Transcriptome and secretome analysis of intra-mammalian life-stages of the emerging helminth pathogen, Calicophoron daubneyi reveals adaptation to a unique host environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock which has seen a rapid rise in prevalence throughout Western Europe in recent years. Following ingestion of metacercariae (parasite cysts) by the mammalian host, newly-excysted juveniles (NEJs) emerge and invade the duodenal submucosa which causes significant pathology in heavy infections. The immature larvae then migrate upwards, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients and to avoid the host immune response. Here, transcriptome analysis of four intra-mammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic disease respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that whilst a family of cathepsins B with varying S2 sub-site residues (indicating distinct substrate specificities) are differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is up-regulated in adult worms, although they are underrepresented in the secretome. The most abundant proteins in adult fluke secretions were helminth defence molecules (HDMs) that likely establish an immune environment permissive to fluke survival and/or neutralise pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognise antigens from other helminths commonly found as co-infections with rumen fluke.

Partial Characterization of Two Cathepsin D Family Aspartic Peptidases of Clonorchis sinensis

Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of <i>Clonorchis</i> <i>sinensis</i> and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of <i>C</i>. <i>sinensis</i>, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of <i>C</i>. <i>sinensis</i>. Sera obtained from rats experimentally infected with <i>C</i>. <i>sinensis</i> reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.

100 BOVINE PREGNANCY-ASSOCIATED GLYCOPROTEINS ARE ALLOCATED TO COTYLEDONARY OR INTERCOTYLEDONARY TROPHOBLAST ACCORDING TO THEIR PHYLOGENETIC ORIGIN

The pregnancy-associated glycoproteins (PAG) form a multigenic family of aspartic peptidases specifically expressed within the trophoblast of the ruminant placenta. In Bos taurus, this family is composed of 21 members segregated into 2 phylogenetic groups. The PAG are mainly produced by the cotyledons, which are discrete areas of massive interdigitations between the maternal endometrium and the foetal trophoblast. Cotyledons are separated by the flat cell layer of intercotyledonary trophoblast areas. According to former studies, modern PAG (PAG I) are produced by binucleate trophoblastic cells of the cotyledons, whereas ancient PAG (PAG II) are reported to be synthesized by both mononucleate and binucleate trophoblastic cells. Binucleate cells migrate into the maternal endometrium and to pour their intracellular content, allowing modern PAG as PAG1 to reach maternal blood circulation where they can be assayed as gestation diagnosis. Investigations about the PAG family are often restricted to a few molecules used for the diagnosis of gestation in blood circulation or in cotyledonary extracts. The aim of this study was to inform PAG I and PAG II expression during gestation in cotyledonary and intercotyledonary tissues. To build strong and innovative results in spite of the high identity sequence between PAG, we designed very specific tools such as RT-PCR primers for the transcripts analysis and antibodies for the proteins studies. Using real-time reverse-transcription PCR, we described the transcript expression of 16 of the 21 bovine PAG. Overall, they showed an increasing expression during gestation. However, we demonstrated a segregation of modern PAG in cotyledon and ancient PAG in the intercotyledonary trophoblast. Belonging to the ancient group, PAG2 was expressed in the cotyledon as a modern PAG. We raised specific antibodies against PAG1 (I), PAG11 (II), and PAG2 (II) that allowed to us confirm transcript data at the protein level using Western blot analysis. Three glycosylation variants of PAG11 were detected along with the gestation: a 45-kDa PAG11 is synthesized from 50 to 220 days of gestation, whereas a 78-kDa and a 70-kDa PAG11 were synthesized from 100 days of gestation. Immunolocalization described specific populations of binucleate cells producing PAG1 or PAG11 or PAG2. The PAG1 is mainly detected in binucleate cells of the whole cotyledon and in a lesser extent in the intercotyledonary trophoblast. The PAG11 was mainly localized in binucleate cells of the intercotyledonary trophoblast but also in a restricted population of binucleate cells of the cotyledon. The PAG2 was only synthesized in the villi of the cotyledon. To conclude, we demonstrated modern and ancient PAG have specific anatomical localization according to their phylogenetic origin. Ancient PAG are specific of nonvillous tissues, whereas modern PAG are mainly synthesized within the villous part of the cotyledon. The PAG2 seem to be an exception, belonging to the ancient group but expressed as a modern PAG and synthesized only in the cotyledon.