Characterization of the endogenous DAF-12 ligand and its use as an anthelmintic agent in Strongyloides stercoralis
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Evaluation Summary:
This work reveals the pathway by which an important human parasite synthesizes a nuclear hormone receptor ligand critical for progression through its life cycle and demonstrates the potential therapeutic implications of perturbing this pathway. The experiments are insightfully and expertly conceived, designed and executed, and the data support the conclusions. This manuscript will be of general interest to parasitologists, nematode biologists, and those studying transcriptional regulatory networks governed by ligand-gated nuclear receptors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
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.
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Author Response:
Reviewer #1:
This work significantly advances our understanding of the role of Strongyloides stercoralis nuclear receptor Ss-DAF-12 in determining parasite life cycle and infection outcomes. Strongyloidiasis is a facultative soil-transmitted nematode that infects hundreds of millions of humans. While infections are typically subclinical, 'hyperinfections' associated with host immunosuppression bring about severe morbidity and mortality that are not well-controlled by existing anthelmintics. DAF-12 signaling in free-living nematodes is known to promote growth and development, lifespan extension, and avoidance of the dauer dormancy state. Some of these findings have been shown to be conserved in parasitic nematodes, where infective stages are argued to be akin to dauer, motivating the study of the orthologous DAF-12 …
Author Response:
Reviewer #1:
This work significantly advances our understanding of the role of Strongyloides stercoralis nuclear receptor Ss-DAF-12 in determining parasite life cycle and infection outcomes. Strongyloidiasis is a facultative soil-transmitted nematode that infects hundreds of millions of humans. While infections are typically subclinical, 'hyperinfections' associated with host immunosuppression bring about severe morbidity and mortality that are not well-controlled by existing anthelmintics. DAF-12 signaling in free-living nematodes is known to promote growth and development, lifespan extension, and avoidance of the dauer dormancy state. Some of these findings have been shown to be conserved in parasitic nematodes, where infective stages are argued to be akin to dauer, motivating the study of the orthologous DAF-12 pathway as an antiparasitic substrate.
Through a lipid fractionation strategy, the authors first show that Δ7-DA is a potent endogenous ligand for Ss-DAF-12 and that Δ7-DA abundance throughout the life cycle is correlated with states of parasite reproductive development. The authors show that S. stercoralis possess the machinery to synthesize Δ7-DA from dietary cholesterol and use an insect cell expression system to identify biosynthetic enzymes in this pathway: Ss-DAF-35, Ss-DHS-16, and the cytochrome P450 Ss-CYP-22a9. CRISPR/Cas9-mediated disruption of Ss-CYP-22a9 is shown to near-completely inhibit cGMP-mediated Δ7-DA production and activation of infective larvae as measured by feeding, which can be rescued by exogenous Δ7-DA. Finally, a gerbil infection model is used to show that Δ7-DA treatment drastically reduces fecal output of larvae in uncomplicated strongyloidiasis and that a combined Δ7-DA/ivermectin regimen can decrease the burden of autoinfective intestinal larvae and prevent death in hyperinfection induced by immune compromise.
Overall, the conclusions are well-supported and this work provides important new insights on a pathway that is of broad relevance to the regulation of the complex and diverse life cycles of parasitic nematodes. The discovery that the combinatorial action of a DAF-12 agonist and ivermectin can synergistically control hyperinfective strongyloidiases is a major and impactful finding. This work will be of great interest to the larger parasitology and nematode biology community. My enthusiasm is only slightly tempered by the acknowledged caveats that currently limit the therapeutic outlook of this approach. The eventual development of therapeutics targeting this pathway could aid the treatment of uncontrolled strongyloides infections and be of potential value for the treatment and control of other parasitic worm infections.
Strengths:
- Experiments are generally well-designed and rigorous, clearly establishing that Δ7-DA is a primary ligand for Ss-DAF-12 and resolving the primary biosynthetic pathway for the production of Δ7-DA in S. stercoralis. While Δ7-DA is the known ligand of C. elegans DAF-12, significant difference in primary sequence justified caution and experimental validation. Similarly, while two of the Δ7-DA biosynthetic pathway enzymes have one-to-one C. elegans orthologs, the role of Ss-CYP-22a9 as the DAF-9 isoenzyme could not have been bioinformatically inferred.
- CRISPR-based knockdowns are not trivial in this system and both the HDR and NHEJ pathways were elegantly leveraged to confirm the in vitro activity of Ss-CYP-22a9 and its essentiality to the life stage responsible for infection.
- Animal studies convincingly reveal the synergistic effect of Δ7-DA and ivermectin in disseminated strongyloidiases. The burdens of intestinal larvae and adults in both uncomplicated and disseminated infection after treatment align with the standing model that Δ7-DA is acting against the intestinal larval stages (L3a) that are naturally deficient in the hormone.
- While there is precedent for combinatorial drug therapies in antifilarial control, there is great novelty in combining drugs that are known to target different stages as opposed to just different molecular targets. This provides the first clear demonstration of this as far as parasitic nematodes are concerned.
Thank you for your enthusiastic and supportive comments.
Weaknesses: Weaknesses are categorically minor.
- As the authors recognize, the animal studies required daily Δ7-DA dosing over a two-week period. While some of this is explained by a short half-life and poor pharmacokinetics, drug was continually delivered directly to the gut at high (uM) concentrations. This is a major hurdle to surmount and it is entirely possible that even if drugs with equivalent potency and more favorable pharmacokinetics were discovered, they would have to be administered in multiple dosing regimens or at prohibitively high concentrations to achieve a curative effect.
We did address this potential limitation in the second to the last paragraph of the Discussion and believe that it should not be an insurmountable task to develop such drugs. The repeated dosing that we used was necessary because the endogenous ligand has poor pharmacokinetic properties, which is a relatively common characteristic of other endogenous nuclear receptor ligands as well. Notably, however, this hurdle has been overcome successfully with the design of potent, long-acting agonists and antagonists (the depot drugs used in reproductive medicine are great examples of this strategy).
- While there is some evidence in other nematode clades that modulation of the DAF-12 pathway can affect developmental phenotypes, many of these parasites have huge phylogenetic separation from strongyloides and lack dormant stages requiring 'activation' or free-living stages. Given the independent evolution of parasitism across the phylum, it is just as likely that drugs acting on DAF-12 will have subtle (and not curative) effects in these other parasite systems.
This will be an important point to address with other parasites. However, we note that a large number of them (including all of the soil-borne nematode parasites that have been surveyed) have a DAF-12 and a similar L3i stage. In fact, the so-called “rule of the infective third stage” is a paradigm that is broadly conserved throughout the nematode phylum.
Reviewer #2:
Mangelsdorf, Kliewer and colleagues here identified the endogenous ligand in Strongyloides stercoralis that governs that parasitic nematode's capacity to autoinfect its mammalian hosts. The ligand, [Delta]7-DA, interacts with nuclear receptor Ss-DAF-12, just as it does with its C. elegans ortholog, Ce-DAF-12, governing transcription of genes essential for metabolism and reproductive growth. Specifically, [Delta]7-DA appears to mediate a switch in DAF-12 function: in unfavorable conditions, [Delta]7-DA is absent and unliganded DAF-12 is said to arrest growth in both species and produces developmentally quiescent infective S. stercoralis larvae; in favorable conditions, the ligand is synthesized and the liganded DAF-12 triggers infection by S. stercoralis and subsequent development and reproduction in both species. The authors determined the ligand's biosynthetic pathway and showed by mutating the rate-limiting enzyme in the pathway that [Delta]7-DA is essential for parasite reproduction, whereas its absence is required for infectious larval development. In an animal model, they demonstrated that administration of [Delta]7-DA suppresses autoinfection and host lethality. Given in combination with an existing drug targeted to actively developing stages, [Delta]7-DA virtually cures the disease.
This work establishes a finding and an implication. The finding: gene circuits for growth and reproduction in nematode species with distinct life cycles -- parasitic vs free-living -- are regulated by a hormonal signal and cognate receptor that are structurally and functionally conserved. This is evolutionarily unremarkable, made mildly surprising because S. stercoralis lacks a cytochrome P450 with strong sequence identity to C. elegans DAF-9, which catalyzes the rate-limiting step in [Delta]7-DA synthesis in C. elegans; a screen of S. stercoralis P-450s demonstrated that Ss-CYP22a9 is the DAF-9 isozyme. The implication: targeting DAF-12 function (either agonism to block lethal hyperinfection or antagonism to prevent development of adult worms) or ligand synthesis may offer a therapeutic route to treating nematode parasitism. This is a valuable implication, identifying three therapeutic target approaches -- Ss-DAF-12 agonism or antagonism, and Ss-CYP22a9 inhibition -- that potentially might be be advanced from these pre-clinical observations. Overall, the manuscript makes a modest yet significant contribution.
Typical of the work from Mangelsdorf and Kliewer, the research plan and experiments are rigorously designed, executed and interpreted. My only quibble is that the requirement for Ss-DAF-12 (unliganded) to produce infectious L3i larvae is claimed (lines 165-167) but not directly demonstrated here. Instead, the authors depend on, and eventually cite in the Discussion (line 340) their nice PNAS paper earlier this year, which makes this case. Because of its importance in rounding out the implication noted above, my preference would be for the authors to add an experiment to this work that documents that Ss-DAF-12 is essential both pre- and post-ligand production.
Thank you for your positive comments and enthusiasm for our work. Regarding your comment on the requirement of Ss-DAF-12 for L3i, in the text we have updated the citation of our previous published results to support our finding that unliganded Ss-DAF-12 is required for L3i formation. Importantly, we also showed this requirement using a different strategy in our present study: we demonstrated that the enzyme that makes the ligand is required for recovery from L3i and that knocking it out results in worms that develop into L3i but do not progress unless exogenous ligand is added back.
Regarding the preference for an experiment that details Ss-DAF-12’s requirement for pre-ligand production, this was shown in our 2021 PNAS paper by Cheong et al. using the Ss-DAF-12 loss of function worms. For post-ligand production, these Ss-DAF-12 KO worms could not be used for the following reason. Recall that the apo-receptor functions as a transcriptional repressor and so loss of Ss-DAF-12 results in de-repression of its targets, thereby phenocopying the presence of the ligand. Thus, adding the ligand to the Ss-DAF-12 KO has no easily discernable phenotype. However, in our PNAS paper we did knock out the essential coactivator (DIP-1) for Ss-DAF-12, which is required for post-ligand activity. This knockout eliminates Ss-DAF-12 ligand transactivation (but not transrepression) and shows the expected phenotype of not being able to recover from L3i.
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Evaluation Summary:
This work reveals the pathway by which an important human parasite synthesizes a nuclear hormone receptor ligand critical for progression through its life cycle and demonstrates the potential therapeutic implications of perturbing this pathway. The experiments are insightfully and expertly conceived, designed and executed, and the data support the conclusions. This manuscript will be of general interest to parasitologists, nematode biologists, and those studying transcriptional regulatory networks governed by ligand-gated nuclear receptors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
-
Reviewer #1 (Public Review):
This work significantly advances our understanding of the role of Strongyloides stercoralis nuclear receptor Ss-DAF-12 in determining parasite life cycle and infection outcomes. Strongyloidiasis is a facultative soil-transmitted nematode that infects hundreds of millions of humans. While infections are typically subclinical, 'hyperinfections' associated with host immunosuppression bring about severe morbidity and mortality that are not well-controlled by existing anthelmintics. DAF-12 signaling in free-living nematodes is known to promote growth and development, lifespan extension, and avoidance of the dauer dormancy state. Some of these findings have been shown to be conserved in parasitic nematodes, where infective stages are argued to be akin to dauer, motivating the study of the orthologous DAF-12 …
Reviewer #1 (Public Review):
This work significantly advances our understanding of the role of Strongyloides stercoralis nuclear receptor Ss-DAF-12 in determining parasite life cycle and infection outcomes. Strongyloidiasis is a facultative soil-transmitted nematode that infects hundreds of millions of humans. While infections are typically subclinical, 'hyperinfections' associated with host immunosuppression bring about severe morbidity and mortality that are not well-controlled by existing anthelmintics. DAF-12 signaling in free-living nematodes is known to promote growth and development, lifespan extension, and avoidance of the dauer dormancy state. Some of these findings have been shown to be conserved in parasitic nematodes, where infective stages are argued to be akin to dauer, motivating the study of the orthologous DAF-12 pathway as an antiparasitic substrate.
Through a lipid fractionation strategy, the authors first show that Δ7-DA is a potent endogenous ligand for Ss-DAF-12 and that Δ7-DA abundance throughout the life cycle is correlated with states of parasite reproductive development. The authors show that S. stercoralis possess the machinery to synthesize Δ7-DA from dietary cholesterol and use an insect cell expression system to identify biosynthetic enzymes in this pathway: Ss-DAF-35, Ss-DHS-16, and the cytochrome P450 Ss-CYP-22a9. CRISPR/Cas9-mediated disruption of Ss-CYP-22a9 is shown to near-completely inhibit cGMP-mediated Δ7-DA production and activation of infective larvae as measured by feeding, which can be rescued by exogenous Δ7-DA. Finally, a gerbil infection model is used to show that Δ7-DA treatment drastically reduces fecal output of larvae in uncomplicated strongyloidiasis and that a combined Δ7-DA/ivermectin regimen can decrease the burden of autoinfective intestinal larvae and prevent death in hyperinfection induced by immune compromise.
Overall, the conclusions are well-supported and this work provides important new insights on a pathway that is of broad relevance to the regulation of the complex and diverse life cycles of parasitic nematodes. The discovery that the combinatorial action of a DAF-12 agonist and ivermectin can synergistically control hyperinfective strongyloidiases is a major and impactful finding. This work will be of great interest to the larger parasitology and nematode biology community. My enthusiasm is only slightly tempered by the acknowledged caveats that currently limit the therapeutic outlook of this approach. The eventual development of therapeutics targeting this pathway could aid the treatment of uncontrolled strongyloides infections and be of potential value for the treatment and control of other parasitic worm infections.
Strengths:
- Experiments are generally well-designed and rigorous, clearly establishing that Δ7-DA is a primary ligand for Ss-DAF-12 and resolving the primary biosynthetic pathway for the production of Δ7-DA in S. stercoralis. While Δ7-DA is the known ligand of C. elegans DAF-12, significant difference in primary sequence justified caution and experimental validation. Similarly, while two of the Δ7-DA biosynthetic pathway enzymes have one-to-one C. elegans orthologs, the role of Ss-CYP-22a9 as the DAF-9 isoenzyme could not have been bioinformatically inferred.
- CRISPR-based knockdowns are not trivial in this system and both the HDR and NHEJ pathways were elegantly leveraged to confirm the in vitro activity of Ss-CYP-22a9 and its essentiality to the life stage responsible for infection.
- Animal studies convincingly reveal the synergistic effect of Δ7-DA and ivermectin in disseminated strongyloidiases. The burdens of intestinal larvae and adults in both uncomplicated and disseminated infection after treatment align with the standing model that Δ7-DA is acting against the intestinal larval stages (L3a) that are naturally deficient in the hormone.
- While there is precedent for combinatorial drug therapies in antifilarial control, there is great novelty in combining drugs that are known to target different stages as opposed to just different molecular targets. This provides the first clear demonstration of this as far as parasitic nematodes are concerned.
Weaknesses: Weaknesses are categorically minor.
- As the authors recognize, the animal studies required daily Δ7-DA dosing over a two-week period. While some of this is explained by a short half-life and poor pharmacokinetics, drug was continually delivered directly to the gut at high (uM) concentrations. This is a major hurdle to surmount and it is entirely possible that even if drugs with equivalent potency and more favorable pharmacokinetics were discovered, they would have to be administered in multiple dosing regimens or at prohibitively high concentrations to achieve a curative effect.
- While there is some evidence in other nematode clades that modulation of the DAF-12 pathway can affect developmental phenotypes, many of these parasites have huge phylogenetic separation from strongyloides and lack dormant stages requiring 'activation' or free-living stages. Given the independent evolution of parasitism across the phylum, it is just as likely that drugs acting on DAF-12 will have subtle (and not curative) effects in these other parasite systems.
-
Reviewer #2 (Public Review):
Mangelsdorf, Kliewer and colleagues here identified the endogenous ligand in Strongyloides stercoralis that governs that parasitic nematode's capacity to autoinfect its mammalian hosts. The ligand, [Delta]7-DA, interacts with nuclear receptor Ss-DAF-12, just as it does with its C. elegans ortholog, Ce-DAF-12, governing transcription of genes essential for metabolism and reproductive growth. Specifically, [Delta]7-DA appears to mediate a switch in DAF-12 function: in unfavorable conditions, [Delta]7-DA is absent and unliganded DAF-12 is said to arrest growth in both species and produces developmentally quiescent infective S. stercoralis larvae; in favorable conditions, the ligand is synthesized and the liganded DAF-12 triggers infection by S. stercoralis and subsequent development and reproduction in both …
Reviewer #2 (Public Review):
Mangelsdorf, Kliewer and colleagues here identified the endogenous ligand in Strongyloides stercoralis that governs that parasitic nematode's capacity to autoinfect its mammalian hosts. The ligand, [Delta]7-DA, interacts with nuclear receptor Ss-DAF-12, just as it does with its C. elegans ortholog, Ce-DAF-12, governing transcription of genes essential for metabolism and reproductive growth. Specifically, [Delta]7-DA appears to mediate a switch in DAF-12 function: in unfavorable conditions, [Delta]7-DA is absent and unliganded DAF-12 is said to arrest growth in both species and produces developmentally quiescent infective S. stercoralis larvae; in favorable conditions, the ligand is synthesized and the liganded DAF-12 triggers infection by S. stercoralis and subsequent development and reproduction in both species. The authors determined the ligand's biosynthetic pathway and showed by mutating the rate-limiting enzyme in the pathway that [Delta]7-DA is essential for parasite reproduction, whereas its absence is required for infectious larval development. In an animal model, they demonstrated that administration of [Delta]7-DA suppresses autoinfection and host lethality. Given in combination with an existing drug targeted to actively developing stages, [Delta]7-DA virtually cures the disease.
This work establishes a finding and an implication. The finding: gene circuits for growth and reproduction in nematode species with distinct life cycles -- parasitic vs free-living -- are regulated by a hormonal signal and cognate receptor that are structurally and functionally conserved. This is evolutionarily unremarkable, made mildly surprising because S. stercoralis lacks a cytochrome P450 with strong sequence identity to C. elegans DAF-9, which catalyzes the rate-limiting step in [Delta]7-DA synthesis in C. elegans; a screen of S. stercoralis P-450s demonstrated that Ss-CYP22a9 is the DAF-9 isozyme. The implication: targeting DAF-12 function (either agonism to block lethal hyperinfection or antagonism to prevent development of adult worms) or ligand synthesis may offer a therapeutic route to treating nematode parasitism. This is a valuable implication, identifying three therapeutic target approaches -- Ss-DAF-12 agonism or antagonism, and Ss-CYP22a9 inhibition -- that potentially might be be advanced from these pre-clinical observations. Overall, the manuscript makes a modest yet significant contribution.
Typical of the work from Mangelsdorf and Kliewer, the research plan and experiments are rigorously designed, executed and interpreted. My only quibble is that the requirement for Ss-DAF-12 (unliganded) to produce infectious L3i larvae is claimed (lines 165-167) but not directly demonstrated here. Instead, the authors depend on, and eventually cite in the Discussion (line 340) their nice PNAS paper earlier this year, which makes this case. Because of its importance in rounding out the implication noted above, my preference would be for the authors to add an experiment to this work that documents that Ss-DAF-12 is essential both pre- and post-ligand production.
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