Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression

The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration.     

Schistosoma mansoni venom allergen like proteins present differential allergic responses in a murine model of airway inflammation

Background

The Schistosoma mansoni Venom-Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in the host-pathogen interaction. Some of these molecules were suggested by us and others as potential immunomodulators and vaccine candidates, due to their functional classification, expression profile and predicted localization. From a vaccine perspective, one of the concerns is the potential allergic effect of these molecules.

Methodology/Principal Findings

Herein, we characterized the putative secreted proteins SmVAL4 and SmVAL26 and explored the mouse model of airway inflammation to investigate their potential allergenic properties. The respective recombinant proteins were obtained in the Pichia pastoris system and the purified proteins used to produce specific antibodies. SmVAL4 protein was revealed to be present only in the cercarial stage, increasing from 0–6 h in the secretions of newly transformed schistosomulum. SmVAL26 was identified only in the egg stage, mainly in the hatched eggs'' fluid and also in the secretions of cultured eggs. Concerning the investigation of the allergic properties of these proteins in the mouse model of airway inflammation, SmVAL4 induced a significant increase in total cells in the bronchoalveolar lavage fluid, mostly due to an increase in eosinophils and macrophages, which correlated with increases in IgG1, IgE and IL-5, characterizing a typical allergic airway inflammation response. High titers of anaphylactic IgG1 were revealed by the Passive Cutaneous Anaphylactic (PCA) hypersensitivity assay. Additionally, in a more conventional protocol of immunization for vaccine trials, rSmVAL4 still induced high levels of IgG1 and IgE.

Conclusions

Our results suggest that members of the SmVAL family do present allergic properties; however, this varies significantly and therefore should be considered in the design of a schistosomiasis vaccine. Additionally, the murine model of airway inflammation proved to be useful in the investigation of allergic properties of potential vaccine candidates.     

Protein Kinase C and Extracellular Signal-Regulated Kinase Regulate Movement,Attachment, Pairing and Egg Release in Schistosoma mansoni

Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis'' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.     

Functional antigens of Trichuris muris. The stimulation of immunity by vaccination of mice with somatic antigen preparations

Functional antigens of Trichuris muris. The stimulation of immunity by vaccination of mice with somatic antigen preparations. International Journal for Parasitology 3: 711–715. Vaccination of mice with somatic antigens of Trichuris muris in Freund's incomplete adjuvant stimulated a high degree of protective immunity and brought about a marked reduction (up to 92 per cent) of larval burdens after infection. Soluble antigens from the anterior (oesophageal) region of adult worms were shown to be most effective in stimulation of immunity, although protection was apparent following vaccination with antigens prepared from the posterior regions of adult worms and from whole larval worms. It is suggested that the functional antigens of the anterior region may originate in the stichosome cells.     

Strongyloides stercoralis: oral transfer of parasitic adult worms produces infection in mice and infection with subsequent autoinfection in gerbils.

Oral transfer of parasitic adult Strongyloides stercoralis produced patent infections in gerbils, C57BL/6J and SCID mice. In gerbils receiving adult worms, 7.3% of the transferred worms established and autoinfective L3 were found beginning on day 5 post-transfer, with peak numbers seen on days 6 and 7 post-transfer and few seen by 9 days post-transfer. These results suggest that development of autoinfective L3 in the gerbil is limited by the immune response of the host. When given orally to mice, between 7.2% (C57BL/6J) and 19.5% (SCID) of the adult worms established. These levels are higher than those previously obtained by the subcutaneous infection of SCID mice with infective larvae. No autoinfective larvae were found in infected mice and the ratio of L1/adult worms was small compared with that seen in gerbils. Thus, mice infected orally can be used as a model to study the interaction between the adult worm and the host, and since autoinfection has not been seen in the murine model, as developed to date, orally infected mice may be useful as a model to study mechanisms preventing autoinfection.     

日本血吸虫SjIrV1的基因特性及免疫保护效果

钙结合蛋白是日本血吸虫生长发育不可缺少的蛋白,具有非常广泛而重要的功能.在课题组日本血吸虫体被表膜蛋白研究基础上,利用PCR技术克隆了中国大陆株日本血吸虫66 kDa钙结合蛋白(SjIrV1)编码基因的cDNA序列,BLAST分析与菲律宾株日本血吸虫SjIrV1 cDNA编码序列一致,荧光定量PCR分析表明该基因在童虫和成虫期不同发育阶段均有表达,其中在35d和42d成虫中表达量较高,在42d雌虫中该基因表达水平远高于42d雄虫.构建重组表达质粒pET28a(+)-SjIrV1,在大肠杆菌中成功诱导表达,重组蛋白主要以可溶性形式存在,通过高效液相色谱法(RP-HPLC)以及串联质谱法(MS/MS)鉴定所获蛋白为目的蛋白SjIrV1.蛋白质印迹(Western blotting)分析结果显示重组蛋白能被感染日本血吸虫鼠血清和免疫鼠血清所识别,SjIrV1蛋白在虫体各发育阶段中均表达.免疫荧光染色实验观察表明SjIrV1主要分布在日本血吸虫成虫的表膜.应用重组蛋白免疫BALB/c小鼠后,免疫鼠血清中检测到较高水平的特异性IgG、IgG1和IgG2a抗体.结果表明SjIrV1可能在日本血吸虫的生长发育过程中起着重要作用.     

Overexpression of phosphatase regenerating liver 3 in oesophageal squamous cell carcinoma associated with metastasis and its comparison with phosphatase regenerating liver 1

Expression of PRL3 (phosphatase of regenerating liver 3) protein was examined with immunohistochemistry in 60 cases of ESCC (oesophageal squamous cell carcinoma) with matched lymph node metastasis (n = 40) and 6 cases of oesophageal adenocarcinoma. Its associations with PRL1 and clinicopathological parameters were analysed. The results showed the frequency of PRL3 protein expression was significantly higher in ESCC (39/60, 65%) than in normal oesophageal mucosa (0/20, P < 0.001); higher in ESCC with lymph node metastasis (30/40, 75%) than in ESCC without lymph node metastasis (9/20, P = 0.022), as well as higher in metastatic ESCC in lymph node (38/40, 95%) than in the primary ESCC (39/60, 65%, P < 0.001). PRL3 was expressed in 1 out of 6 oesophageal adenocarcinomas, but showed no nuclear staining of PRL1. Expression of PRL3 protein was positively associated with the grade and partially with the stage of ESCC. These results suggest that expression of PRL3 protein may be involved in the metastasis of ESCC and serve as a biomarker for prediction of ESCC metastasis.     

Morphological and histochemical studies on the prostate gland of developing and adult Paramphistomum cervi (Digenea: Paramphistomatidae)

Morphological and histochemical studies have been made on the development of the prostate gland of the digenetic trematode, Paramphistomum cervi during the course of its infection in sheep; histochemical characterization of prostate gland secretion in adult worms and its functional relationship with the transport of spermatozoa have also been studied. In 6-wk-old worms, the terminal portion of the male genital duct is formed of what appears to be a syncytial epithelium containing a relatively large number of nuclei compared to the rest of the male duct. Cellular organization of the prostate gland becomes conspicuous in 8-wk-old worms and the prostate gland is fully developed by 16 wk. Two types of prostate gland cells, characteristic of the adult, become distinct in 16-wk-old worms which contain spermatozoa in the lumen of their vas deferens and pars prostatica. Adult worms show two types of prostate gland cells: type I containing mainly glycoprotein and type II mainly phospholipid granules. Weak-to-strong activities of acid phosphatase, alkaline phosphatase, esterase, lipase, adenosine triphosphatase, tetrazolium reductase, NAD-diaphorase, isocitrate dehydrogenase, glyceraldehyde dehydrogenase, α-glycerophosphate dehydrogenase and glucose-6-phosphate dehydrogenase have been observed in the prostate gland. The release of prostate gland secretion and the passage of spermatozoa through the lumen of pars prostatica appear to be synchronized events.     

Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells

Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.     

Schistosoma mansoni encodes SMT3B and SMT3C molecules responsible for post-translational modification of cellular proteins

The sumoylation pathway is a post-translational modification of nuclear proteins widespread among several organisms. SMT3C is the main protein involved in this process and it is covalently conjugated to a diverse assortment of nuclear protein targets. To date, 3 SUMO paralogues (SMT3C, A/B) have been characterized in mammals and plants. In this work we characterized two SUMO related genes, named SMT3B and SMT3C throughout Schistosoma mansoni life cycle. The SmSMTB/C encodes for proteins sharing significant amino acid homology with SMT3. Phylogenetical analyses revealed that both SmSMT3B/C are distinct proteins. Additionally, SmSMT3B and C are expressed in cercariae, adult worms, eggs and schistosomula however SmSMT3C gene showed an expression level 7 to 9 fold higher than SmSMT3B in eggs, schistosomula and adult worms. The comparison between the SmSMT3C genomic and cDNA sequences established that the encoding sequence is interrupted by 3 introns of 70, 37 and 36 bp. Western Blot has shown SMT3 conjugates are present in nuclear and total protein fractions of adults and cercariae. Therefore our results suggest a functional sumoylation pathway, and the presence of two paralogues also suggests the specificity of substrates for SMT3 in S. mansoni.     

The anthelmintic activity of a novel organic arsenical, R7/45, upon Brugia pahangi in Meriones unguiculatus

The new organic arsenical R7/45 is a rapidly acting and very potent anthelmintic against adult Brugia pahangi in jirds. Against adult worms implanted into the peritoneal cavity 5 subcutaneous (SC) injections at 2.5 mg/kg of R7/45 killed 100% of adult worms. A single dose SC of 20 mg/kg was 100% effective and 10 mg/kg 76% effective against adult worms. When jirds were autopsied at different times after treatment at 20 mg/kg SC 89% of worms were dead within three days. R7/45 was not active when given by stomach intubation. Pretreatment of jirds with R7/45 had no effect on adult worms subsequently implanted into jirds. R7/45 was highly active against third and fourth stage larvae of B. pahangi in jirds.     

Red blood cell lysate modulates the expression of extracellular matrix proteins in dermal fibroblasts

Trichinella spiralis is a zoonotic nematode and food borne parasite and infection with T. spiralis leads to suppression of the host immune response and other immunopathologies. The excretory/secretory (ES) products of T. spiralis play important roles in the process of immunomodulation. However, the mechanisms and related molecules are unknown. Macrophages, a target for immunomodulation by the helminth parasite, play a critical role in initiating and modulating the host immune response to parasite infection. In this study, we examined the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor α, interleukin-1β , interleukin-6 , and interleukin-12) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms and 5-day-old adult worms/new-born larvae significantly inhibited inducible nitric oxide synthase gene expression in LPS-induced macrophages. In addition, ES products alone boosted the expression of anti-inflammatory cytokines interleukin-10 and transforming growth factor-β and effector molecule arginase 1 in J774A.1 macrophages. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in LPS-stimulated J774A.1 macrophages. These results suggest that ES products regulate host immune response at the macrophage level through inhibition of pro-inflammatory cytokines production and induction of macrophage toward the alternative phenotype, which maybe important for worm survival and host health.     

First identification of a phosphorylcholine-substituted protein from Caenorhabditis elegans: isolation and characterization of the aspartyl protease ASP-6

Caenorhabditis elegans is a widely accepted model system for parasitic nematodes, drug screening and developmental studies. Similar to parasitic worms, C. elegans expresses glycosphingolipids and glycoproteins carrying, in part, phosphorylcholine (PCho) substitutions, which might play important roles in nematode development, fertility and, at least in the case of parasites, survival within the host. With the exception of a major secretory/excretory product from Acanthocheilonema viteae (ES-62), no protein carrying this epitope has been studied in detail yet. Here we report on the identification, characterization and localization of the aspartyl protease ASP-6 of C. elegans, which is excreted by the nematode in a PCho-substituted form. Within the worm, most prominent expression of the protein is observed in the intestine, while muscle and epithelial cells express asp-6 to a lesser extent. In animals harboring an ASP-6::GFP fusion protein, diffuse fluorescence throughout the body cavity of adult worms indicates that the chimeric protein is secreted.     

Expression of retinaldehyde dehydrogenase 1 in the anterior pituitary glands of adult rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development and is also a regulatory factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine factor is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases that catalyze the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. Recently, we demonstrated that RALDH2 and RALDH3, but not RALDH1, were expressed in the developing anterior pituitary gland of rats, but the expression of RALDHs in the adult pituitary gland was not determined. Therefore, we have now examined the expression of RALDH1, RALDH2, and RALDH3 mRNAs in the pituitary gland of adult rats. Analysis by quantitative real-time polymerase chain reaction of adult pituitary glands has revealed a high level of RALDH1 mRNA but not of RALDH2 mRNA or RALDH3 mRNA. We have also detected mRNA expression for RALDH1 in the anterior pituitary gland by in situ hybridization with digoxigenin-labeled cRNA probes. Double-staining for RALDH1 mRNA and pituitary hormones or S-100 protein, a marker of folliculo-stellate cells (FS-cells), has revealed RALDH1 mRNA expression in a portion of prolactin-producing cells, marginal layer cells, and FS-cells. Our results suggest that RA is generated in the adult anterior pituitary gland, and that it may act locally on pituitary cells. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (18790149) and by the Foundation of Growth Science.