key: cord-0256013-twoh0t19
authors: Wang, Zhu; Cheong, Mi Cheong; Tsien, Jet; Deng, Heping; Qin, Tian; Stoltzfus, Jonathan D. C.; Jaleta, Tegegn G.; Li, Xinshe; Lok, James B.; Kliewer, Steven A.; Mangelsdorf, David J.
title: Characterization of the Endogenous DAF-12 Ligand and Its Use as an Anthelmintic Agent in Strongyloides stercoralis
date: 2021-09-08
journal: bioRxiv
DOI: 10.1101/2021.09.07.459359
sha: 439a2aceb605e2057191c87db18f932646ebdaa1
doc_id: 256013
cord_uid: twoh0t19

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 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.

In C. elegans, favorable environmental cues induce DAF-9 expression and DAF-12 ligand 211 synthesis through a cGMP signaling pathway, leading to reproductive development (Hu, 2007) . 212 Likewise in S. stercoralis, favorable host conditions act through a similar pathway to stimulate 213 feeding, a sign of reactivated development in L3i (Stoltzfus et al., 2014) . To study the role of D7-214 DA synthesis in parasite reproductive development, we first asked whether D7-DA is made in 215 and then tested endogenous D7-DA levels in the L3i larvae. In the presence of 8-Br-cGMP, we 218 found that endogenous D7-DA was increased progressively over a 3-day period to levels (~100 219 nM) that would saturate Ss-DAF-12 occupancy ( Figure 5A ). Furthermore, this stimulation of D7- Figure 5B ). To evaluate the phenotype due to loss of Ss-CYP22a9, we assayed cGMP-induced 231 feeding behavior in L3i, which as noted above is an early marker of the transition to reproductive 232 development (Lok, 2007; Stoltzfus et al., 2014; Viney & Lok, 2015) . In both wildtype larvae and 233 CRISPR-control larvae in which a gene unrelated to development (Ss_unc-22) was targeted, 8-Br-234 cGMP strongly induced the feeding behavior as expected ( Figure 5C ). In contrast, in Ss-cyp22a9 function of Ss-CYP22a9, we also analyzed the production of D7-DA in Ss-cyp22a9 knockout L3i 241 larvae. For this experiment, we targeted the Ss-cyp22a9 gene using non-homologous end joining 242 CRISPR with the same sgRNA as in Figure 5C and In S. stercoralis, uncomplicated infections are frequently asymptomatic but can last for 251 years due to the persistence of L3a worms that escape the host immune system and continually re-252 infect the host at a low level. However, when the host is immune suppressed and the intestinal 253 barrier is compromised, autoinfection accelerates out of control as more and more L3a worms 254 invade the host, leading to a lethal hyperinfection. The results above suggested that targeting the 255 DAF-12 pathway might provide a novel therapeutic opportunity for treating this disease. This 256 strategy is based on the idea that like their L3i counterparts in the environment, the lack of D7-DA 257 is required for intestinal larvae to become infectious L3a ( Figure 3C) vehicle versus 32 ± 6 for DA, q < 0.03; Figure 6A ). In contrast, the numbers of parasitic adults, 270 which produce intestinal larvae, were not significantly changed by D7-DA treatment ( Figure 6B ). Similar to what is observed in immunocompromised humans, this results in the rapid progression 286 of disease to a disseminated hyperinfection that is lethal to all of the animals, with >90% of them 287 dying within 40 days ( Figure 7A , black line). In contrast, when a matched group of these animals 288 was treated with D7-DA starting at 10 days after the hyperinfection was initiated, there was an 289 impressive 70% survival rate that lasted for at least 90 days at which point we arbitrarily terminated An intriguing aspect of this study was the finding that D7-DA is the endogenous ligand for 377 Ss-DAF12. Although D7-DA was first identified as the endogenous DAF-12 ligand in C. elegans, 378 its presence in S. stercoralis was unexpected. In comparison to its C. elegans homolog, the Ss- The findings discussed above underscore the potential of developing agonists that would 398 precociously activate unliganded Ss-DAF-12 and kill L3a larvae, a therapy that would be 399 propitious and specific to S. stercoralis. However, the observation that liganded DAF-12 also is 400 required at a completely different but essential stage of the lifecycle to promote reproductive 401 growth raises the possibility of also targeting DAF-12 with an antagonist. Given that DAF-12 has 23  21  19  17  15  13  11  9  7  5  3  1  47  45  43  41  39  37  35  33  31  29  27  25  69  67  65  63  61  59  57  55  53  51 (A) Ss-cyp22a9 mRNA expression is increased by cGMP in L3i larvae. L3i larvae were treated as in Figure 5 and Ss-cyp22a9 mRNA levels were measured by qPCR and compared to 18S rRNA levels. Results expressed as means ± S.D. from technical triplicates. *, p < 0.01 by t-test. Duplicate experiments were performed with similar results. (B) CRISPR knockout strategy for targeting Ss-cyp22a9 in S. stercoralis: Free-living adult females were microinjected with CRISPR targeting vectors (for either NHEJ or HDR gene editing) and allowed to mate. F1 progeny were grown to the L3i stage and GFP-positive worms were phenotyped and genotyped. (C) Homology-directed repair targeting vector for Ss-cyp22a9 CRISPR. Red line indicates position of the insertion repair template in the P450 domain of the Ss-cyp22a9 gene (SSTP_0001032100). The guide sequence and PAM site (underlined) are shown. The recombinant locus expresses a GFP marker from the Sr-eef-1 promoter fused to the 3'-UTR of the Ss-era-1 gene. F, R, forward and reverse primers, respectively; HA, homology arms. WT and recombinant alleles produce 1052 bp and 823 bp products with F1/R1 and F1/R2, respectively. feeding behaviors were then visualized as described above and genotyped individually by single 782 worm PCR with the primers listed in Table 3 .

In a second set of experiments, NHEJ CRISPR was used to produce enough Ss-cyp22a9 784 knock-out worms for detection of endogenous D7-DA. As described above, 50 free-living females of S. stercoralis were microinjected with plasmids expressing Cas9 (pPV540, 20 ng/ul) and 786 sgRNAs against Ss-cyp22a9 or Ss-unc-22 (60 ng/ul). Following a fecal culture with free-living 787 males at 25⁰C for 7 days, the resulting L3i larvae were collected by the Baermann technique and 788 treated by 0.5 mM 8-Br-cGMP in M9 buffer for 3 days. Levels of D7-DA were analyzed by UPLC-789 MS as described above. 

RNAseq analysis of 967 the parasitic nematode Strongyloides stercoralis reveals divergent regulation of canonical 968 dauer pathways

The biology of Strongyloides spp

Nuclear receptors: 973 emerging drug targets for parasitic diseases

The nuclear receptor DAF-12 regulates nutrient metabolism and 977 reproductive growth in nematodes

Identification of the nuclear receptor DAF-12 as a therapeutic 982 target in parasitic nematodes

A novel 3-hydroxysteroid dehydrogenase that 986 regulates reproductive development and longevity

The Rieske oxygenase DAF-36 functions as 990 a cholesterol 7-desaturase in steroidogenic pathways governing longevity

The somatic reproductive tissues of C. elegans promote 994 longevity through steroid hormone signaling

Nuclear Receptors: Recent Drug Discovery for 997

This work was supported 1004 by the National Institutes of Health (grant AI105856 to

UT Southwestern Eugene McDermott Scholarship 1007 (T.Q.), and the Howard Hughes Medical Institute

AUTHOR CONTRIBUTIONS

conceived and designed the study

optimized the synthesis and provided 1012 dafachronic acids