Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi - PubMed (original) (raw)
Development of an in vivo RNAi protocol to investigate gene function in the filarial nematode, Brugia malayi
Chuanzhe Song et al. PLoS Pathog. 2010.
Abstract
Our ability to control diseases caused by parasitic nematodes is constrained by a limited portfolio of effective drugs and a paucity of robust tools to investigate parasitic nematode biology. RNA interference (RNAi) is a reverse-genetics tool with great potential to identify novel drug targets and interrogate parasite gene function, but present RNAi protocols for parasitic nematodes, which remove the parasite from the host and execute RNAi in vitro, are unreliable and inconsistent. We have established an alternative in vivo RNAi protocol targeting the filarial nematode Brugia malayi as it develops in an intermediate host, the mosquito Aedes aegypti. Injection of worm-derived short interfering RNA (siRNA) and double stranded RNA (dsRNA) into parasitized mosquitoes elicits suppression of B. malayi target gene transcript abundance in a concentration-dependent fashion. The suppression of this gene, a cathepsin L-like cysteine protease (Bm-cpl-1) is specific and profound, both injection of siRNA and dsRNA reduce transcript abundance by 83%. In vivo Bm-cpl-1 suppression results in multiple aberrant phenotypes; worm motility is inhibited by up to 69% and parasites exhibit slow-moving, kinked and partial-paralysis postures. Bm-cpl-1 suppression also retards worm growth by 48%. Bm-cpl-1 suppression ultimately prevents parasite development within the mosquito and effectively abolishes transmission potential because parasites do not migrate to the head and proboscis. Finally, Bm-cpl-1 suppression decreases parasite burden and increases mosquito survival. This is the first demonstration of in vivo RNAi in animal parasitic nematodes and results indicate this protocol is more effective than existing in vitro RNAi methods. The potential of this new protocol to investigate parasitic nematode biology and to identify and validate novel anthelmintic drug targets is discussed.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Dissemination and persistence of intrathoracically injected Cy 3-labelled Brugia malayi Cathepsin-L1 siRNAs in Aedes aegypti.
Midgut and Malpighian tubule tissues are shown in light (upper panel) and fluorescence (lower panel) micrographs from 1 to 9 days post-injection (scale bar 100 µm).
Figure 2. Concentration-dependent, in vivo suppression of Brugia malayi Cathepsin-L1 (Bm-cpl-1) using siRNA (Top) or dsRNA (Bottom) RNAi triggers.
Micrograph shows ethidium bromide stained agarose gel electrophoresis of relative RT-PCR analysis of individual, _B. malayi_-infected mosquitoes 48 h post-injection of RNAi trigger at 10 d post-infection. Amplified product for the target gene, _Bm_-cpl-1, is shown above a neuropeptide reference gene (Bm-flp-14).
Figure 3. Quantitative PCR demonstrates significant reduction in Bm-cpl-1 transcript levels as a result of siRNA and dsRNA RNAi trigger injection into _B. malayi_-infected Ae. aegypti.
Both siRNA (A) and dsRNA (B) injection reduces Bm-cpl-1 transcript by 83% compared to controls (saline injected Ae. aegypti infected with B. malayi). Bm-cpl-1 and control gene, Bm-flp-14, are normalized to a reference gene Bm-thp-1. qPCR was performed 48 h post-injection of RNAi trigger at 10 d post-infection. Each bar represents 13 mosquitoes from three biological replicates.
Figure 4. Aberrant motility of dsRNA _Bm-cpl-1_-exposed B. malayi.
Frequency distribution for motility of L3 stage B. malayi recovered from Ae. aegypti showing significantly reduced motility of Bm-cpl-1 suppressed worms. Parasitized mosquitoes were injected with saline (control), 150 ng eGFP dsRNA, or 150 ng Bm-cpl-1 dsRNA at 7 or 10 d post-infection (dpi), then dissected to obtain parasites at 14 dpi. Parasite motility was scored on a 1–5 scale, with 1 = immobile and 5 = all parts of worm in constant motion (control n = 101, ds_eGFP_ n = 68, ds_Bm-cpl-1_ 10 dpi n = 70, ds_Bm-cpl-1_ 7 dpi n = 48, P<0.001).
Figure 5. Disrupted motile phenotypes of dsRNA _Bm-cpl-1_-exposed B. malayi.
Frequency distribution for parasites exhibiting rigorous knotting behavior at both ends of worm, one end, or not at all showing normal terminal curvature is inhibited by Bm-cpl-1 suppression. Parasitized mosquitoes were injected with saline (control), 150 ng eGFP dsRNA, or 150 ng Bm-cpl-1 dsRNA at 7 or 10 d post-infection (dpi), then dissected to obtain parasites at 14 dpi (control n = 101, ds_eGFP_ n = 68, ds_Bm-cpl-1_ 10 dpi n = 70, ds_Bm-cpl-1_ 7 dpi n = 48).
Figure 6. The frequency of caudal paralysis and kinked posture of dsRNA _Bm-cpl-1_-exposed B. malayi.
The frequency of both caudal paralysis and kinked posture is significantly increased with Bm-cpl-1 suppression. Parasitized mosquitoes were injected with saline (control), 150 ng eGFP dsRNA, or 150 ng Bm-cpl-1 dsRNA at 7 or 10 d post-infection (dpi), then dissected to obtain parasites at 14 dpi (control n = 101, ds_eGFP_ n = 68, ds_Bm-cpl-1_ 10 dpi n = 70, ds_Bm-cpl-1_ 7 dpi n = 48, P<0.001).
Figure 7. dsRNA _Bm-cpl-1_-exposed B. malayi fail to migrate to the head of the mosquito.
Frequency distribution of infectious (L3) stage B. malayi recovered from Ae. aegypti in the head, thorax or abdomen of the mosquito host. Parasites were dissected from Ae. aegypti mosquitoes 14 d post-infection and 4 d post-injection of saline (control), 150 ng eGFP dsRNA or 150 ng Bm-cpl-1 dsRNA. One worm was recovered from each mosquito. Numbers of mosquitoes dissected from three biological replicates: n = 18 (control), n = 20 (ds_eGFP_), and n = 31 (ds_Bm-cpl_-1).
Figure 8. _Bm-cpl-1_-suppressed B. malayi are significantly shorter than control worms.
Parasites were dissected from Ae. aegypti mosquitoes 14 days post-infection and 4 d post-injection of saline (control, A) or 150 ng Bm-cpl-1 dsRNA (B). Scale bar 250 µm. (C) RNAi-exposed parasites are significantly shorter in length, but not width, than control worms. Numbers of parasites from three biological replicates: n = 19 (control) and n = 13 (ds_Bm-cpl_-1) (P<0.001).
Figure 9. Ae. aegypti survival significantly increases as infection prevalence decreases in _Bm-cpl-1_-dsRNA exposed mosquitoes.
The effects on survival (A) and infection prevalence (B) are most profound in parasites exposed to Bm-cpl-1 dsRNA at the L2/L3 transition (7 d post-infection, dpi). Parasites were exposed to saline (control), 150 ng eGFP dsRNA or 150 ng Bm-cpl-1 dsRNA at 7 or 10 dpi, then were dissected from Ae. aegypti mosquitoes at 14 dpi.
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