cultivation-heligmosomoides-polygyrus-an-immunomodulatory-nematode

Cultivation of <em>Heligmosomoides Polygyrus:</em> An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

University of Edinburgh

Inspection of the nucleotide sequence of bacterial artificial chromosome number 49_J_14 [Le Paslier, M.C., Pierce, R.J., Merlin, F., Hirai, H., Wu, W., Williams, D.L., Johnston, D., LoVerde, P.T., Le Paslier, D., 2000. Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library. Genomics 65, 87-94] from chromosome 1 of the genome of Schistosoma mansoni (GenBank ) revealed the likely presence of a proviral form of a novel schistosome retrotransposon. The novel element, which we named the fugitive, belonged to the mag-like family of the gypsy-Ty3 clade of long terminal repeat retrotransposons. It was closely related to the mag-like retrotransposon Gulliver from Schistosoma japonicum, but was dissimilar to several other long terminal repeat retrotransposons known from S. mansoni including Boudicca, Saci-1, Saci-2 and Saci-3. The full length fugitive element was 4811 bp constituted of a single read-through open reading frame of 4134 bp flanked at both ends by identical long terminal repeat sequences of 273 bp. The open reading frame encoded retroviral-like gag, with a distinctive double Cys-His motif, and pol polyprotein, with a pol domain order of protease, reverse transcriptase, RNaseH and integrase. Examination of schistosome transcriptome sequences in the public domain revealed that the fugitive was transcribed in at least six developmental stages of S. mansoni, while bioinformatics approaches and Southern hybridisation analysis indicated that as many as 2000 copies of the fugitive were interspersed throughout the schistosome genome.

The fugitive LTR retrotransposon from the genome of the human blood fluke, Schistosoma mansoni.

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells.

In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni.

We describe two homologues of the mammalian divalent metal transporter (DMT1) for Schistosoma mansoni, a pathogenic intravascular parasite of humans. Schistosomes have a high nutritional and metabolic demand for iron. Nucleotide sequences of the parasite homologues, designated SmDMT1A and -B, are identical in all but the 5'-regions. The predicted amino acid sequences share at least 60% identity with DMT1 (=Nramp2) of humans, mice, and rats, and at least 55% identity with Nramp1 from mice, humans and Caenorhabditis elegans. SmDMT1A is expressed in differentiating eggs, miracidia, cercariae, schistosomula, and adults, whereas SmDMT1B is expressed in all but the miracidium and occurs at lower levels than SmDMT1A in differentiating eggs and cercariae. An iron-responsive element, present at the 3'-untranslated region of many DMT1 molecules, is not present in schistosome mRNAs studied here. A Western blot analysis of adult worm preparations using a homologous rabbit serum raised against a schistosome DMT1 peptide and a heterologous serum raised against mammalian DMT1, revealed a band approximating 115 kDa. By immunofluorescence microscopy, the schistosome DMT1s localize primarily to the tegument. Iron uptake assays demonstrated that SmDMT1s were able to rescue yeast growth in ferrous iron-transport deficient yeast (fet3fet4). The results suggest that schistosomes express molecules for ferrous iron transport in their tegument, suggesting trans-tegumental transport as one means of iron acquisition for these parasites.

Two isoforms of a divalent metal transporter (DMT1) in Schistosoma mansoni suggest a surface-associated pathway for iron absorption in schistosomes.

Schistosomes are blood-dwelling flukes that infect 200 million people worldwide and are responsible for hundreds of thousands of deaths annually. Using a signal sequence trap, we cloned from Schistosoma mansoni two cDNAs, Sm-tsp-1 and Sm-tsp-2, encoding the tetraspanin (TSP) integral membrane proteins TSP-1 and TSP-2. We raised antibodies to recombinant TSP fusion proteins and showed that both proteins are exposed on the surface of S. mansoni. Recombinant TSP-2, but not TSP-1, is strongly recognized by IgG1 and IgG3 (but not IgE) from naturally resistant individuals but is not recognized by IgG from chronically infected or unexposed individuals. Vaccination of mice with the recombinant proteins followed by challenge infection with S. mansoni resulted in reductions of 57% and 64% (TSP-2) and 34% and 52% (TSP-1) for mean adult worm burdens and liver egg burdens, respectively, over two independent trials. Fecal egg counts were reduced by 65-69% in both test groups. TSP-2 in particular provided protection in excess of the 40% benchmark set by the World Health Organization for progression of schistosome vaccine antigens into clinical trials. When coupled with its selective recognition by naturally resistant people, TSP-2 seems to be an effective vaccine antigen against S. mansoni.

Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis.

Developmentally arrested infective larvae of strongylid nematodes are activated to resume growth by host-derived cues encountered during invasion of the mammalian host. Exposure of Nippostrongylus brasiliensis infective larvae to elevated temperature (37°C) is sufficient to activate signalling pathways which result in resumption of feeding and protein secretion. This occurs independently of exposure to serum or glutathione, in contrast to the hookworm Ancylostoma caninum, and is not initiated by chemical exsheathment. No qualitative differences in protein secretion were induced by host serum as visualised by two-dimensional SDS-PAGE, although exposure of larvae to an aqueous extract of rat skin did stimulate secretion of a small pre-synthesised bolus of proteins. Infective larvae began feeding after a lag period of 3-4 h at 37°C, reaching a maximum of 90% of the population feeding by 48 h. Neither a membrane permeant analogue of cyclic GMP nor muscarinic acetylcholine receptor agonists stimulated feeding at 20°C, and high concentrations of both compounds inhibited temperature-induced activation. LY294002, an inhibitor of phosphatidylinositol 3-kinase, Akt inhibitor IV, an inhibitor of Akt protein kinase, and ketoconazole, an inhibitor of cytochrome P450, all blocked resumption of feeding and protein secretion at 37°C. Serotonin increased the rate of feeding assessed by uptake of radiolabelled BSA, but could not initiate feeding independently of elevated temperature. Collectively, the data suggest that the early signalling events for larval activation in N. brasiliensis differ substantially from A. caninum, but that they may converge at pathways downstream of phosphatidylinositol 3-kinase involving steroid hormone synthesis.

Activation of Nippostrongylus brasiliensis infective larvae is regulated by a pathway distinct from the hookworm Ancylostoma caninum.

Iron has an integral role in numerous cellular reactions and is required by virtually all organisms. In physiological conditions, iron is abundant in a largely insoluble ferric state. Ferric reductases are an essential component of iron uptake by cells, reducing iron to the soluble ferrous form. Cytochromes b561 (cyts-b561) are a family of ascorbate reducing transmembrane proteins found in most eukaryotic cells. The identification of the ferric reductase duodenal cytochrome b (dcytb) and recent observations that other cyts-b561 may be involved in iron metabolism have opened novel perspectives for elucidating their physiological function.

A cytochrome b561 with ferric reductase activity from the parasitic blood fluke, Schistosoma japonicum.

Integrative conjugative elements (ICEs) are mobile genetic elements (MGEs) that possess all genes necessary for excision, transfer and integration into recipient genome. They also carry accessory genes that impart new phenotypic features to recipient strains. ICEs therefore play an important role in genomic plasticity and population structure. We previously characterised ICESde3396, the first ICE identified in the β-hemolytic Streptococcus dysgalactiae subsp equisimilis (SDSE) and demonstrated its transfer to single isolates of Streptococcus pyogenes (group A streptococcus, GAS) and Streptococcus agalactiae (group B streptococcus, GBS). While molecular studies found the ICE in multiple SDSE and GBS isolates, it was absent in all GAS isolates examined.

Danielle J. Smyth

Institute of Immunology and Infection Research

University of Edinburgh

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products