Fig 1.
Hypothesized regulation of the Strongyloides stercoralis life cycle by the nuclear hormone receptor DAF-12.
The S. stercoralis parasitic female (P Female) produces larval progeny by mitotic parthenogenesis, and these progeny have several possible developmental fates. A female post-parasitic first-stage larva (PP L1) can either precociously develop inside the host to an autoinfective third-stage larva (L3a), which develops to a second-generation parasitic female, or be passed in the feces to develop outside the host by one of two routes: a homogonic route directly to a developmentally arrested infectious third-stage larva (iL3), which is favored at host-like temperatures (e.g., 37°C), or a heterogonic route to a free-living adult female (FL Female), which is favored at lower temperatures (e.g., 22°C). We hypothesize that this developmental checkpoint is regulated by dafachronic acid ligands for the nuclear hormone receptor Ss-DAF-12, with liganded Ss-DAF-12 favoring heterogonic development. Larval progeny of the single free-living generation of females and males invariably form iL3, and this developmental arrest is hypothesized to be governed by the absence of Ss-DAF-12 signaling. Once inside a host, the third-stage larva resumes development and feeding, resulting in a form designated the L3+. We hypothesize that resumption of development by iL3 entering the host and maturation to the P Female requires an increase in signaling by Ss-DAF-12, stimulated by increased biosynthesis of its steroid ligand.
Fig 2.
Δ7-Dafachronic acid regulates the developmental switch controlling the homogonic or heterogonic fates of post-parasitic female larvae of Strongyloides stercoralis.
The frequency distribution of homogonically (red bar) and heterogonically (green bars) developing S. stercoralis post-parasitic females in cultures maintained at 22°C or at 37°C in the presence or absence of 1 μM Δ7-dafachronic acid (Δ7-DA) was plotted. Data are based on counts of worms, explanted to culture as hatchling first-stage larvae from the intestines of experimentally infected gerbils, scored for development at 24 and 48 hours of culture and assigned to one of two developmental classes representing larvae developing by the homogonic route to infectious third-stage larvae (iL3) or larvae developing via the heterogonic route to free-living females via rhabditiform second, third, and fourth larval stages (L4-FL Adult). Post-parasitic males were excluded from the analysis, as males only develop heterogonically. The bar height represents the mean of five biological replicates and the error bar +1 standard deviation. Brackets indicate statistical comparisons of iL3 frequency at 37°C to frequencies at both 22°C and 37°C with 1 μM Δ7-DA; *** indicates P < 0.001.
Fig 3.
Exogenous Δ7-dafachronic acid promotes formation of Strongyloides stercoralis second-generation free-living larvae and blocks the formation of infective third-stage larvae.
Post-free-living larvae of S. stercoralis developing synchronously in agar plate cultures were exposed to increasing concentrations of Δ7-dafachronic acid (Δ7-DA). A) Frequency of development to rhabditiform post-free-living third- and fourth-stage larvae (FL L3-L4) as a function of Δ7-DA concentration. The sample denoted 0 nM Δ7-DA is the ethanol control. ** Positive correlation of FL L3-L4 frequency with Δ7-DA concentration is significant (P = 0.001; R2 = 0.902). B) Frequency of development to infective third-stage larvae (iL3) as a function of Δ7-DA concentration, with 0 nM Δ7-DA as the ethanol control. Negative correlation of iL3 frequency with Δ7-DA concentration is significant (P = 0.0135; R2 = 0.736). In both panels, bar height represents the mean of three biological replicates; error bars represent +1 standard deviation.
Fig 4.
Formation of second-generation free-living larvae in Strongyloides stercoralis requires 24–48 hours of exposure to Δ7-dafachronic acid.
Frequency of development to second-generation free-living rhabditiform L3 and L4 (FL L3-L4) in S. stercoralis was plotted as a function of duration of exposure to increasing concentrations of Δ7-dafachronic acid (Δ7-DA). Ascending shaded triangles indicate that developmental frequencies were determined at Δ7-DA concentrations of 125 nM (lightest), 250 nM, 500 nM, and 1,000 nM (darkest). Bar height represents the mean of three biological replicates; error bars represent +1 standard deviation. The overall effects of Δ7-DA exposure duration and concentration were significant, P < 0.0001. For all possible pairwise statistical comparisons of FL L3-L4 frequency with DA exposure durations of 24, 48, and 72 hours, ** P < 0.01; *** P < 0.001.
Fig 5.
Ketoconazole inhibits developmental activation of Strongyloides stercoralis iL3 in host-like culture conditions; this inhibition is rescued by Δ7-dafachronic acid.
Frequency of feeding by cultured larvae, as reflected by ingestion of fluorescein isothiocyanate (FITC), was used as an index of developmental activation of infectious third-stage larvae (iL3). Larval mortality was scored as the percentage of larvae classed as non-motile and therefore “dead.” (A) Frequency of iL3 feeding and larval mortality as a function of ketoconazole concentration in Dulbecco’s modified Eagle’s Medium (DMEM), a permissive culture medium. DMEM without ketoconazole was the positive control. M9 buffer, a non-permissive medium, was used without ketoconazole as the negative control. ** Negative correlation of iL3 feeding and ketoconazole concentration (bracketed) is significant (P = 0.0028; Spearman r = -1.000). (B) Frequency of iL3 feeding and larval mortality as a function of Δ7-dafachronic acid (Δ7-DA) concentration in DMEM cultures containing an inhibitory concentration of ketoconazole (35 μM). Cultures of larvae in DMEM alone or in DMEM with 800 nM Δ7-DA were positive controls. Cultures of larvae in M9 buffer constituted the negative control. * Positive correlation of iL3 feeding in 35 μM ketoconazole and Δ7-DA concentration (bracketed) is significant (P = 0.0167; Spearman r = 1.000). In both panels, bar heights represent the mean percentage of iL3 feeding (blue bars) or the mean percentage of dead iL3 (white bars) in four biological replicates. Error bars indicate +1 standard deviation.