Nuclear hormone receptors (NHRs) are emerging target candidates against nematode infection and resistance. However, there is a lack of comprehensive information on NHR-coding genes in parasitic nematodes. In this study, we curated the nhr gene family for 60 major parasitic nematodes from humans and animals. Using a high-throughput RNA interference platform, we knocked down 43 nhr genes of H. contortus and identified at least two genes that are required for the viability (i.e., nhr-105) and development (i.e., nhr-17) of the infective larvae of this parasitic nematode in vitro. Harnessing this preliminary functional atlas of nhr genes for H. contortus will prime the biological studies of this gene family in nematode genetics, infection, and anthelmintic metabolism within host animals, as well as the promising discovery of novel intervention targets.
Methods
RNAi: A soaking method was used for the small interfering RNA (siRNA)- mediated gene knockdown assay in H. contortus (Khan et al., 2020). siRNAs targeting specific nhr gene were designed using the BLOCK-iT™ RNAi Designer (https://rnaidesigner.thermofisher.com/rnaiexpress/) and checked for specificity by BLASTN searching against H. contortus gene sets. Gene-specific siRNA was synthesised and packed with a cationic lipid formulation Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Packed siRNA (200 pM) was added into the DMEM containing xL3s of H. contortus, 10% sheep serum and 1× antibiotic-antimycotic (Thermo Fisher Scientific, USA), and incubated for 24, 48 or 72 h, at 38 ◦C and 10% v/v CO2. qRT-PCR was used for gene knockdown analysis of RNAi treated larvae as described above.
Motility assay Motility of RNAi-treated and untreated larvae were measured using an established method (Herath et al., 2022; Taki et al., 2021). In brief, xL3s of H. contortus were dispensed into 96-well cell culture plates at a density of 300 worms per well, and incubated in DMEM (Thermo Fisher Scientific, USA) containing 10% sheep serum, 1× antibiotic-antimycotic (Thermo Fisher Scientific, USA), lipofectamine-packed siRNAs targeting specific nhr gene (test) or same volume of DMEM (control) for 48 h at 38 ◦C and 10% v/v CO2. The larval motility of test and control groups (four replicates were included in each group) was measured using the WMicrotracker ONE in vitro biosystem (Phylumtech, Argentina), according to the manufacturer’s instructions. Death ratios of RNAi-treated and -untreated larvae were calculated after 72 h of incubation. Motility and death ratio of RNAi-treated larvae were calculated and normalised to that of untreated larvae.
Results
Genome-wide RNAi indicates two nhr genes crucial for larval viability and development. We screened nhr genes that are important for larval viability and development, by soaking the xL3s of H. contortus with specific siRNA targeting certain nhr gene in vitro. RNAi-mediated gene knockdown was detected for 43 nhr genes in this parasitic nematode (P ≤ 0.05; Fig. 4A), resulting in phenotypes of decreased motility (e.g., nhr-105, nhr-119 and nhr-173) or larval development (e.g., nhr-17, nhr-25.2 and nhr-121). In particular, compared with untreated larvae, RNAi (nhr-105) resulted in a 60% decrease of larval motility after 24 h of treatment in vitro (Fig. 4B), whereas RNAi (nhr-17) resulted in a 55% compromise of the development ratio from xL3s to L4s in vitro after seven days of treatment (Fig. 4C). Neither the markedly decreased larval motility nor the significantly compromised larval development was a result of worm death (Supplementary Fig. 2). Compared with negative control, transcript levels of several nhr genes were significantly upregulated after soaking with specific siRNA. These nhr genes include nhr-14 (P ≤ 0.001), nhr-43 (P ≤ 0.05), fax-1 (P ≤ 0.05), unc-55 (P ≤ 0.001), and HCON_00107920 (P ≤ 0.01) (Fig. 4A). However, no obvious phenotypic changes in terms of larval viability or development were found.
Using barber’s pole worm as a gastrointestinal nematode model, we demonstrated that some nhr genes are responsive to host serum and/or anthelmintic treatment, including nhr-64 which might be involved in anthelmintic stress response, nhr-105 that was demonstrated important in larval motility, and nhr-17 that was required for larval development. These novel findings provide key molecules involved in drug resistance and target candidates for drug discovery.
Infection, Genetics and Evolution, Volume 122, 2024, https://doi.org/10.1016/j.meegid.2024.105609
Zhendong Du, Danni Tong, Xueqiu Chen, Fei Wu, Shengjun Jiang, Jingju Zhang, Yi Yang, Rui Wang, Sambuu Gantuya, Tserennyam Davaajargal, Sukhbaatar Lkhagvatseren, Zayat Batsukh, Aifang Du, Guangxu Ma