated NOD2 and its downstream adaptor protein kinase, Receptor-interacting protein 2 (Rip2), in mouse macrophages. Blocking NOD2-Rip2 signaling significantly reduced the production of ROS and subsequently decreased the phosphorylation of nuclear factor-κB p65 and extracellular signal-regulated kinase, which in turn inhibited the production of four proinflammatory cytokines, namely, interleukin (IL)-1β, IL-6, IL-12p40 and tumor necrosis factor-α. In summary, our results indicate that the NOD2-Rip2 signal, which is activated by G. lamblia, contributes to the production of proinflammatory cytokines and ROS in mouse macrophages.
- Published
- 2022
45. The Autophagy Induced by Cryptosporidium Parvum Via Mtor Pathway in Hct-8 Cells
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Qile Yu, Zhipeng Li, Nan Zhang, Jianhua Li, Pengtao Gong, Xin Li, Xiaocen Wang, and Xichen Zhang
- Published
- 2023
46. Host defense against Neospora caninum infection via IL-12p40 production through TLR2/TLR3-AKT-ERK signaling pathway in C57BL/6 mice
- Author
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Xin Li, Xichen Zhang, Xiaocen Wang, Mengge Chen, Xu Zhang, Nan Zhang, Jianhua Li, and Pengtao Gong
- Subjects
MAPK/ERK pathway ,C57BL/6 ,MAP Kinase Signaling System ,Immunology ,chemical and pharmacologic phenomena ,Biology ,Microbiology ,Mice ,Immune system ,parasitic diseases ,Animals ,Molecular Biology ,Protein kinase B ,Coccidiosis ,Interleukin-12 Subunit p40 ,Macrophages ,Intracellular parasite ,fungi ,Neospora ,hemic and immune systems ,biology.organism_classification ,Toll-Like Receptor 2 ,Neospora caninum ,Toll-Like Receptor 3 ,Mice, Inbred C57BL ,Oncogene Protein v-akt ,TLR2 ,Phosphorylation ,Cattle ,Signal Transduction - Abstract
Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum-induced IL-12p40 production and the role of TLR2/TLR3 in anti-N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2-/- mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3-/- mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2-/- and TLR3-/- mouse macrophages. In N. caninum-infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2-/- and TLR3-/- mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2-/-, and TLR3-/- mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum, and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production.
- Published
- 2021
47. Development of a double antibodies sandwich ELISA for the detection of avian leukosis virus subgroup J based on monoclonal antibodies against gp85
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Pengtao Gong, Qinlei Yu, Ding He, Nan Wang, Lili Cao, Guo Yanbing, Dong Hang, Shao Hongze, Shuxian Yuan, Yao Xinhua, Panpan Zhao, and Xue Zhang
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Mice, Inbred BALB C ,Avian Leukosis Virus ,biology ,medicine.drug_class ,Biophysics ,Antibodies, Monoclonal ,Enzyme-Linked Immunosorbent Assay ,General Medicine ,Avian leukosis ,Antibodies, Viral ,Monoclonal antibody ,Sensitivity and Specificity ,Biochemistry ,Virology ,Virus ,Avian Leukosis ,Viral Envelope Proteins ,medicine ,biology.protein ,Animals ,Female ,Antibody ,Chickens - Published
- 2021
48. Protective efficacy of Toxoplasma gondii bivalent MAG1 and SAG1 DNA vaccine against acute toxoplasmosis in BALB/c mice
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Lili Cao, Juan Liu, Songgao Cao, Panpan Zhao, He Ding, Shuxian Yuan, Xingzhong Sun, Hang Dong, Yanbing Guo, Nan Wang, and Pengtao Gong
- Abstract
Background: Toxoplasma gondii is capable of infecting a wide range of warm-blooded animals, causing a worldwide epidemic of zoonotic toxoplasmosis. SAG1 protein is expressed at the proliferative tachyzoite stage, whereas MAG1 is expressed at the bradyzoite and tachyzoite stages. These two proteins display protective roles in previous studies, however, there synergetic protective efficacy as a DNA vaccine against toxoplasmosis has not been clarified.Methods: In this study, we amplified TgMAG1 and TgSAG1 genes and inserted into eukaryotic expression vector pcDNA3.1(+). The pcDNA3.1(+)-TgMAG1 (pMAG1), pcDNA3.1(+)-TgSAG1 (pSAG1), pcDNA3.1(+)-TgMAG1-TgSAG1 (pMAG1-SAG1) plasmids were transfected into HEK-293 cells and each protein was verified in vitro through western blot. Then, mice were intramuscularly immunized with pMAG1, pSGA1 or pMAG1-SGA1, and anti-T. gondii IgG levels were measured in the serum. Cytokines levels of IL-4, IL-10 and IFN-γ in mice splenocytes culturing supernatants were measured using commercial ELISA kits. Immunized mice were challenged with T. gondii tachyzoites with lethal doses followed by determination of mortality, whereas mice infected with low dose tachyzoites followed by monitoring the parameters of survival rate and parasites burden analysis of brains and livers.Results: The pMAG1, pSGA1 or pMAG1-SGA1 exhibited well reactogenicity with expected band sizes of 17.843 kDa, 15.572 kDa, 33.415 kDa, respectively. The immunized mice triggered significantly high levels of anti-T. gondii IgG antibodies in comparison with that in the negative control groups, moreover pMAG1-SGA1 immune achieved the highest levels. The DNA vaccines also led to obvious IFN-γ release from splenocytes culturing supernatants, whereas had no role in the IL-4 and IL-10. The protective efficacy results showed that DNA vaccines immunization prolonged the acute infection mice survival time to 14 d, 16 d, 32 d. Consistently, the liver and brain parasites in each immunization group were significantly reduced comparing with PBS control group.Conclusions: This study revealed that bivalent TgMAG1 and TgSAG1 DNA vaccine displayed excellent protective immunity against toxoplasmosis in mice. These data provide new sight into the development of Toxoplasma gondii vaccines.
- Published
- 2022
49. Trichomonas gallinae induces heterophil extracellular trap formation in pigeons
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Hongyu Wang, Yuru Wang, Xuehan Wang, Ran Wei, Xiaocen Wang, Pengtao Gong, Nan Zhang, Xichen Zhang, Xin Li, and Jianhua Li
- Subjects
Infectious Diseases ,General Veterinary ,Insect Science ,Parasitology ,General Medicine - Abstract
Avian trichomonosis is a worldwide and cross-species epidemic, and the infection in pigeons is particularly severe. Although the disease causes a serious threat to poultry health resulting in significant economic losses, the relationship between Trichomonas gallinae (T. gallinae) and host innate immunity is still not clear. Extracellular traps (ETs) are an innate immunity response to parasitic infections. However, whether host cells can produce ETs after T. gallinae infection has not yet been reported. In the present study, the ability of T. gallinae to induce the production of heterophil extracellular traps (HETs) in pigeons was examined. T. gallinae-induced HETs were observed by scanning electron microscopy (SEM) and the main components of HETs were detected by fluorescence confocal microscopy. Changes in reactive oxygen species (ROS) and lactate dehydrogenase (LDH) were tested during the HETosis. A quantitative analysis of T. gallinae-induced HETs, the role of myeloperoxidase (MPO), store-operated Ca (2+) entry (SOCE), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in T. gallinae-induced HET formation were conducted by inhibitor assays. The results showed that T. gallinae induced ET formation in pigeon heterophils. ETs consisted of a DNA skeleton, neutrophil elastase (NE), MPO, and Histone3 (H3). T. gallinae-induced HETs formation in a dose- and time-dependent process. The release of T. gallinae-induced HETs depends on MPO, SOCE, and NADPH oxidase. Furthermore, after T. gallinae stimulated pigeon heterophils, ROS production was significantly increased, while no significant differences in the LDH activity were observed.
- Published
- 2022
50. Antitumor effect of invasive Lactobacillus plantarum delivering associated antigen gene sHSP between Trichinella spiralis and Lewis lung cancer cells
- Author
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Taotao Yue, Xichen Zhang, Pengtao Gong, Jianhua Li, Xiaocen Wang, Xin Li, Yeting Ma, Xuejiao Chen, Xu Zhang, Shuqin Cheng, Hongbo Zhang, and Nan Zhang
- Subjects
Pharmacology ,Immunology ,Immunology and Allergy - Published
- 2023
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