Recent advances in functional genomics for parasitic nematodes of mammals

Abstract

Human-parasitic nematodes infect over a quarter of the world's population and are a major cause of morbidity in low-resource settings. Currently available treatments have not been sufficient to eliminate infections in endemic areas, and drug resistance is an increasing concern, making new treatment options a priority. The development of new treatments requires an improved understanding of the basic biology of these nematodes. Specifically, a better understanding of parasitic nematode development, reproduction and behavior may yield novel drug targets or new opportunities for intervention such as repellents or traps. Until recently, our ability to study parasitic nematode biology was limited because few tools were available for their genetic manipulation. This is now changing as a result of recent advances in the large-scale sequencing of nematode genomes and the development of new techniques for their genetic manipulation. Notably, skin-penetrating gastrointestinal nematodes in the genus Strongyloides are now amenable to transgenesis, RNAi and CRISPR/Cas9-mediated targeted mutagenesis, positioning the Strongyloides species as model parasitic nematode systems. A number of other mammalian-parasitic nematodes, including the giant roundworm Ascaris suum and the tissue-dwelling filarial nematode Brugia malayi, are also now amenable to transgenesis and/or RNAi in some contexts. Using these tools, recent studies of Strongyloides species have already provided insight into the molecular pathways that control the developmental decision to form infective larvae and that drive the host-seeking behaviors of infective larvae. Ultimately, a mechanistic understanding of these processes could lead to the development of new avenues for nematode control.

Keywords: CRISPR; Parasitic helminth; Parasitic nematode; RNAi; Strongyloides; Transgenesis.

Fig. 1.

The life cycles of parasitic nematode species that are currently models for genetic transformation. (A) The life cycle of the skin-penetrating gastrointestinal nematodes Strongyloides stercoralis and Strongyloides ratti. Parthenogenetically reproducing parasitic adult females reside in the host intestinal tract. Parasitic females excrete eggs (in the case of S. ratti) and larvae (in the case of S. stercoralis) into the environment in host feces. Post-parasitic L1 larvae then follow one of two distinct developmental trajectories: (1) direct development into infective third-stage larvae (iL3s) that must infect a host to continue the life cycle (blue arrows) or (2) indirect development through a single free-living generation in the environment (red arrows). Free-living male and female adults mate, and all of their progeny develop into iL3s that must infect a host. Free-living adult females of S. stercoralis and S. ratti are amenable to gonadal microinjection techniques and thus are well suited for genetic intervention. For simplicity, the indirect developmental cycle does not show post-parasitic L2–L4 larval stages or post-free-living L1–L2 larval stages. (B) The life cycle of the human-parasitic filarial nematode Brugia malayi. Male and female parasitic adults reside in the host lymphatic system. After mating, parasitic females release microfilariae that enter the host's bloodstream. A mosquito vector takes a bloodmeal from the infected host, resulting in ingestion of microfilariae. The microfilariae develop into L3s within the mosquito vector. When the mosquito takes another bloodmeal, the L3s are deposited into a new host, resume development, and eventually become male and female parasitic adults. Genetic transformation has been achieved in B. malayi female parasitic adults, microfilariae and L3s using a variety of different techniques including microinjection, microparticle bombardment and chemical transformation.

Fig. 2.

Transfection of B. malayi using DNA–liposome complexes. (A) Schematic diagram of the transfection procedure used to make transgenic B. malayi L3s. Each well contained a layer of feeder bovine embryo skeletal muscle cells, a transwell insert and ∼100 B. malayi L3s in RPMI media. Lipofectamine was added to plasmid DNA to produce DNA–lipid complexes (micelles), and the complexes were then added to the larvae in the wells. Every day for 8 days, the medium was changed and fresh DNA–lipid complex was added. On day 5, ascorbic acid was included to induce molting, as molting larvae are more likely to take up DNA. On day 8, worms were collected and screened for transgene expression. (B) Fluorescence images of B. malayi L3s expressing green fluorescent protein (GFP), yellow fluorescent protein (YFP) or cherry red (CHR) under the control of a ubiquitous B. malayi promoter (adapted from Liu et al., 2018). Scale bar: 300 nm.

Fig. 3.

RNAi-mediated knockdown of daf-12 in S. ratti defines a role for daf-12 in infective larval development. (A) Schematic diagram of early-stage RNA interference (RNAi) treatment of S. ratti. Post-parasitic L1 larvae were isolated and incubated in RNAi culture medium with either small interfering RNA (siRNA) targeting the S. ratti daf-12 gene or a scramble control. Following incubation, nematodes were transferred to agar plates and their developmental trajectory was assessed. (B) Early-stage RNAi suppressing S. ratti daf-12 reduces the proportion of nematodes that undergo direct development into iL3s. **P<0.001, Mann–Whitney test. (C) Schematic diagram of late-stage RNAi treatment of S. ratti. A mixed culture of free-living larvae and adults was incubated in RNAi culture medium with either siRNA targeting S. ratti daf-12 or a scramble control. Following incubation, adults were transferred to agar plates supplemented with Escherichia coli HB101 to grow and reproduce. iL3 progeny were then collected and used to infect rat hosts. (D) Late-stage RNAi suppressing S. ratti daf-12 results in a decreased worm burden in feces collected from infected rat hosts over the course of 7–17 days post-infection. **P<0.001, Student's t-test. (B and D adapted from Dulovic and Streit, 2019.)

Fig. 4.

CRISPR/Cas9-mediated targeted mutagenesis in S. stercoralis. (A) A pipeline for CRISPR/Cas9-mediated targeted mutagenesis in S. stercoralis. Individual plasmid vectors for Cas9, a single guide RNA (sgRNA) for the target of interest and a repair template for homology-directed repair (HDR) encoding an mRFPmars reporter are microinjected into S. stercoralis free-living adult females. iL3 progeny are screened for expression of the reporter indicating possible disruption of the target locus. Individual iL3 progeny are then tested for phenotypes. The iL3s are genotyped post hoc for disruption of the gene of interest by splitting the genomic DNA from each iL3 into four equal PCR reactions: C, control reaction confirming successful genomic DNA isolation; wt, reaction amplifying the wild-type locus of interest; 5′, reaction for HDR at the 5′ border of cassette integration; 3′, reaction for HDR at the 3′ border of cassette integration. 5′ and 3′ reactions only amplify if a successful HDR event has occurred at the target. Note the absence of a wt band for the knockout iL3 shown, suggesting a homozygous disruption of the gene of interest for this iL3. (B) Targeted mutagenesis of S. stercoralis unc-22. Free-living females were injected with CRISPR/Cas9 constructs targeting unc-22 and F₁ iL3s were collected. F₁ iL3s were subjected to 1% nicotine treatment, which induces twitching in unc-22 iL3s but results in paralysis in wild-type iL3s. Approximately 40% of F₁ iL3s displayed nicotine-induced twitching following CRISPR/Cas9 targeting of unc-22. Twitching, mRFPmars-expressing iL3s were collected and genotyped post hoc to confirm HDR at the unc-22 target locus. As a control, the Cas9 construct was omitted from the pipeline shown in A to generate ‘no-Cas9 control’ iL3s; no nicotine-induced twitching was observed in these F₁ iL3s. ****P<0.0001, Fisher's exact test. (C) Targeted mutagenesis of S. stercoralis tax-4. Free-living adult females were injected with CRISPR/Cas9 constructs targeting tax-4, and mRFPmars-expressing F₁ iL3s were collected. Individual iL3s were then placed in a thermal gradient and allowed to crawl freely. The tax-4 iL3s failed to migrate up the thermal gradient. By contrast, the no-Cas9 control iL3s engaged in robust positive thermotaxis. ****P<0.0001, Mann–Whitney test. (A and B adapted from Gang et al., 2017; C adapted from Bryant et al., 2018.)