Huanqin Zheng

Clonorchis sinensis enolase: Identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products

Enolase plays a key role in energy metabolism and development of most organisms. We isolated a gene encoding enolase from Clonorchis sinensis (C. sinensis) adult cDNA library and expressed the recombinant protein in Escherichia coli. C. sinensis enolase (Csenolase) was identified as both an excretory/secretory product and a tegumental component of C. sinensis by western blot analysis. The transcriptional level of Csenolase was examined at adult worm, metacercaria, cercaria and egg of C. sinensis, and results showed that Csenolase is transcribed at the four life stages of C. sinensis while showing a significant higher expression level at the stage of adult worm. Immunohistochemical localization indicated that Csenolase was specifically deposited on the tegument of adult worm and cyst wall of metacercaria. Ligand blot assay revealed a specific characteristic of dose-dependent plasminogen-binding activity of Csenolase and kinetic parameters were explored using 2-phospho-D-glycerate (2-PGA) as the primary substrate by monitoring the conversion of nicotinamide-adenine dinucleotide (NADH) into nicotinamide adenine dinucleotide (NAD). In addition, Csenolase exhibited active enzyme activity in catalytic reactions while the anti-Csenolase serum inhibited the enzyme activity. In vitro incubation experiments revealed that Csenolase might play key roles in the growth of the parasites. In conclusion, Csenolase is an important glycolytic enzyme required for the development of C. sinensis, and may be a potential vaccine candidate and drug target against C. sinensis infection.

rSj16, a recombinant protein of Schistosoma japonicum‐derived molecule, reduces severity of the complete Freund’s adjuvant‐induced adjuvant arthritis in rats’ model

Sj16, a 16‐kDa protein produced by Schistosoma japonicum, has been demonstrated to have anti‐inflammatory effect. However, the possible mechanism of these phenomena has not been discovered. Here, we tried to touch it with arthritis rats’ model induced by injection of complete Freund’s adjuvant (CFA). A set of pathogenic characters were observed in CFA‐treated rat, including local and systematic read‐out, which showed the model successfully set up. After administration of rSj16 (recombinant Sj16) in vivo, paw swelling reduced significantly and in a dose‐dependent manner, the level of TNF‐α, IL‐1β and NO decreased and IL‐10 in the serum increased. In vitro, rSj16 reversed the augmented surface expression of CD80, CD86, CD54 and OX6 induced by lipopolysaccharide (LPS) in bone marrow–derived DCs (BMDCs), whereas endocytotic capacity of rSj16‐treated dendritic cell (DC) was profoundly increased. IL‐12p70 released from rSj16‐treated BMDC was decreased but IL‐10 increased. Further, following incubation with rSj16 primed BMDCs, the sensitized T cells exhibited increased production of anti‐inflammatory IL‐10 and IL‐4 and decreased production of IL‐12p70 and IFN‐γ. Collectively, these results implied that rSj16 alleviated CFA‐induced arthritis, and the possible mechanisms may be its interruption of maturation and function of DCs. rSj16 could be a potential therapeutic agent against rheumatoid arthritis.

Schistosoma japonicum : proteomics analysis of differentially expressed proteins from ultraviolet-attenuated cercariae compared to normal cercariae

Schistosomiasis is considered the most important human helminthiasis in terms of morbidity and mortality. In this study, comparative soluble proteomic analysis of normal cercariae and ultraviolet-irradiated attenuated cercariae (UVAC) from Schistosoma japonicum were carried out in view of the high efficiency of irradiation-attenuated cercariae vaccine. The results revealed that some proteins showed significant differential expression in the parasite after treatment with ultraviolet light. Total 20 protein spots were identified by mass spectrometry, corresponded to five groups according to their functions in the main that were structural and motor proteins (actin, et al.), energy metabolism associated enzymes (glyceraldehydes-3-phosphage dehydrogenase, et al.), signaling transduction pathway-associated molecules (14-3-3 protein, et al.), heat shock protein families (HSP 70 family, et al.), and other functional proteins (20S proteasome). Furthermore, our results indicated that the differential expression of the proteins by ultraviolet irradiation may be, at least partially, acquired by regulating the mRNA levels of corresponding proteins. These results may provide new clues for further exploring the mechanism of protective immunity induced by UVAC and may shed some light on the development of vaccines against schistosomiasis.

Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis.

Cysteine proteases, a superfamily of hydrolytic enzymes, have numerous functions in parasites. Here, we reported the cloning and characterization of a cDNA encoding a cathepsin B (AcCPB) from Angiostrongylus cantonensis fourth-stage larvae cDNA library. The deduced amino acid sequence analysis indicated AcCPB is related to other cathepsin B family members with an overall conserved architecture. AcCPB is evolutionarily more close to other parasitic nematode cathepsin B than the ones from hosts, sharing 43–53% similarities to the homologues from other organisms. Real-time quantitative PCR analysis revealed that AcCPB was expressed significantly higher in the fourth-stage larvae (L4) and the fifth-stage larvae (L5) than that in the third-stage larvae (L3) and adult worms (Aw). Unexpectedly, AcCPB was expressed at a higher level in L4 and L5 derived from mice than the larvae at the same stages derived from rats. The protease activity of recombinant AcCPB (rAcCPB) expressed in Escherichia coli showed high thermostability and acidic pH optima. The role in ovalbumin digestion and enzyme activity of rAcCPB could be evidently inhibited by cystatin from A.cantonensis. Furthermore, we found rAcCPB increased the expression levels of CD40, MHC II, and CD80 on LPS-stimulated dendritic cells (DCs). In this study, we provided the first experimental evidence for the expression of cathepsin B in A.cantonensis. Besides its highly specific expression in the stages of L4 and L5 when the worms cause dysfunction of the blood–brain barrier of hosts, AcCPB displayed different expression profiles in non-permissive host- and permissive host-derived larval stages and was involved in the maturation of DCs, suggesting a potential role in the central nervous system invasion and the immunoregulation during parasite–host interactions.

Genome-wide identification and functional annotation of Plasmodium falciparum long noncoding RNAs from RNA-seq data

The life cycle of Plasmodium falciparum is very complex, with an erythrocytic stage that involves the invasion of red blood cells and the survival and growth of the parasite within the host. Over the past several decades, numbers of studies have shown that proteins exported by P. falciparum to the surface of infected red blood cells play a critical role in recognition and interaction with host receptors and are thus essential for the completion of the life cycle of P. falciparum. However, little is known about long noncoding RNAs (lncRNAs). In this study, we designed a computational pipeline to identify new lncRNAs of P. falciparum from published RNA-seq data and analyzed their sequences and expression features. As a result, 164 novel lncRNAs were found. The sequences and expression features of P. falciparum lncRNAs were similar to those of humans and mice: there was a lack of sequence conservation, low expression levels, and high expression coefficient of variance and co-expression with nearby coding sequences in the genome. Next, a coding/noncoding gene co-expression network for P. falciparum was constructed to further annotate the functions of novel and known lncRNAs. In total, the functions of 69 lncRNAs, including 44 novel lncRNAs, were annotated. The main functions of the lncRNAs included metabolic processes, biosynthetic processes, regulation of biological processes, establishment of localization, catabolic processes, cellular component organization, and interspecies interactions between organisms. Our results will provide clues to further the investigation of interactions between human hosts and parasites and the mechanisms of P. falciparum infection.

Differences in microglia activation between rats-derived cell and mice-derived cell after stimulating by soluble antigen of IV larva from Angiostrongylus cantonensis in vitro

Angiostrongylus cantonensis is a rodent nematode. Adult worms of A. cantonensis live in the pulmonary arteries of rats. Humans and mice are accidental hosts or named nonpermissive hosts. The larva cannot develop into an adult worm and only causes serious eosinophilic meningitis or meningoencephalitis if humans or mice eat food containing larva of A. cantonensis in the third stage. The differing consequences largely depend on differing immune responses of the host to parasite during A. cantonensis invasion and development. Microglia is considered to be the key immune cell in the central nervous system like macrophage. To further understand the reasons for why mice and rats attain different outcomes in A. cantonensis infection, we set up the method to isolate and culture newborn rats’ primary microglia and observe the activation of the microglia cells, comparing with mice microglia cell line N9. We treated cells with soluble antigen of the fourth larva of A. cantonensis (L4 larva) and measured mRNA levels of IL-1β, IL-5, IL-6, IL-13, eotaxin, iNOS, and TNF-α by real-time PCR. The results showed that N9 expressed high mRNA level of IL-6, IL-1β, TNF-α, iNOS, IL-5, IL-13, and eotaxin, but primary microglia only had IL-5, IL-13, and eotaxin mRNA level. It implies that microglia from rats and mice had different reaction to soluble antigen of A. cantonensis. Therefore, we supposed that microglia may play an immune modulation role during the brain inflammation induced by A. cantonensis.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-γ activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

Linalool, derived from Cinnamomum camphora (L.) Presl leaf extracts, possesses molluscicidal activity against Oncomelania hupensis and inhibits infection of Schistosoma japonicum

BackgroundSchistosomiasis japonicum remains a considerable economic and public health concern in China, the Philippines and Indonesia. Currently available measures to control the unique intermediate host Oncomelania hupensis are frequently associated with severe side effects. Previous studies have demonstrated that linalool-rich extracts from various plants exhibited promising biological activities including cytotoxic, anti-microbial and anti-parasitic properties.MethodsWe identified the components of leaf extracts from Cinnamomum camphora by gas chromatography coupled to mass spectrometry (GC-MS) and investigated molluscicidal and larvicidal effects of linalool against O. hupensis and Schistosoma japonicium. The ultrastructural alterations in gills, salivary gland, stomach and hepatopancreas of snails were observed under the light microscope and transmission electron microscope, and lesions to tegument of cercaria were examined under a light microscope and fluorescence microscope. We then evaluated the effects of linalool on skin penetration and migration of schistosomula and adult survival by measurement of worm burden and egg counts in Balb/C mice infected with linalool-treated cercariae.ResultsIn the present work, 44 components were identified from the leaf extracts of C. camphora, of which linalool was the most abundant constituent. Linalool exhibited the striking molluscicidal and larvicidal effects with LC50 = 0.25 mg/L for O. hupensis and LC50 = 0.07 mg/L for cercaria of S. japonicium. After exposure to linalool, damage to the gills and hepatopancreas of the snails, and to the tegument and body-tail joint of cercariae was apparent. In addition, linalool markedly reduced the recovered schistosomulum from mouse skin after challenge infection, and therefore decreased the worm burden in infected animals, but not fecundity of female adults of the parasite.ConclusionsOur findings indicated that linalool might be a novel chemotherapeutic agent against S. japonicium and the snail intermediate host.

Interleukin 33 Mediates Type 2 Immunity and Inflammation in the Central Nervous System of Mice Infected With Angiostrongylus cantonensis

Angiostrongylus cantonensis can induce central nervous system (CNS) injury and cause human eosinophilic meningitis. The CNS has been found to have high expression of interleukin 33 (IL-33), which promotes the pathogenesis of T-helper 2 (Th2)-related disease. Given the predominantly type 2 response induced by A. cantonensis-infected mice and human, it is likely that IL-33 may play a role in aiding this process. We report here that IL-33 protein and ST2L messenger RNA (mRNA) transcripts in the brains were upregulated during A. cantonensis infection and that both splenocytes and brain mononuclear cells became IL-33 responsive and produced interleukin 5 and interleukin 13. Furthermore, administration of IL-33 to A. cantonensis-infected mice enhanced ST2L expression and cytokine production. Interestingly, brain IL-33 protein and ST2L mRNA levels were elevated 14-21 days after infection in BALB/c mice, compared with C57BL/6 mice. Thus, our data indicate that IL-33 produced in the brain may function as an inflammatory mediator in eosinophilic meningitis induced by A. cantonensis.

Mast cells modulate acute toxoplasmosis in murine models.

The role of mast cells (MCs) in Toxoplasma gondii infection is poorly known. Kunming outbred mice were infected intraperitoneally with RH strain T. gondii, either treated with compound 48/80 (C48/80, MC activator) or disodium cromoglycate (DSCG, MC inhibitor). Compared with infected controls, infected mice treated with C48/80 exhibited significantly increased inflammation in the liver (P < 0.01), spleen (P < 0.05), and mesentery (P < 0.05) tissues, higher parasite burden in the peritoneal lavage fluids (P < 0.01), and increased levels of mRNA transcripts of T. gondii tachyzoite surface antigen 1 (SAG1) gene in the spleen and liver tissues (P < 0.01), accompanied with significantly increased Th1 cytokine (IFN-γ, IL-12p40, and TNF-α) (P < 0.01) and decreased IL-10 (P < 0.01) mRNA expressions in the liver, and increased IFN-γ (P < 0.01) and IL-12p40 (P < 0.01) but decreased TNF-α (P < 0.01) and IL-4 (P < 0.01) in the spleens of infected mice treated with C48/80 at day 9-10 p.i. Whereas mice treated with DSCG had significantly decreased tissue lesions (P < 0.01), lower parasite burden in the peritoneal lavage fluids (P < 0.01) and decreased SAG1 expressions in the spleen and liver tissues (P < 0.01), accompanied with significantly increased IFN-γ (P < 0.01) and IL-12p40 (P < 0.05) in the liver, and decreased IFN-γ (P < 0.05) and TNF-α (P < 0.01) in the spleens; IL-4 and IL-10 expressions in both the spleen and liver were significantly increased (P < 0.01) in the infected mice treated with DSCG. These findings suggest that mediators associated with the MC activation may play an important role in modulating acute inflammatory pathogenesis and parasite clearance during T. gondii infection in this strain of mice. Thus, MC activation/inhibition mechanisms are potential novel targets for the prevention and control of T. gondii infection.