Identification of a nuclear receptor/coactivator developmental signaling pathway in the nematode parasite Strongyloides stercoralis.

DAF-12 is nematode-specific nuclear receptor that has been proposed to govern development of the infectious stage of parasitic species, including Strongyloides stercoralis Here, we identified a parasite-specific coactivator, called DAF-12 interacting protein-1 (DIP-1), that is required for DAF-12 ligand-dependent transcriptional activity. DIP-1 is found only in Strongyloides spp. and selectively interacts with DAF-12 through an atypical receptor binding motif. Using CRISPR/Cas9-directed mutagenesis, we demonstrate that DAF-12 is required for the requisite developmental arrest and the ligand-dependent reactivation of infectious S. stercoralis infective third-stage larvae, and that these effects require the DIP-1 coactivator. These studies reveal the existence of a distinct nuclear receptor/coactivator signaling pathway that governs parasite development.

Full mitochondrial and nuclear genome comparison confirms that Onchocerca sp. "Siisa" is Onchocerca ochengi.

Onchocerca ochengi is a nodule-forming filarial nematode parasite of cattle. It is the closest known relative of the human parasite Onchocerca volvulus, with which it shares the black fly vector Simulium damnosum. Onchocerca sp. "Siisa" was described in black flies and in cattle and, based on limited mitochondrial sequence information, appeared to be about equally phylogenetically distant from O. ochengi and O. volvulus. Based on molecular genetic markers and apparent interbreeding, we later proposed that O. sp. "Siisa" belongs to the species O. ochengi. However, we did not demonstrate directly that the hybrids were fertile, and we were still unable to resolve the phylogenetic relationship of O. ochengi, O. sp. "Siisa," and O. volvulus, leaving some concerns with the conclusion mentioned above. Here, we present fully assembled, manually curated mitochondrial genomes of O. ochengi and O. sp. "Siisa," and we compare multiple individuals of these two taxa with respect to their whole mitochondrial and nuclear genomes. Based on the mitochondrial genomes, O. ochengi and O. sp. "Siisa" are phylogenetically much closer to each other than to O. volvulus. The differences between them are well within the range of what is expected for within-species variation. The nuclear genome comparison provided no indication of genetic separation of O. ochengi and O. sp. "Siisa." From this, in combination with the earlier literature, we conclude that O. ochengi and O. sp. "Siisa" should be considered one species.

Parasitological and transcriptomic comparison of Strongyloides ratti infections in natural and in suboptimal permissive hosts.

The nematode genus Strongyloides consists of fairly species-specific small intestinal parasites of various vertebrates, among them the human pathogen S. stercoralis. Between the parthenogenetic parasitic generations these worms can also form single facultative sexual free-living generations. In addition to their primary hosts, several species can also live more or less well in other permissive hosts, which are sometimes not very closely related with the normal host. For example, S. stercoralis can also infect dogs and non-human primates. Here we compare the infection and reproductive success over time and the gene expression profiles as determined by quantitative sequencing of S. ratti parasitizing in its natural host rat and in the permissive host gerbil. We show that in gerbils fewer infective larvae successfully establish in the host, but those that do accomplish this survive and reproduce for longer and produced a higher proportion of males during the first two month of infection. Globally, the gene expression profiles in the two hosts are very similar. Among the relatively few differentially expressed genes, astacin-like and acetylcholinesterase genes are prominently represented. In the future it will be interesting to see if these changes in the suboptimal host are indeed ecologically sensible responses to the different host.

Characterization of the endogenous DAF-12 ligand and its use as an anthelmintic agent in Strongyloides stercoralis.

A prevalent feature of Strongyloides stercoralis is a life-long and potentially lethal infection that is due to the nematode parasite's ability to autoinfect and, thereby, self-replicate within its host. Here, we investigated the role of the parasite's nuclear receptor, Ss-DAF-12, in governing infection. We identified Δ7-DA as the endogenous Ss-DAF-12 ligand and elucidated the hormone's biosynthetic pathway. Genetic loss of function of the ligand's rate-limiting enzyme demonstrated that Δ7-DA synthesis is necessary for parasite reproduction, whereas its absence is required for the development of infectious larvae. Availability of the ligand permits Ss-DAF-12 to function as an on/off switch governing autoinfection, making it vulnerable to therapeutic intervention. In a preclinical model of hyperinfection, pharmacologic activation of DAF-12 suppressed autoinfection and markedly reduced lethality. Moreover, when Δ7-DA was administered with ivermectin, the current but limited drug of choice for treating strongyloidiasis, the combinatorial effects of the two drugs resulted in a near cure of the disease.

Advances in the Molecular and Cellular Biology of Strongyloides spp.

PURPOSE OF REVIEW: This paper constitutes an update of recent studies on the general biology, molecular genetics, and cellular biology of Strongyloides spp. and related parasitic nematodes. RECENT FINDINGS: Increasingly, human strongyloidiasis is considered the most neglected of neglected tropical diseases. Despite this, the last 5 years has seen remarkable advances in the molecular biology of Strongyloides spp. Genome sequences for S. stercoralis, S. ratti, S. venezuelensis, S. papillosus, and the related parasite Parastrongyloides trichosuri were created, annotated, and analyzed. These genomic resources, along with a practical transgenesis platform for Strongyloides spp., aided a major achievement, the advent of targeted mutagenesis via CRISPR/Cas9 in S. stercoralis and S. ratti. The genome sequences have also enabled significant molecular epidemiologic and phylogenetic findings on human strongyloidiasis, including the first genetic evidence of zoonotic transmission of S. stercoralis between dogs and humans. Studies of molecular signaling pathways identified the nuclear receptor Ss-DAF-12 as one that can be manipulated in the parasite by exogenous application of its steroid ligands. The chemotherapeutic implications of this were unscored by a study in which a Ss-DAF-12 ligand suppressed autoinfection by S. stercoralis in a new murine model of human strongyloidiasis. SUMMARY: Seminal advances in genomics of Strongyloides spp. have transformed research into strongyloidiasis, facilitating fundamental phylogenetic and epidemiologic studies and aiding the deployment of CRISPR/Cas9 gene disruption and editing as functional genomic tools in Strongyloides spp. Studies of Ss-DAF-12 signaling in S. stercoralis demonstrated the potential of this pathway as a novel chemotherapeutic target in parasitic nematodes.

Bacillus thuringiensis Cry5B is Active against Strongyloides stercoralis in vitro.

Strongyloidiasis, caused by Strongyloides stercoralis infection, is an important neglected tropical disease that causes significant public health problems in the tropics and subtropics. The disease can persist in hosts for decades and may be life-threatening because of hyperinfection and dissemination. Ivermectin (mostly) and albendazole are the most common anthelmintics used for treatment. Albendazole is suboptimal for this parasite, and although ivermectin is quite effective in immunocompromised patients, a multiple-course regimen is required. Furthermore, reliance on a single drug class for treating intestinal nematodes is a recipe for future failure. Therefore, it is important to discover new anthelmintics to treat or prevent human strongyloidiasis. One promising candidate is the Bacillus thuringiensis crystal protein Cry5B. Cry5B is highly potent against parasitic nematodes, for example, hookworms and Ascaris suum. Here, we investigated the potential of Cry5B against S. stercoralis. Multiple stages of S. stercoralis, including the first larval stage (L1s), infective stage (iL3s), free-living adult stage, and parasitic female stage, were all susceptible to Cry5B as indicated by impairment of motility and decreased viability in vitro. In summary, Cry5B demonstrated strong potential as an effective anthelmintic for treatment and transmission control of human strongyloidiasis, justifying further experiments to investigate in vivo therapeutic efficacy.

Evaluation of DNA Extraction Methods on Individual Helminth Egg and Larval Stages for Whole-Genome Sequencing.

Whole-genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative; however, these samples typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole-genome sequencing of individual immature helminth samples. These approaches do not use whole-genome amplification, a common but costly approach to increase the yield of low-input samples. We first tested individual parasites from two species spotted onto FTA cards-egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni-before further testing on an additional five species-Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Strongyloides stercoralis, and Trichuris muris-with an optimal protocol. A sixth species-Dracunculus medinensis-was included for comparison. Whole-genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation both between species and between different life stages from some species described. These results demonstrate the feasibility of whole-genome sequencing of individual parasites, and highlight a new avenue toward generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

Duplications and Positive Selection Drive the Evolution of Parasitism-Associated Gene Families in the Nematode Strongyloides papillosus.

Gene duplication is a major mechanism playing a role in the evolution of phenotypic complexity and in the generation of novel traits. By comparing parasitic and nonparasitic nematodes, a recent study found that the evolution of parasitism in Strongyloididae is associated with a large expansion in the Astacin and CAP gene families.To gain novel insights into the developmental processes in the sheep parasite Strongyloides papillosus, we sequenced transcriptomes of different developmental stages and sexes. Overall, we found that the majority of genes are developmentally regulated and have one-to-one orthologs in the diverged S. ratti genome. Together with the finding of similar expression profiles between S. papillosus and S. ratti, these results indicate a strong evolutionary constraint acting against change at sequence and expression levels. However, the comparison between parasitic and free-living females demonstrates a quite divergent pattern that is mostly due to the previously mentioned expansion in the Astacin and CAP gene families. More detailed phylogenetic analysis of both gene families shows that most members date back to single expansion events early in the Strongyloides lineage and have undergone subfunctionalization resulting in clusters that are highly expressed either in infective larvae or in parasitic females. Finally, we found increased evidence for positive selection in both gene families relative to the genome-wide expectation.In summary, our study reveals first insights into the developmental transcriptomes of S. papillosus and provides a detailed analysis of sequence and expression evolution in parasitism-associated gene families.

Different but overlapping populations of Strongyloides stercoralis in dogs and humans-Dogs as a possible source for zoonotic strongyloidiasis.

Strongyloidiasis is a much-neglected soil born helminthiasis caused by the nematode Strongyloides stercoralis. Human derived S. stercoralis can be maintained in dogs in the laboratory and this parasite has been reported to also occur in dogs in the wild. Some authors have considered strongyloidiasis a zoonotic disease while others have argued that the two hosts carry host specialized populations of S. stercoralis and that dogs play a minor role, if any, as a reservoir for zoonotic S. stercoralis infections of humans. We isolated S. stercoralis from humans and their dogs in rural villages in northern Cambodia, a region with a high incidence of strongyloidiasis, and compared the worms derived from these two host species using nuclear and mitochondrial DNA sequence polymorphisms. We found that in dogs there exist two populations of S. stercoralis, which are clearly separated from each other genetically based on the nuclear 18S rDNA, the mitochondrial cox1 locus and whole genome sequence. One population, to which the majority of the worms belong, appears to be restricted to dogs. The other population is indistinguishable from the population of S. stercoralis isolated from humans. Consistent with earlier studies, we found multiple sequence variants of the hypervariable region I of the 18 S rDNA in S. stercoralis from humans. However, comparison of mitochondrial sequences and whole genome analysis suggest that these different 18S variants do not represent multiple genetically isolated subpopulations among the worms isolated from humans. We also investigated the mode of reproduction of the free-living generations of laboratory and wild isolates of S. stercoralis. Contrary to earlier literature on S. stercoralis but similar to other species of Strongyloides, we found clear evidence of sexual reproduction. Overall, our results show that dogs carry two populations, possibly different species of Strongyloides. One population appears to be dog specific but the other one is shared with humans. This argues for the strong potential of dogs as reservoirs for zoonotic transmission of S. stercoralis to humans and suggests that in order to reduce the exposure of humans to infective S. stercoralis larvae, dogs should be treated for the infection along with their owners.