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

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

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

Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis.

We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40-64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington's disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered

<i>Ss-age-1</i> is expressed in amphidial neurons, the intestine, and other tissues.

<p>Fluorescence (A,C) and DIC (B,D) images of transgenic <i>S. stercoralis</i> post-free-living first-stage larvae expressing <i>Ss-age-1p::egfp::Ss-era-1t</i> from an extra-chromosomal array. (A,B) Expression of the EGFP reporter was present in the intestine (i), gonadal primordium (g), amphidial/head neuron (a), hypodermis (h), and phasmidial/tail neuron (p). (C,D) Expression of the EGFP reporter was present in an amphidial neuron (long arrow), with positional homology to AWC in <i>C. elegans</i>. The other cell body of the amphidial neuron pair is out of the plane of focus (short arrow). Cell bodies of the amphidial neurons align just lateral to the black lines in panel D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038587#pone.0038587-Srinivasan1" target="_blank">[74]</a>. Scale bars = 100 µm.</p

<i>Ce-age-1</i> is expressed in amphidial neurons and other tissues.

<p>Fluorescence (A,C) and DIC (B,D) images of transgenic <i>C. elegans</i> first-stage larvae expressing <i>Ce-age-1p::Ce-age-1(102bp) ::egfp::Ce-age-1t</i> from an extra-chromosomal array. (A,B) Strong expression of the EGFP reporter was present in amphidial neurons (a), a neuron or support cell anterior to the nerve ring (c), and the sphincter connecting the pharynx to the intestine (s). Weak expression was present in the intestine (i), hypodermis (h), and a phasmidial neuron (p). (C,D) EGFP reporter expression was present in the amphidial neurons AWC (short arrow) and ASJ (long arrow). Cell bodies of the amphidial neurons align just lateral to the black lines in panel D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038587#pone.0038587-Srinivasan1" target="_blank">[74]</a>. Scale bars = 100 µm.</p

Sites of <i>Ss-age-1</i> expression in transgenic <i>S. stercoralis</i> post-free-living first-stage larvae.

<p>Sites of <i>Ss-age-1</i> expression in transgenic <i>S. stercoralis</i> post-free-living first-stage larvae.</p

<i>Ss</i>-AGE-1 is a homolog of the <i>Ce</i>-AGE-1 PI3 kinase catalytic subunit.

<p>(A) Intron-exon structure of the <i>Ss-age-1</i> unspliced mRNA sequence. Grey boxes indicate the three exons, with the numbers above indicating the first and last base pairs of the exon. Introns are indicated by slanted lines between the exons, with the numbers indicating the total intron length. The 5′ and 3′ untranslated regions (UTR) are indicated with horizontal lines, with the italicized numbers indicating the total length of the UTR in base-pairs (bp). (B) Domain structure of the <i>Ss</i>-AGE-1 predicted protein. Shaded boxes represent the five protein family domains, with the numbers indicating the first and last amino acids of the domain. (C) Phylogenetic analysis of <i>Ss</i>-AGE-1. The predicted <i>Ss</i>-AGE-1 protein groups with other class I PI3 kinase catalytic subunits, including <i>Ce</i>-AGE-1. Abbreviations: <i>Strongyloides stercoralis</i> (Ss), <i>Strongyloides ratti</i> (Sr), <i>Parastrongyloides trichosuri</i> (Pt), <i>Brugia malayi</i> (Bm), <i>Caenorhabditis briggsae</i> (Cb), <i>Caenorhabditis elegans</i> (Ce), <i>Homo sapiens</i> (Hs), <i>Drosophila melanogaster</i> (Dm), and <i>Saccharomyces cerevisiae</i> (Sc). Accession numbers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038587#s2" target="_blank">Methods</a>.</p

<i>S. stercoralis</i> L3i activation is attenuated by the PI3 kinase inhibitor LY294002.

<p><i>In vitro</i> activation of <i>S. stercoralis</i> L3i under host-like culture conditions by incubation in DMEM, 10% canine serum, and 12.5 mM reduced glutathione for 24 hours at 37°C and 5% CO₂. The percentage of L3i that resumed feeding, a hallmark of activation, was scored by ingestion of FITC into the pharynx. Conditions included DMSO (carrier) positive control and M9 buffer negative control. The PI3 kinase inhibitor LY294002 was evaluated at 100 µM, 50 µM, and 10 µM, with each condition compared to the DMSO control using a logistic regression analysis. Error bars represent +1 SEM and parenthetical integers show the total number of L3i evaluated for each condition.</p

Stable transgenic lines of <i>S. ratti</i> are established by microinjection of nucleic acid constructs followed alternating rounds of host and culture passage, selecting on expression of a fluorescent reporter gene product.

<p><b>A</b>) Diagram of major steps in isolation of stable transgenic lines of <i>S. ratti</i>. Initial gene transfer into parental (P0) free-living females is by gonadal microinjection of donor vectors alone or in combination with helper vector or capped transposase encoding mRNA. F1 larvae are derived in culture and screened for GFP expression. Transgene expressing individuals are reared to infective L3 (L3i) and inoculated into rats to establish as parasitic females. F2 progeny released in the feces of these rats are screened for transgene expression and positive larvae reared in culture to free-living males and females and allowed to mate. F3 progeny arising from these crosses are reared to L3i in culture in inoculated into rats. F4 progeny arising in the feces are screened for reporter transgene expression and used for further alternating rounds of culture and host passage with continued selection on GFP. The timeline indicates the intervals in days following microinjection of parental worms in which each generation is isolated up to the F5 when transmission and expression are generally stabilized. <b>B</b>) Frequency of transgene expressing progeny by generation during selection of stable transgenic lines of <i>S. ratti</i>. Data are mean (± one standard deviation) percentages of reporter transgene-expressing individuals in each generation of alternating host and culture passage with selection on GFP (see panel A). Means are derived from three independently established lines (PV2, PV3 and PV4). Sample sizes (<b>n</b>) indicated below the abscissa are totals of worms examined from all three lines. Statistics: in panel B, 2-way ANOVA, performed using Prism ver. 5.0c (GraphPad Software, Inc., La Jolla, California, USA), revealed a highly significant effect due to generation (P<0.0001) but no significant effect (P = 0.2) due to donor plasmid.</p