Proteomics and phosphoproteomics reveal novel proteins involved in Cipangopaludina chinensis carcasses.

Cipangopaludina chinensis is a common freshwater mollusk that is widely distributed worldwide, especially in China. In our research, 1,382 proteins and 1,039 phosphorylated proteins were identified from C. chinensis carcasses, and 690 differentially expressed proteins (DEPs) were quantified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the DEPs are involved in cellular processes, single-organism processes, metabolic processes, developmental processes, localization, and biological regulation. The phosphorylated proteins were found to be related to the Rap1 signaling pathway, Ras signaling pathway, calcium signaling pathway, and longevity-regulating pathways. Moreover, we also identified important regulatory enzymes, such as guanylate cyclase, tyrosine protein kinase, receptor protein tyrosine kinase, and glyoxylate reductase/hydroxypyruvate reductase. Notably, we found guanylate cyclase to be present in multiple signaling pathways, including the Rap1 signaling pathway, calcium signaling pathway, Ras signaling pathway, insulin secretion, longevity regulating pathway, glutamatergic synapse, circadian entrainment, and gap junction. This enzyme may play a crucial role in regulating molecular mechanisms in C. chinensis. In summary, proteomic and phosphoproteomic analyses of C. chinensis carcasses displayed significant differences among different geographical isolates, which helps enhance our understanding of food nutrition, signaling pathways, and metabolic mechanisms in C. chinensis.

First reported Porrocaecum angusticolle infection in Griffon vulture (Gyps fulvus) in China.

This present study is the first case of a Porrocaecum angusticolle (P. angusticolle) infection reported in Griffon vulture (Gyps fulvus) in China. This study aimed to identify the nematode species and explore the genetic evolution of worms infecting Gyps fulvus (G.fulvus). Clinical examination revealed several milky white parasites in the stomach and intestinal tract. Polymerase chain reaction and partial 18S gene sequencing analyses identified these worms to be P. angusticolle (SD isolates). Further phylogenetic analyses revealed that they shared the highest genetic identity (99.9%) with a P. angusticolle isolate (EU004820.1) from Germany. Our study is the first report on the identification and characterization of P. angusticolle infecting G.fulvus in China, based on clinical findings and molecular diagnosis. Therefore, our study provides novel insights for the diagnosis of P. angusticolle infections and the prevention of nematode transmission in wild and domestic animals.

Comparative proteomics analysis of adult Haemonchus contortus isolates from Ovis ammon.

Haemonchus contortus is an important parasite that causes disease that seriously endangers ruminant animals cattle, sheep, goat, and camel. Here, we compared the proeomics analysis of three adult Haemonchus contortus isolates from mouflons (Ovis ammon). A total of 1,299 adult worm proteins were identified, and 461 proteins were quantified, of which 82 (108), 83 (97), and 97 (86) significantly upregulated (downregulated) differentially expressed proteins (DEPs) were detected among pairwise comparisons (1-vs.-3, 2-vs.-3, and 2-vs.-1). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatic analysis indicated that these DEPs are mainly concentrated in cellular composition, molecular function, biological function, and catabolism pathways. In addition, Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out to screen the DEPs. The main biological processes involved were nucleotide, nucleotide phosphate, ribonucleotide, purine-containing compound, purine ribonucleotide, single-organism, oxoacid, organic, carboxylic, oxoacid metabolic processes and single-organism catabolic processes. The majority of KEGG pathways were found to be related to metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, carbon metabolism, and microbial metabolism in diverse environments. Moreover, we also found differences in the expression of some important or novel regulatory proteases, such as serine hydroxymethyl transferase (SHMT), dihydrolipoyl dehydrogenase (DLD), and transket pyr domain-containing protein (TKPD). In summary, label-free proteomic analysis of adult H. contortus worms displayed significant differences in three different individual isolates, which helps to improve our understanding of the growth and metabolic mechanisms of H. contortus in different individuals and relative natural environments and provides novel drug targets for the treatment of parasitic diseases.

Metabolism Profile of Mequindox in Sea Cucumbers In Vivo Using LC-HRMS.

In this work, the metabolism behavior of mequindox (MEQ) in sea cucumber in vivo was investigated using LC-HRMS. In total, nine metabolites were detected and identified as well as the precursor in sea cucumber tissues. The metabolic pathways of MEQ in sea cucumber mainly include hydrogenation reduction, deoxidation, carboxylation, deacetylation, and combinations thereof. The most predominant metabolites of MEQ in sea cucumber are 2-iso-BDMEQ and 2-iso-1-DMEQ, with deoxidation and carbonyl reduction as major metabolic pathways. In particular, this work first reported 3-methyl-2-quinoxalinecarboxylic acid (MQCA) as a metabolite of MEQ, and carboxylation is a major metabolic pathway of MEQ in sea cucumber. This work revealed that the metabolism of MEQ in marine animals is different from that in land animals. The metabolism results in this work could facilitate the accurate risk assessment of MEQ in sea cucumber and related marine foods.

Comparative proteomics analysis of Schistosoma japonicum developed in different Oncomelania snails as intermediate hosts.

Schistosomiasis is a tropical parasitic disease that seriously endangers humans and animals. In this study, two Oncomelania snails, Oncomelania hupensis (O. hupensis) and Oncomelania weishan (O. weishan), were infected with Schistosoma japonicum (S. japonicum) cercariae during the early period, and ICR mice were subsequently infected with two kinds of miracidia that developed in male and female adult worms. In this study, isobaric tags for relative and absolute quantification (iTRAQ) were used to identify four channels: 113, 115, 117, and 119. A total of 2364 adult schistosome proteins were identified, and 1901 proteins were quantitative. Our results revealed 68 differentially expressed proteins (DEPs) in female adult worms, including 24 upregulated proteins and 44 downregulated proteins, and 55 DEPs in male adult worms, including 25 upregulated proteins and 30 downregulated proteins. LC-MS/MS and bioinformatics analysis indicated that these DEPs are mainly concentrated in cellular composition, molecular function, biological function and catabolism pathways. In summary, this proteomics analysis of adult schistosomes that hatched in two intermediate hosts helps to improve our understanding of the growth and developmental mechanisms of S. japonicum.

Corrigendum: Comparative proteomics analysis of Schistosoma japonicum developed in different Oncomelania snails as intermediate hosts.

[This corrects the article DOI: 10.3389/fcimb.2022.959766.].

Comparative Proteomics Analysis for Elucidating the Interaction Between Host Cells and Toxoplasma gondii.

Toxoplasma gondii, a representative model organism belonging to the phylum Apicomplexa, can infect almost all warm-blooded organisms, including humans. The invasion of host cells via host-parasite interaction is the key step for T. gondii to complete its life cycle. Herein we performed tandem mass tag analysis to investigate global proteomic changes in host cells (human foreskin fibroblasts, HFFs) [HFFs infected with T. gondii (HT) vs. HFFs (H)] and T. gondii [HT vs. T. gondii (T)] during intracellular infection. Overall, 3477 and 1434 proteins were quantified, of which 375 and 1099 proteins were differentially expressed (adjusted p-value < 0.05 and >1.5 or <0.67-fold change) in host cells and T. gondii, respectively. T. gondii invasion relies on the secretion of numerous secretory proteins, which originate from three secretory organelles: micronemes, rhoptries, and dense granules. In the HT vs. T group, few secretory proteins were upregulated, such as microneme proteins (MICs: MIC6, MIC10), rhoptry bulb proteins (ROPs: ROP5, ROP17), and dense granule proteins (GRAs: GRA4, GRA5, GRA12). In contrast, dozens of known secretory proteins were significantly downregulated in T. gondii-infected HFFs. In HFFs, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed a large number of differentially expressed proteins (DEPs) enriched in metabolic processes and immune-associated signaling pathways, such as NF-κB, cAMP, and Rap1 signaling pathways. Further, in case of T. gondii, DEPs were involved in ribosome biogenesis, citrate cycle, and galactose metabolism, indicating that cell biosynthesis and metabolism of T. gondii were altered after host cell invasion. These findings reveal novel modifications in the proteome of host cells as well as T. gondii, helping us better understand the mechanisms underlying host-parasite interaction.

Protective immunity induced by a DNA vaccine cocktail expressing TgSAG1, TgROP2, and the genetic adjuvant HBsAg against Toxoplasma gondii infection.

Toxoplasma gondii is an intracellular obligate parasitic protozoon that can infect all warm-blooded animals, causing zoonotic toxoplasmosis. So far, there is no commercial toxoplasmosis vaccine for human use. In the present study, we constructed a DNA vaccine cocktail which includes the surface protein (SAG1) and the rhoptry protein ROP2 denoted as pEGFP-N1-SAG1-ROP2. In order to improve the efficacy, HBsAg was used as a genetic adjuvant to construct pEGFP-N1-HBsAg-SAG1-ROP2. Two eukaryotic plasmids were transiently transfected into HEK293T cells and the expression was examined using fluorescence microscopy and western blotting. We then immunized Kunming mice intramuscularly with the DNA vaccine. After three immunizations, the immune response was evaluated by measuring antibody levels, cytokine production, percentages of CD4+ and CD8+ T lymphocytes, and the survival times of the T. gondii RH strain challenged mice. The results showed that the two DNA vaccines stimulated Th1 responses, and had a higher antibody titer, IL-2, IL-12, and IFN-γ levels, and percentage of CD4+ and CD8+ T lymphocytes than the control group. In addition, mice immunized with the pEGFP-N1-HBsAg-SAG1-ROP2 vaccine showed increased survival times compared with pEGFP-N1-SAG1-ROP2.

Understanding the regulation of overwintering diapause molecular mechanisms in Culex pipiens pallens through comparative proteomics.

To reveal overwintering dormancy (diapause) mechanisms of Culex pipiens pallens (L.), global protein expression differences at three separate time points represent nondiapause, diapause preparation and overwintering diapause phases of Cx. pipiens pallens were compared using iTRAQ. Cx. pipiens pallens females accumulate more lipid droplets during diapause preparation and overwintering diapause maintenance than during the nondiapause phase. A total of 1030 proteins were identified, among which 1020 were quantified and compared. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), Domain and Clusters of Orthologous Groups (COG) analyses revealed key groups of proteins, pathways and domains differentially regulated during diapause preparation and overwintering diapause maintenance phases in this mosquito, including major shifts in energy production and conversion, fatty acid metabolism, the citrate (TCA) cycle, and the cytoskeletal reorganization pathway. Our results provide novel insight into the molecular bases of diapause in mosquitoes and corroborate previously reported diapause-associated features in invertebrates. More interestingly, the phototransduction pathway exists in Cx. pipiens pallens, in particular, actin, rather than other proteins, appears to have substantial role in diapause regulation. In addition, the differential changes in calmodulin protein expression in each stage implicate its important regulatory role of the Cx. pipiens pallens biological clock. Finally, 24 proteins were selected for verification of differential expression using a parallel reaction monitoring strategy. The findings of this study provide a unique opportunity to explore the molecular modifications underlying diapause in mosquitoes and might therefore enable the future design and development of novel genetic tools for improving management strategies in mosquitoes.

Prediction of Toxoplasma gondii virulence factor ROP18 competitive inhibitors by virtual screening.

BACKGROUND: Rhoptry protein 18 (ROP18) is a key virulence factor of Toxoplasma gondii. The host's immune responses mediated by immune-related GTPases (IRGs) could be blocked by ROP18's kinase activity. ROP18 also interacts with various substrates, such as activating transcription factor 6 beta (ATF6ß) and affects multiple physiological functions within host cells, thereby inducing intense virulence. In this study, competitive inhibitors targeted to ROP18 were subjected to virtual screening based on the principle of structure-based drug design (SBDD). METHODS: The preparation of the ROP18 structure was conducted using the "Structure Prepare" function of Molecular Operating Environment (MOE) software. The ATP-binding pocket was selected as the starting point for virtual screening. Construction of the pharmacophore model used Extended Hückel Theory (EHT) half-quantitative measurement and construction, as well as the characteristics of Type I kinase inhibitors. The pharmacophore model of ROP18 was imported into the Specs database for small molecule similarity screening using EHT pharmacophore measurement. Hit compounds were selected using the functions of London dG and generalized-born volume integral/weighted surface area (GBVI/WSA) scoring. The top 100 hits were analyzed by molecular docking and structure activity relationships (SAR) analysis. RESULTS: The final pharmacophore comprised three typical characteristics: three hydrogen bond acceptors/donors, two ring aromatic features occupying the hydrophobic core, and one cation group feature targeted to the terminus of ATP. A total of 1314 hit compounds analogous to ROP18 pharmacophore were passed through the Specs. After two rounds of docking, 25 out of 100 hits were identified as belonging to two main scaffold types: phthalimide ring structure, thiazole ring and styrene structure. Additionally, the screen also identified 13 inhibitors with distinct scaffold types. The docking models and SAR analysis demonstrated that these hits could engage in multiple hydrogen bonds, salt bridges halogen bonds, and hydrophobic interactions with ROP18, and para-position halo substituents on the benzene ring may enhance their affinity scoring. CONCLUSIONS: A pharmacophore against the ROP18 ATP-binding pocket was successfully constructed, and 25 representative inhibitors from 15 scaffold clusters were screened using the Specs database. Our results provide useful scaffold types for the chemical inhibition of ROP18 or alternative conformations to develop new anti-toxoplasmosis drug leads.

Mutation Profile of pfdhfr and pfdhps in Plasmodium falciparum among Returned Chinese Migrant Workers from Africa.

We evaluated markers of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum among 254 returned migrant workers in China from Africa from 2013 to 2016. High prevalences of pfdhfr (97.2%) and pfdhps (96.5%) mutations were observed. The partially resistant genotype was homogeneously distributed in Africa with a modestly high prevalence (48%), whereas the super resistant genotype was only found in West Africa with a very low frequency (1.2%). The findings provided baseline data about the molecular markers of SP resistance.

Protective immune response against Toxoplasma gondii elicited by a novel yeast-based vaccine with microneme protein 16.

Toxoplasma gondii is an obligate intracellular protozoan that can invade all eukaryotic cells and infect all warm-blood animals, causing the important zoonosis toxoplasmosis. Invasion of host cells is the key step necessary for T. gondii to complete its life cycle and microneme proteins play an important role in attachment and invasion of host cells. Microneme protein 16 (TgMIC16) is a new protective protein in T. gondii and belongs to transmembrane microneme proteins (TM-MIC). The TM-MICs are released onto the parasite's surface as complexes capable of interacting with host cell receptors. In the present study, we expressed the TgMIC16 protein on the surface of Saccharomyce cerevisiae (pCTCON2-TgMIC16/EBY100) and evaluated it as a potential vaccine for BALB/c mice against challenge infection with the RH strain of T. gondii. We immunized BALB/c mice both orally and intraperitoneally. After three immunizations, the immune response was evaluated by measuring antibody levels, lymphocyte proliferative responses, percentages of CD4+ and CD8+ T lymphocytes, cytokine production, and the survival times of challenged mice. The results showed that the pCTCON2-TgMIC16/EBY100 vaccine stimulated humoral and cellular immune responses. In addition, mice immunized with the pCTCON2-TgMIC16/EBY100 vaccine showed increased survival times compared with non-immunized controls. In summary, TgMIC16 displayed on the cell surface of S. cerevisiae could be used as potential vaccine against toxoplasmosis.

Cysticercosis in Shandong Province, Eastern China.

We analyzed demographic and clinical data and estimated the incidence of cysticercosis in Shandong Province, China, during 1975-2014. Our analyses showed that a cysticercosis-endemic area is present in Shandong Province, especially in its western regions. Improved surveillance and control are needed to address the elevated risk for cysticercosis in this region.

Neospora caninum ROP16 play an important role in the pathogenicity by phosphorylating host cell STAT3.

Neospora caninum is a common cause of abortions in cattle and nervous system dysfunctions in dogs. Our analysis shows that NcROP16 and TgROP16 have similar structures and may have similar functions. To our surprise, we found that similar to the T. gondii RH strain, the N. caninum Nc-1 strain could phosphorylate STAT3Y705, but in contrast to T. gondii, N. caninum Nc-1 could not phosphorylate STAT6Y641. We constructed a gene-knockout plasmid and screened ΔNcROP16 strains at the gene, protein and transcription levels. Plaque assays, invasion assays and intracellular proliferation tests indicated that the ΔNcROP16 strain phenotypes had changed, resulting in smaller plaques and slower intracellular growth. A virulence analysis showed that the cerebral loads of the parasite in mice infected with the ΔNcROP16 strain were significantly reduced compared to the loads in mice infected with the Nc-1 strain. In contrast, the overexpression of ROP16 led to the largest number of parasites observed in the mouse brains. Similarly, the overexpression of ROP16 caused the most powerful virulence in mice. In addition, NcROP16 takes part in the STAT3 signaling pathway in different host cells. This occurs by the secretion of NcROP16 into the host cell, where it phosphorylates STAT3, and phosphorylated STAT3 then migrates to the cell nucleus. NcROP16 can enter the host nucleus and continuously phosphorylate STAT3, resulting in the induction of host cell apoptosis. The parasites engineered to over express the NcROP16 induce the increased transcription of apoptotic-related genes, such as Fas, FasL and Bax and enhanced ANA1 cell apoptosis. The results show that NcROP16 is a key virulence factor in N. caninum, promoting the host cell apoptosis and enhancing the pathogenicity of the parasites for the host by phosphorylating STAT3.

Expression of codon-optimized TgMIC16 in three Escherichia coli strains.

In a previous study, we found that rabbit anti-Toxoplasma gondii serum was capable of recognizing truncated T. gondii microneme protein 16 (TgMIC16), indicating that TgMIC16 is an essential antigenic T. gondii protein. However, the broad application of this recombinant protein is limited by its low expression level. In this study, we performed codon optimization of TgMIC16 by changing the codon-adaptation index from 0.22 to 1.0 without altering the amino acid sequence and expressed the optimized gene in three different Escherichia coli strains, followed by comparison of soluble recombinant-protein expression and yield. Our results showed that the recombinant protein rTgMIC16 was expressed as inclusion bodies in all three strains following optimization of induction parameters, and western blot analysis revealed the presence of a ~72-kD recombinant protein as a specific band following purification. A shuffle-expression strain was selected to amplify incubation products and induce expression, resulting in an overall rTgMIC16 yield of ~20 mg/L. These findings provide a basis for further investigation of TgMIC16 to elucidate its functions and interaction partners.

High-sensitivity chemiluminescent immunoassay investigation and application for the detection of T-2 toxin and major metabolite HT-2 toxin.

BACKGROUND: T-2 toxin is a widely distributed mycotoxin in cereals. HT-2 toxin is the major metabolite, which is also a contaminant in cereals. T-2 toxin and HT-2 toxin have been identified as having carcinogenic, hepatotoxic, teratogenic and immunotoxic properties. To reduce the risk of contamination, a rapid, highly sensitive and inexpensive assay for the detection is required. RESULTS: In this study a high-sensitivity chemiluminescent enzyme-linked immunoassay (CL-ELISA) of T-2 toxin and HT-2 toxin was developed. With the help of the chemiluminescent substrate, this protocol showed a highly sensitive character with an IC50 as low as 33.28 ng mL-1 and 27.27 ng mL-1 for T-2 and HT-2, respectively. In addition, this method had no cross-reaction with other structurally related mycotoxins. CONCLUSION: These results indicated that the developed CL-ELISA could be applied for the detection of T-2 toxin and HT-2 toxin in actual samples without complicated steps. © 2016 Society of Chemical Industry.

Epidemiological Investigation of Asymptomatic Dogs with Leishmania Infection in Southwestern China Where Visceral Leishmaniasis is Intractable.

Heishui county, located in northwest Sichuan province, southwestern China, is an endemic area of zoonotic visceral leishmaniasis (VL) and is the most intractable area. VL is never destroyed in it. Asymptomatic dogs (Leishmania parasites have been diagnosed but clinically healthy) are considered to be a potential reservoir host in zoonotic VL area, and most can lead to infection of individuals, that is a new challenge for controlling VL in humans. The present study aimed to assess the Leishmania infection rate of asymptomatic dogs in Heishui county. Total 105 asymptomatic domestic dogs were gathered from 4 districts in Heishui county to investigate the infection rate with serological and molecular methods based on ELISA and kinetoplast minicircle DNA(kDNA) PCR, respectively. Out of 105 dogs, 44 (41.9%) were positive by more than 1 method; 21 (20.0%) were positive by ELISA, and 30 (28.6%) were positive by kDNA-PCR. Our study showed that Leishmania infection of domestic dogs which is clinically healthy is prevalent in the studied district, and the asymptomatic dogs infected by Leishmania may be the primary reason for the prevalence of visceral leishmaniasis in the area.

[Prokaryotic Expression, Purification and Immunological Characterization of Micronemal Protein 16 of Toxoplama gondii].

Objective: To prokaryotically express three gene fragments of micronemal protein 16 （TgMIC16） of Toxoplasma gondii, and analyze the immunoreactivity of the three recombinant protein products. Methods: Primers were designed for three fragments of TgMIC16 gene which encode proteins within the functional domain. Reverse-transcription PCR was used to generate cDNA from RNA, and the three fragments were amplified on the cDNA by PCR using the designed primers. The PCR products were double-digested, inserted into the pET-32a（+） plasmid, and transformed into Escherichia coli TOP10 cells. Plasmids extracted from positive clones were confirmed by BamHⅠ/HindⅢ double digestion and sequencing, and further transformed into E. coli Rosetta cells. Protein expression was induced by IPTG, and confirmed by SDS-PAGE. The expressed recombinant proteins were purified with Ni-NTA affinity chromatography and their immunoreactivity analyzed with Western blotting. Results: The amplified three fragments were 1 806, 1 290 and 855 bp in size. Double digestion and sequencing results confirmed the successful construction of the three recombinant plasmids. SDS-PAGE analysis showed successful expression of the three recombinant proteins (M(r) 88 000, 68 000 and 52 000, respectively）, in the form of inclusion bodies. Western blotting showed that the three purified recombinant proteins reacted with His monoclonal antibody and rabbit anti-T. gondii antibody. Conclusion: The three fragments within the functional domain of TgMIC16 are successfully expressed in prokaryotic expression system and show immunoreactivity.