Albarqi Mennatallah M Y, Stoltzfus Jonathan D, Pilgrim Adeiye A, Nolan Thomas J, Wang Zhu, Kliewer Steven A, Mangelsdorf David J, Lok James B
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Department of Biology, Hollins University, Roanoke, Virginia, United States of America.
PLoS Pathog. 2016 Jan 4;12(1):e1005358. doi: 10.1371/journal.ppat.1005358. eCollection 2016 Jan.
The complex life cycle of the parasitic nematode Strongyloides stercoralis leads to either developmental arrest of infectious third-stage larvae (iL3) or growth to reproductive adults. In the free-living nematode Caenorhabditis elegans, analogous determination between dauer arrest and reproductive growth is governed by dafachronic acids (DAs), a class of steroid hormones that are ligands for the nuclear hormone receptor DAF-12. Biosynthesis of DAs requires the cytochrome P450 (CYP) DAF-9. We tested the hypothesis that DAs also regulate S. stercoralis development via DAF-12 signaling at three points. First, we found that 1 μM Δ7-DA stimulated 100% of post-parasitic first-stage larvae (L1s) to develop to free-living adults instead of iL3 at 37°C, while 69.4±12.0% (SD) of post-parasitic L1s developed to iL3 in controls. Second, we found that 1 μM Δ7-DA prevented post-free-living iL3 arrest and stimulated 85.2±16.9% of larvae to develop to free-living rhabditiform third- and fourth-stages, compared to 0% in the control. This induction required 24-48 hours of Δ7-DA exposure. Third, we found that the CYP inhibitor ketoconazole prevented iL3 feeding in host-like conditions, with only 5.6±2.9% of iL3 feeding in 40 μM ketoconazole, compared to 98.8±0.4% in the positive control. This inhibition was partially rescued by Δ7-DA, with 71.2±16.4% of iL3 feeding in 400 nM Δ7-DA and 35 μM ketoconazole, providing the first evidence of endogenous DA production in S. stercoralis. We then characterized the 26 CYP-encoding genes in S. stercoralis and identified a homolog with sequence and developmental regulation similar to DAF-9. Overall, these data demonstrate that DAF-12 signaling regulates S. stercoralis development, showing that in the post-parasitic generation, loss of DAF-12 signaling favors iL3 arrest, while increased DAF-12 signaling favors reproductive development; that in the post-free-living generation, absence of DAF-12 signaling is crucial for iL3 arrest; and that endogenous DA production regulates iL3 activation.
寄生线虫粪类圆线虫复杂的生命周期会导致感染性第三期幼虫（iL3）发育停滞或发育为可繁殖成虫。在自由生活的线虫秀丽隐杆线虫中，滞育停滞和生殖生长之间的类似决定是由一类类固醇激素即法尼醇（DAs）控制的，这类激素是核激素受体DAF - 12的配体。DAs的生物合成需要细胞色素P450（CYP）DAF - 9。我们从三个方面验证了DAs也通过DAF - 12信号通路调节粪类圆线虫发育这一假设。首先，我们发现1 μM Δ7 - DA能刺激100%的寄生后第一期幼虫（L1s）在37°C时发育为自由生活成虫而非iL3，而对照组中69.4±12.0%（标准差）的寄生后L1s发育为iL3。其次，我们发现1 μM Δ7 - DA可防止自由生活后的iL3停滞，并刺激85.2±16.9%的幼虫发育为自由生活的杆状第三和第四阶段，而对照组为0%。这种诱导需要Δ7 - DA暴露24 - 48小时。第三，我们发现CYP抑制剂酮康唑在类似宿主的条件下可阻止iL3取食，在40 μM酮康唑中只有5.6±2.9%的iL3取食，而阳性对照组为98.8±0.4%。这种抑制作用可被Δ7 - DA部分挽救，在400 nM Δ7 - DA和35 μM酮康唑中71.2±16.4%的iL3取食，这为粪类圆线虫内源性DA的产生提供了首个证据。然后我们对粪类圆线虫中26个编码CYP的基因进行了表征，并鉴定出一个与DAF - 9序列和发育调控相似的同源物。总体而言，这些数据表明DAF - 12信号通路调节粪类圆线虫的发育，表明在寄生后一代中，DAF - 12信号通路缺失有利于iL3停滞，而DAF - 12信号通路增强有利于生殖发育；在自由生活后一代中，DAF - 12信号通路缺失对iL3停滞至关重要；内源性DA的产生调节iL3的激活。
