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Efforts to identify new drugs for therapeutic and preventive treatments against parasitic nematodes have gained increasing interest with expanding pathogen omics databases and drug databases from which new anthelmintic compounds might be identified. Here, a novel approach focused on integrating a pan-Nematoda multi-omics data targeted to a specific nematode organ system (the intestinal tract) with evidence-based filtering and chemogenomic screening was undertaken. Based on de novo computational target prioritization of the 3,564 conserved intestine genes in A. suum, exocytosis was identified as a high priority pathway, and predicted inhibitors of exocytosis were tested using the large roundworm (Ascaris suum larval stages), a filarial worm (Brugia pahangi adult and L3), a whipworm (Trichuris muris adult), and the non-parasitic nematode Caenorhabditis elegans. 10 of 13 inhibitors were found to cause rapid immotility in A. suum L3 larvae, and five inhibitors were effective against the three phylogenetically diverse parasitic nematode species, indicating potential for a broad spectrum anthelmintics. Several distinct pathologic phenotypes were resolved related to molting, motility, or intestinal cell and tissue damage using conventional and novel histologic methods. Pathologic profiles characteristic for each inhibitor will guide future research to uncover mechanisms of the anthelmintic effects and improve on drug designs. This progress firmly validates the focus on intestinal cell biology as a useful resource to develop novel anthelmintic strategies. Author summary: The intestinal cells of parasitic nematodes are not known to regenerate, therefore disruption of essential processes that cause irreparable damage to intestinal cells is expected to promote worm expulsion. To facilitate improved methods of therapy we need to better understand the basic intestinal cell and tissue functions of this critical organ. To that end have undertaken a comprehensive analysis of multi-omics omics data and identify and prioritize intestinal genes/pathways with essential functions and associated drugs and established a foundational model of the STH intestinal system using the large roundworm Ascaris suum to test and validate inhibitors of these functions. We found 10 inhibitors to impacted motility, and seven of those showed severe pathology and an apparent irreparable damage to intestinal cells. Furthermore, five inhibitors were effective against the three phylogenetically diverse parasitic nematode species, indicating potential for a broad spectrum anthelmintics. Our results firmly validate the focus on intestinal cell biology as a useful resource to develop novel anthelmintic strategies. [ABSTRACT FROM AUTHOR]

The biological and molecular complexity of nematodes has impeded research on development of new therapies for treatment and control. We have focused on the versatility of the nematode intestine as a target for new therapies. To that end, it is desirable to establish a broad and deep understanding of the molecular architecture underlying intestinal cell functions at the pan-Nematoda level. Multiomics data were generated to uncover the evolutionary principles underlying both conserved and adaptable features of the nematode intestine. Whole genomes were used to reveal the functional potential of the nematodes, tissue-specific transcriptomes provided a deep assessment of genes that are expressed in the adult nematode intestine, and comparison of selected core species was used to determine a first approximation of the pan-Nematoda intestinal transcriptome. Differentially expressed transcripts were also identified among intestinal regions, with the largest number expressed at significantly higher levels in the anterior region, identifying this region as the most functionally unique compared to middle and posterior regions. Profiling intestinal miRNAs targeting these genes identified the conserved intestinal miRNAs. Proteomics of intestinal cell compartments assigned proteins to several different intestinal cell compartments (intestinal tissue, the integral and peripheral intestinal membranes, and the intestinal lumen). Finally, advanced bioinformatic approaches were used to predict intestinal cell functional categories of seminal importance to parasite survival, which can now be experimentally tested and validated. The data provide the most comprehensive compilation of constitutively and differentially expressed genes, predicted gene regulators, and proteins of the nematode intestine. The information provides knowledge that is essential to understand molecular features of nematode intestinal cells and functions of fundamental importance to the intestine of many, if not all, parasitic nematodes. [ABSTRACT FROM AUTHOR]

The intestine of parasitic nematodes has proven an important target for therapies aimed at prevention and treatment of diseases caused by these pathogens in humans, animals and plants. We have developed a unique research model with the intestine ofAscaris suum, the large round worm of swine and humans, that will enhance biological research on this tissue. To expand utility of this model, we quantitatively compared expression of 15,382 coding RNAs and 277 noncoding, micro RNAs (miRNAs) among 3 contiguous regions of the adultA. suumintestine. Differentially expressed transcripts were identified among regions, with the largest number expressed at significantly higher levels in the anterior region, identifying this region as the most functionally unique compared to middle and posterior regions. We further identified 64 exon splice variants (from 47 genes) that are differentially expressed among these regions. A total of 2,063 intestinal mRNA transcripts were predicted to be targeted by intestinal miRNA, and negative correlation coefficients for miRNA:mRNA abundances predicted 22 likely influential miRNAs and 503 likely associated miRNA:mRNA pairs.A. suumintestinal miRNAs were identified that are conserved with intestinal miRNAs fromC. elegans(10 mature sequences and 13 seed sequences conserved), and prospective intestinal miRNAs from the murine gastrointestinal nematode,Heligmosomoides polygyrus(5 mature and 11 seeds). Most of the conserved intestinal miRNAs were also high abundance miRNAs. The data provide the most comprehensive compilation of constitutively and differentially expressed genes along the length of the intestine for any nematode species. The information will guide prospective development of many hypotheses on nematode intestinal functions encoded by mRNAs, miRNAs and interactions between these RNA populations. [ABSTRACT FROM PUBLISHER]

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine. Author Summary: Past research has demonstrated that the nematode intestine has value for developing new methods of therapy and control of parasitic nematodes, as related to both vaccines and other anthelmintics. Yet, information related to basic intestinal cell biology is very limited. Research progress reported here moves towards the comprehensive identification of proteins (peptidases and others), and hence functions, that are sited on the apical intestinal membrane and within the intestinal lumen of adult female Ascaris suum. These advances provide an unprecedented research model to determine critical functions sited at these locations and to develop approaches to inhibit those functions. Comparative analysis among diverse parasitic species raises expectations that the results from A. suum can be applied to many parasitic nematodes for which similar research is technically impossible to perform. [ABSTRACT FROM AUTHOR]

Background: Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine) in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus). We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism. Methodology/Principal Findings: Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female. Conclusions/Significance: The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a valuable resource for studying tissue-specific biological functions in nematodes. Author Summary: Tissue-specific gene expression provides fundamental information about the biology of diverse cell types within an organism and interactions among tissues within multicellular organisms. However, such studies are experimentally challenging in smaller organisms such as many nematodes species, including the species (Caenorhabditis elegans) that is widely used in biomedical research. Ascaris suum (the large roundworm of swine), however, is of particular interest as a model nematode because it is large enough to allow for the dissection of individual tissues, and equally important because it is closely related to A. lumbricoides, which infects ∼1 billion people worldwide. Here, we build significantly on the previous tissue-specific gene expression research in A. suum by producing the first nematode RNA-seq dataset that spans multiple specific tissues, including three non-reproductive and two reproductive tissues in both male and female A. suum worms. This analysis provides significant details on the biological functions occurring within each of these tissues, which has not been previously explored. It also provides insight into specific gene regulation pathways active in each of the tissues, which have broad applicability across other nematodes, including both non-parasitic and parasitic species. [ABSTRACT FROM AUTHOR]

Background: Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine) in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus). We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism. Methodology/Principal Findings: Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female. Conclusions/Significance: The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a valuable resource for studying tissue-specific biological functions in nematodes. [ABSTRACT FROM AUTHOR]

Background: Caenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine) in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus). We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism. Methodology/Principal Findings: Overexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female. Conclusions/Significance: The expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a valuable resource for studying tissue-specific biological functions in nematodes. [ABSTRACT FROM AUTHOR]

Genome evolution studies for the phylum Nematoda have been limited by focusing on comparisons involving Caenorhabditis elegans. We report a draft genome sequence of Trichinella spiralis, a food-borne zoonotic parasite, which is the most common cause of human trichinellosis. This parasitic nematode is an extant member of a clade that diverged early in the evolution of the phylum, enabling identification of archetypical genes and molecular signatures exclusive to nematodes. We sequenced the 64-Mb nuclear genome, which is estimated to contain 15,808 protein-coding genes, at ∼35-fold coverage using whole-genome shotgun and hierarchal map-assisted sequencing. Comparative genome analyses support intrachromosomal rearrangements across the phylum, disproportionate numbers of protein family deaths over births in parasitic compared to a non-parasitic nematode and a preponderance of gene-loss and -gain events in nematodes relative to Drosophila melanogaster. This genome sequence and the identified pan-phylum characteristics will contribute to genome evolution studies of Nematoda as well as strategies to combat global parasites of humans, food animals and crops. [ABSTRACT FROM AUTHOR]

Parasitic nematodes that infect humans, animals, and plants cause serious diseases that are deleterious to human health and agricultural productivity. Chemical and biological control methods have reduced the impact of these parasites. However, surviving environmental stages lead to persistent reinfection of host species. In addition, development of resistance to nematicides and anthelmintics by these parasites and reduced availability of some nematicides, for environmental protection, pose significant obstacles for current and future prospects of effective parasite control. Due to marked differences in host species, research on animal and plant parasitic nematodes often proceeds independently. Despite the differences between animals and plants, basic cellular properties are shared among these host organisms. Some common properties may be important for mechanisms [homologous or convergent (homoplastic)] by which nematodes successfully infect these diverse hosts or by which animal and plant hosts resist infections by these pathogens. Here we compare host/parasite interactions between plant parasitic nematodes (PPN) and animal parasitic nematodes, with an emphasis on mammalian hosts (MPN). Similarities and differences are considered in the context of progress on molecular dissection of these interactions. A comprehensive coverage is not possible in the space allotted. Instead, an illustrative approach is used to establish examples that, it is hoped, exemplify the value of the comparative approach. [ABSTRACT FROM AUTHOR]

SUMMARY Immune responses to Haemonchus contortus intestinal antigens were evaluated using abomasal lymph node (ALN) lymphocytes from kid goats protected against challenge infection by immunization with parasite intestinal antigen, and from kids that were challenged after immunization with ovalbumin. ALN lymphocytes from the intestinal antigen-immunized group produced significantly higher antibody levels against intestinal antigens than the ovalbumin group, supporting the theory that immunization contributed to that ALN response. In contrast, intestinal lysates and membrane enriched preparations from intestinal cells stimulated significant proliferation of ALN lymphocytes in both groups. The proliferation was antigen-dependent, since intestinal antigens failed to stimulate proliferation in ALN lymphocytes from unimmunized and uninfected kids. For both the intestinal antigen and ovalbumin immunized groups, CD4+ T lymphocytes predominated in ALN lymphocytes that were stimulated to proliferate by intestinal antigens. The results indicate that H. contortus infection alone can induce ALN lymphocyte responses to intestinal antigens. In contrast to ALN lymphocyte responses, serum antibody against intestinal antigens was generally low to undetectable in ovalbumin-immunized kids following infection. Abomasal mucus from an H. contortus infected lamb was probed with a monoclonal antibody that binds to a periodate sensitive determinant on numerous H. contortus intestinal membrane and secreted proteins. Numerous bands of reactivity were detected, indicating that multiple parasite intestinal antigens were released into abomasal mucus during infection. The results, challenge the general concept that H. contortus intestinal antigens are ‘hidden’ from the host immune system during an infection. On the contrary, parasite intestinal proteins may be relatively abundant antigens presented to the host during infection. In addition, ALN T lymphocytes appear to provide a... [ABSTRACT FROM AUTHOR]

Establishing methods to investigate treatments that induce cell death in parasitic nematodes will promote experimental approaches to elucidate mechanisms and to identify prospective anthelmintics capable of inducing this outcome. Here, we extended recent progress on a method to monitor cell death and to identify small molecule inhibitors in Ascaris suum to Haemonchus contortus, a phylogenetically distant parasitic nematode of significance for both human and agricultural animal health. We utilized a diverse group of small molecule inhibitors referred to as nematode intestinal toxins/toxicants (NITs) coupled with motility, cytological and cell death assays to resolve gross effects on motility and individual cells and organ systems of two H. contortus larval stages in culture. Early transcriptional response evaluation identified NIT-responsive genes and pathways. The scope of death among cells in larvae varied among NITs but shared patterns with A. suum, despite the approach having some limitations due to characteristics of H. contortus larvae. Gene response patterns varied among NITs tested and provided information on the cell targets and pathways affected. Experimental NIT assays provide tools capable of inducing cell death in larval stages of parasitic nematodes, and can resolve many individual cells and organ systems in which cell death can be induced. [ABSTRACT FROM AUTHOR]