Morphogenesis of Strongyloides stercoralis Infective Larvae Requires the DAF-16 Ortholog FKTF-1

Based on metabolic and morphological similarities between infective third-stage larvae of parasitic nematodes and dauer larvae of Caenorhabditis elegans, it is hypothesized that similar genetic mechanisms control the development of these forms. In the parasite Strongyloides stercoralis, FKTF-1 is an ortholog of DAF-16, a forkhead transcription factor that regulates dauer larval development in C. elegans. Using transgenesis, we investigated the role of FKTF-1 in S. stercoralis' infective larval development. In first-stage larvae, GFP-tagged recombinant FKTF-1b localizes to the pharynx and hypodermis, tissues remodeled in infective larvae. Activating and inactivating mutations at predicted AKT phosphorylation sites on FKTF-1b give constitutive cytoplasmic and nuclear localization of the protein, respectively, indicating that its post-translational regulation is similar to other FOXO-class transcription factors. Mutant constructs designed to interfere with endogenous FKTF-1b function altered the intestinal and pharyngeal development of the larvae and resulted in some transgenic larvae failing to arrest in the infective stage. Our findings indicate that FKTF-1b is required for proper morphogenesis of S. stercoralis infective larvae and support the overall hypothesis of similar regulation of dauer development in C. elegans and the formation of infective larvae in parasitic nematodes

Structural and functional characterisation of the fork head transcription factor-encoding gene, Hc-daf-16, from the parasitic nematode Haemonchus contortus (Strongylida)

Despite their phylogenetic diversity, parasitic nematodes share attributes of longevity and developmental arrest (=hypobiosis) with free-living nematodes at key points in their life cycles, particularly in larval stages responsible for establishing infection in the host. Insulin-like signalling plays crucial roles in the regulation of life span and arrest (=dauer formation) in the free-living nematode, Caenorhabditis elegans. Insulin-like signalling in C. elegans negatively regulates the fork head boxO (FoxO) transcription factor encoded by daf-16, which is linked to initiating a dauer-specific pattern of gene expression. Orthologues of daf-16 have been identified in several species of parasitic nematode. Although function has been demonstrated for an orthologue from the parasitic nematode Strongyloides stercoralis (Rhabditida), the functional capabilities of homologues/orthologues in bursate nematodes (Strongylida) are unknown. In the present study, we used a genomic approach to determine the structures of two complete daf-16 orthologues (designated Hc-daf-16.1 and Hc-daf-16.2) and their transcripts in the parasitic nematode Haemonchus contortus, and assessed their function(s) using C. elegans as a genetic surrogate. Unlike the multiple isoforms of Ce-DAF-16 and Ss-DAF-16, which are encoded by a single gene and produced by alternative splicing, mRNAs encoding the proteins Hc-DAF-16.1 and Hc-DAF-16.2 are transcribed from separate and distinct loci. Both orthologues are transcribed in all developmental stages and both sexes of H. contortus, and the inferred proteins (603 and 556 amino acids) each contain a characteristic, highly conserved fork head domain. In spite of distinct differences in genomic organisation compared with orthologues in C. elegans and S. stercoralis, genetic complementation studies demonstrated here that Hc-daf-16.2, but not Hc-daf-16.1, could restore daf-16 function to a C. elegans strain carrying a null mutation at this locus. These findings are consistent with previous results for S. stercoralis and demonstrate functional conservation of the daf-16b orthologue between key parasitic nematodes from two different taxonomic orders and C. elegans. We conclude from these experiments that the fork head transcription factor DAF-16 and, by inference, other insulin-like signalling elements, are conserved in H. contortus, a parasitic nematode of paramount economic importance. We demonstrate that functionality is sufficiently conserved in Hc-DAF-16.2 that it can replace Ce-DAF-16 in promoting dauer arrest in C. elegans

Strongyloides stercoralis age-1: A Potential Regulator of Infective Larval Development in a Parasitic Nematode

Infective third-stage larvae (L3i) of the human parasite Strongyloides stercoralis share many morphological, developmental, and behavioral attributes with Caenorhabditis elegans dauer larvae. The ‘dauer hypothesis’ predicts that the same molecular genetic mechanisms control both dauer larval development in C. elegans and L3i morphogenesis in S. stercoralis. In C. elegans, the phosphatidylinositol-3 (PI3) kinase catalytic subunit AGE-1 functions in the insulin/IGF-1 signaling (IIS) pathway to regulate formation of dauer larvae. Here we identify and characterize Ss-age-1, the S. stercoralis homolog of the gene encoding C. elegans AGE-1. Our analysis of the Ss-age-1 genomic region revealed three exons encoding a predicted protein of 1,209 amino acids, which clustered with C. elegans AGE-1 in phylogenetic analysis. We examined temporal patterns of expression in the S. stercoralis life cycle by reverse transcription quantitative PCR and observed low levels of Ss-age-1 transcripts in all stages. To compare anatomical patterns of expression between the two species, we used Ss-age-1 or Ce-age-1 promoter::enhanced green fluorescent protein reporter constructs expressed in transgenic animals for each species. We observed conservation of expression in amphidial neurons, which play a critical role in developmental regulation of both dauer larvae and L3i. Application of the PI3 kinase inhibitor LY294002 suppressed L3i in vitro activation in a dose-dependent fashion, with 100 µM resulting in a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for treated L3i in comparison to the control. Together, these data support the hypothesis that Ss-age-1 regulates the development of S. stercoralis L3i via an IIS pathway in a manner similar to that observed in C. elegans dauer larvae. Understanding the mechanisms by which infective larvae are formed and activated may lead to novel control measures and treatments for strongyloidiasis and other soil-transmitted helminthiases

Microarray-Based Analysis of Differential Gene Expression between Infective and Noninfective Larvae of Strongyloides stercoralis

Strongyloides stercoralis is a soil-transmitted helminth that affects an estimated 30–100 million people worldwide. Chronically infected persons who are exposed to corticosteroids can develop disseminated disease, which carries a high mortality (87–100%) if untreated. Despite this, little is known about the fundamental biology of this parasite, including the features that enable infection. We developed the first DNA microarray for this parasite and used it to compare infective third-stage larvae (L3i) with non-infective first stage larvae (L1). Using this method, we identified 935 differentially expressed genes. Functional characterization of these genes revealed L3i biased expression of heat shock proteins and genes with products that have previously been shown to be immunoreactive in infected humans. Genes putatively involved in transcription were found to have L1 biased expression. Potential chemotherapeutic and vaccine targets such as far-1, ucr 2.1 and hsp-90 were identified for further study

Reconstruction of the insulin-like signalling pathway of Haemonchus contortus

Background: In the present study, we reconstructed the insulin/insulin-like growth factor 1 signalling (IIS) pathway for Haemonchus contortus, which is one of the most important eukaryotic pathogens of livestock worldwide and is related to the free-living nematode Caenorhabditis elegans. Methods: We curated full-length open-reading frames from assembled transcripts, defined the complement of genes that encode proteins involved in this pathway and then investigated the transcription profiles of these genes for all key developmental stages of H. contortus. Results: The core components of the IIS pathway are similar to their respective homologs in C. elegans. However, there is considerable variation in the numbers of isoforms between H. contortus and C. elegans and an absence of AKT-2 and DDL-2 homologs from H. contortus. Interestingly, DAF-16 has a single isoform in H. contortus compared with 12 in C. elegans, suggesting novel functional roles in the parasitic nematode. Some IIS proteins, such as DAF-18 and SGK-1, vary in their functional domains, indicating distinct roles from their homologs in C. elegans. Conclusions: This study paves the way for the further characterization of key signalling pathways in other socioeconomically important parasites and should help understand the complex mechanisms involved in developmental processes

Molecular taxonomic characterization of the human parasitic population of the nematode Strongyloides stercoralis in Cambodia and development and evaluation of methods for the genetic study of Strongyloides ratti

Nematodes of the genus Strongyloides are common small intestinal parasites of vertebrates. They have a complex life cycle, which in addition to parthenogenetic parasitic adults also contains a facultative free-living adult generation, with males and females. The presence of sexually reproducing adults outside of the host offers opportunities for genetic research, which are quite unique for an endo-parasitic organism. Accordingly, Strongyloides spp. is developing into a model system for parasitological, basic biological and evolutionary studies. In the first part of my thesis, I examined the Small Ribosomal Subunit rDNA (SSU) sequences from S. stercoralis larvae isolated from human patients in Cambodian highly S. stercoralis prevalent areas. Three polymorphic positions and three different haplotypes were identified within a region of the SSU normally considered to be essentially invariable within a nematode species. Interestingly, no hybrid individuals were found. These results suggested a low frequency of interbreeding between the different haplotypes in this area, either because S. stercoralis in this region reproduces only asexually or because crossing happens only within rather than between haplotypes. Many research tools and techniques routinely used in model organisms like Caenorhabditis elegans are not yet available for Strongyloides spp. One of these methods is mutagenesis using chemical mutagens. In the second part of my thesis I devised a protocol to mutagenize S. ratti with the chemical mutagen Ethyl Methanosulfonate (EMS). Using this protocol, I generated S. ratti mutants with a higher proportion of animals developing into the parasitic form. As a control, I also attempted to obtain the same effect by selection only, in absence of EMS. Next I evaluate the possibility of identifying the mutated genes by whole genome sequencing of multiple mutagenized and selected strains. While this approach appeared promising, I also found that the currently used laboratory strain is not sufficiently isogenic such that the number of resulting candidate mutations, which need to be tested is rather high. The strong population bottlenecks associatiated with the mutagenesis and selection procedures reduced the genetic complexity of the populations significantly. This demonstrated that generating a more isogenic S. ratti strain for genetic work is possible. In the third part of my thesis I isolated and characterized Strongyloides mariner-like transposons (SMARTs). Contrary to S. ratti, in S. papillosus several copies of SMART appeared potentially active. These transposons have the potential to be used as genetic tools as it has been demonstrated for the related Tc1 transposons in C. elegans.Nematoden der Gattung Strongyloides sind weit verbreitete Dünndarmparasiten von Wirbeltieren. Der komplexe Lebenszyklus beinhaltet neben parthenogenetischen parasitischen Adulttieren auch eine fakultative frei lebende Generation mit Männchen und Weibchen. Die Existenz von Geschlechtstieren außerhalb des Wirts eröffnet Möglichkeiten für genetische Studien, die für Endoparasiten außergewöhnlich sind. Dementsprechend entwickelt sich Strongyloides spp. zu einem Modellsystem für parasitologische, grundlagenbiologische und evolutionsbiologische Studien. Im ersten Teil meiner Doktorarbeit untersuchte ich die Sequenzen der rDNA für die kleine ribosomale Untereinheit (SSU) von S. stercoralis Larven aus Patienten in Regionen Kambodschas mit hoher S. stercoralis Prävalenz. Ich identifizierte drei polymorphe Positionen und drei verschiedene Haplotypen in einem Abschnitt der SSU, der normalerweise als praktisch invariable innerhalb einer bestimmten Art gilt. Interessanterweise gab es keine Hybride. Dieses Resultat lässt vermuten, dass sich S. stercoralis in dieser Gegend ausschließlich parthenogenetisch vermehrt, oder dass Kreuzung nur innerhalb, aber nicht zwischen den Haplotypen vorkommt. Viele Methoden, die in Modellorganismen wie C. elegans routinemäßig angewandt werden, sind für Strongyloides spp. noch nicht verfügbar. Eine davon ist die Mutagenese mittels chemischer Mutagene. Im zweiten Teil meiner Doktorarbeit erarbeitete ich ein Protokoll zur Mutagenese von S. ratti mit Ethyl Methanosulfonat (EMS). Ich isolierte mutante S. ratti Linien, die sich zu einem größeren Anteil zu parasitischen Individuen entwickelten. Als Kontrolle versuchte ich den gleichen Effekt auch durch reine Selektion, ohne Mutagen zu erzeugen. Ich testete ich, ob es möglich ist die mutierten Gene durch Genomsequenzierung der mutanten und selektierten Linien zu identifizieren. Der Ansatz erwies sich als vielversprechend, Allerdings fand ich, dass der momentan verwendete Laborstamm nicht ausreichend isogen ist. Die Anzahl der isolierten Kandidatenmutationen, die getestet werden müssten, stellte sich deshalb als recht hoch heraus. Die mutagenisierten und selektierten Linien, die in Folge des Experiments durch starke "population bottlenecks" gegangen waren zeigten eine deutlich reduzierte genetische Variabilität. Dies zeigte, dass es möglich ist, einen mehr isogenen S. ratti Stamm für genetische Arbeiten zu erzeugen. Im dritten Teil meiner Doktorarbeit isolierte und charakterisierte ich "Strongyloides mariner-like Transposons" (SMARTs). Anders als in S. ratti fand ich in S. papillosus mehrere möglicherweise aktive SMART Kopien. Diese Transposone haben das Potential als genetischen Werkzeugen benutzt zu werden, wie dies für die verwandten Tc1 Transposone in C. elegans der Fall ist

On the role of dauer in the adaptation of nematodes to a parasitic lifestyle

Abstract Nematodes are presumably the most abundant Metazoa on Earth, and can even be found in some of the most hostile environments of our planet. Various types of hypobiosis evolved to adapt their life cycles to such harsh environmental conditions. The five most distal major clades of the phylum Nematoda (Clades 8–12), formerly referred to as the Secernentea, contain many economically relevant parasitic nematodes. In this group, a special type of hypobiosis, dauer, has evolved. The dauer signalling pathway, which culminates in the biosynthesis of dafachronic acid (DA), is intensively studied in the free-living nematode Caenorhabditis elegans, and it has been hypothesized that the dauer stage may have been a prerequisite for the evolution of a wide range of parasitic lifestyles among other nematode species. Biosynthesis of DA is not specific for hypobiosis, but if it results in exit of the hypobiotic state, it is one of the main criteria to define certain behaviour as dauer. Within Clades 9 and 10, the involvement of DA has been validated experimentally, and dauer is therefore generally accepted to occur in those clades. However, for other clades, such as Clade 12, this has hardly been explored. In this review, we provide clarity on the nomenclature associated with hypobiosis and dauer across different nematological subfields. We discuss evidence for dauer-like stages in Clades 8 to 12 and support this with a meta-analysis of available genomic data. Furthermore, we discuss indications for a simplified dauer signalling pathway in parasitic nematodes. Finally, we zoom in on the host cues that induce exit from the hypobiotic stage and introduce two hypotheses on how these signals might feed into the dauer signalling pathway for plant-parasitic nematodes. With this work, we contribute to the deeper understanding of the molecular mechanisms underlying hypobiosis in parasitic nematodes. Based on this, novel strategies for the control of parasitic nematodes can be developed

Infective Larvae Production and Development in Strongyloides and its role in the dauer hypothesis

The dauer hypothesis for the evolution of parasitism in nematodes, states that dauer larvae, a specialised third larval stage present in many free living nematodes, served as a pre adaptation to parasitism and evolved into infective larvae (L3i). Unique biological features like alternating free-living sexual and parasitic parthenogenetic generations make Strongyloides spp., which includes the human pathogen S. stercoralis, an attractive model to study parasitism and the evolution thereof. One of the most striking features is that all L3is are female, although they can be produced from sexual reproduction. The main aims of this thesis were to test the dauer hypothesis for the specific example of Strongyloididae and to understand how female only progeny are formed from sexual reproduction. While Strongyloides ratti is an attractive model species as it can be maintained within its natural host, its free living stages can only be extracted from fecal cultures and many tools that are standard in model organisms are not yet available, making experimentation difficult. Therefore, I firstly set out to increase the toolkit within S. ratti. I strongly improved the protocol to culture S. ratti on bacteria seeded agar plates, which led to highly increased fecundity and survival, allowing easier experimentation. Further I devised the first working procedures for RNAi in any species of Strongyloides. As S. ratti appears refractive to microinjection, I developed an RNAi by soaking protocol which works across multiple life stages and has minimal off-target effects. Using the aforementioned RNAi technique, I studied daf-12 natively within S. ratti (the first study of its kind within Strongyloides). By suppressing DAF-12 I found that many functions of this nuclear hormone receptor are conserved between C. elegans and S. ratti. In particular, daf-12(RNAi) inhibited the formation of infective larvae as it does for dauer larvae in C. elegans. This demonstrates that the molecular mechanisms underlying dauer formation and L3i formation are conserved between species as phylogenetically distant as C. elegans and S. ratti , suggesting that DAF-12 it is a promising target for novel antihelminthics and providing support for the dauer hypothesis. In order to study the transition from free-living to parasitic, we need well-studied closely related free-living species to parasites of interest, of which we are currently lacking. After noticing that Rhabditophanes KR3021 (the closest known free living species to Strongyloides) produces dauer larvae and arrested J2 larvae (both previously unreported) under starvation conditions, I aimed to characterise this species and provide clearly lacking basic information. In addition to providing a detailed description of life cycle and morphology, I showed that the daf-12 pathway is also involved in dauer (but not arrested J2) formation making Rhabditophanes/Strongyloides a highly attractive system for the further evaluation of the dauer hypothesis. Next, by characterising spermatogenesis in S. ratti, S. papillosus and P. trichosuri (which does make males), I found that there is an uneven division resulting in viable X-bearing and non-viable nullo-X sperm. I showed that spermatogenesis itself is highly conserved within the Strongyloididae, from the presence of giant nuclei in the distal gonad, to expression and production of major sperm protein