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Effect of in vitro gene silencing on the microfilariae released by female B. malayi adult worm. a, b depict the healthy and motile microfilariae of medium control (a) and negative control (scrambled siRNA) (b), dead and severely sluggish microfilariae released by siRNA1 (c), siRNA2 (d), siRNA1 + 2-treated female parasites in vitro (e)
Source publication
Background
Galactofuranose is an essential cell surface component present in bacteria, fungi and several nematodes such as Caenorhabditis spp., Brugia spp., Onchocerca spp. and Strongyloides spp. This sugar maintains the integrity of parasite surface and is essential for virulence. UDP-Galactopyranose mutase (bmugm) plays a key role in Galf biosynt...
Contexts in source publication
Context 1
... to ~84-86% at 72 h and ~90% after further 48 h of transfer to siRNA- free medium ( Table 5). As expected, targeting of bmugm by two distinct sequences specific siRNAs at any time point proved to be marginally better. No off- target effects were noticed in presence of scrambled siRNA. Mf released in control cultures were healthy, actively motile (Fig. 4a, b) unlike those targeted specif- ically. Significant reduction (65-75%) in the motility of released mf was observed within 24 h which was exem- plified with increased exposure time (>90% after 48 h). Majority of mf released at 72 h were dead (Fig. 4c-e and Table 6) with visible phenotypic deformities ( Fig. 5a-d) as compared to intact mf ...
Context 2
... in presence of scrambled siRNA. Mf released in control cultures were healthy, actively motile (Fig. 4a, b) unlike those targeted specif- ically. Significant reduction (65-75%) in the motility of released mf was observed within 24 h which was exem- plified with increased exposure time (>90% after 48 h). Majority of mf released at 72 h were dead (Fig. 4c-e and Table 6) with visible phenotypic deformities ( Fig. 5a-d) as compared to intact mf from controls (Fig. ...
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Citations
... Según los estudiosde Spracklin et al. (2017), el efecto del ARNi se hereda hasta por cinco generaciones en C. elegans.El silenciamiento de genes a través del ARNi es muy utilizado en un número importante de parásitos de interés veterinario y humano. Dentro de ellos, Ascaris suum(McCoy et al., 2015), Brugia malayi(Misra, Gupta, & Misra-Bhattacharya, 2017), Leishmania spp.(Brettmann et al., 2016), Fasciola hepatica(Dell'Oca et al., 2014), Entamoeba histolytica(Khalil, Foda, Suresh, & Singh, 2016), Schistosoma mansoni(Gava et al., 2017), Strongyloides stercoralis(Lei et al., 2017) y Toxocara canis(Ma et al., 2015). Parásitos con informes de silenciamiento de genes.En H. contortus el silenciamiento de genes es una herramienta muy utilizada. ...
... Unlike trematode parasites, RNAi in nematode is seen with a lack of visible phenotypic or morphological changes and variation in susceptibility of certain target genes, besides, some worm like Heligmosomoides polygyrus, murine intestinal nematode, are refractile to RNAi pathways (Lendner et al. 2008). RNAi has been validated in Nippostrongylus braziliensis (Hussein et al. 2002), Brugia malayi (Aboobaker and Blaxter 2003;Ford et al. 2009;Song et al. 2010;Misra et al. 2017), Onchocercus volvulus (Lustigman et al. 2004;Ford et al. 2005), Trichostrongylus colubriformis (Issa et al. 2005), Oestertagia ostertagi , Haemonchus contortus Kotze and Bagnall 2006;Samarasinghe 2010;Naqvi et al. 2020) and Ascaris suum (Xu et al. 2010;Chen et al. 2011;McCoy et al. 2015). Most of these studies used dsRNA as RNAi inducer in animal and human parasitic nematode in vitro and in vivo as well. ...
Screening of vaccine or drug target in parasitic helminth is hindered by lack of robust tool for functional studies of parasite protein which account for the availability of only a few anti-helminthic vaccines, diagnostic assay and slower pace of development of an anthelmintic drug. With the piling up of parasite transcriptomic and genomic data, in silico screening for possible vaccine/drug target could be validated by functional characterization of proteins by RNA interference or CRISPR/Cas9. These reverse genetic engineering tools have opened up a better avenue and opportunity for screening parasitic proteins in vitro as well as in vivo. RNA interference provides a technique for silencing targeted mRNA transcript for understanding a gene function in helminth as evidence by work in Caenorhabditis elegans. Recent genetic engineering tool, CRISPR/Cas9 allows knock-out/deletion of the desired gene in parasitic helminths and the other provision it provides in terms of gene knock-in/insertion in parasite genome is still to be explored in future. This manuscript discussed the work that has been carried out on RNAi and CRISPR/Cas9 for functional studies of helminth parasitic proteins.
... In the case of S. ratti, RNAi-enhancing genes, double-stranded DNA uptake-associated genes, and other RNAi-affiliated genes have not been identified [103]. To bypass these complications, a small interfering RNA soaking method devised in Brugia malayi has been adopted and utilized in S. ratti to knock down the phenotypically well-defined daf-12 gene [103,104]. Thus, RNAi provides a promising alternative to the CRISPR/Cas9 system for studying gene function and will be particularly useful in situations when large numbers of iL3s are required, genes of interest lack efficient Cas9 target sites, functional redundancy among genes necessitates the simultaneous knockdown of large numbers of genes, and/or complete knockout of a gene is lethal. ...
The oft-neglected human-parasitic threadworm, Strongyloides stercoralis, infects roughly eight percent of the global population, placing disproportionate medical and economic burden upon marginalized communities. While current chemotherapies treat strongyloidiasis, disease recrudescence and the looming threat of anthelminthic resistance necessitate novel strategies for nematode control. Throughout its life cycle, S. stercoralis relies upon sensory cues to aid in environmental navigation and coordinate developmental progression. Odorants, tastants, gases, and temperature have been shown to shape parasite behaviors that drive host seeking and infectivity; however, many of these sensory behaviors remain poorly understood, and their underlying molecular and neural mechanisms are largely uncharacterized. Disruption of sensory circuits essential to parasitism presents a promising strategy for future interventions. In this review, we describe our current understanding of sensory behaviors – namely olfactory, gustatory, gas sensing, and thermosensory behaviors – in Strongyloides spp. We also highlight the ever-growing cache of genetic tools optimized for use in Strongyloides that have facilitated these findings, including transgenesis, CRISPR/Cas9-mediated mutagenesis, chemogenetic neuronal silencing, and the use of fluorescent biosensors to measure neuronal activity. Bolstered by these tools, we are poised to enter an era of rapid discovery in Strongyloides sensory neurobiology, which has the potential to shape pioneering advances in the prevention and treatment of strongyloidiasis.
... For each time point, we incubated 5 adult female worms for 24 h in an equal mixture of the siRNAs (Bma-LAD-2, Bma-Fukutin, Bma-ShTK, and Bma-Serpin) or one siRNA (Bma-EGF-like-02820, Bma-EGF-like-48010, Bma-Peptidase, and Bma-Reprolysin) at a total concentration of 5 mM in 850 mL of medium in a 5,000 molecular-weight-cutoff Pur-A-Lyzer dialysis tube (Sigma-Aldrich). Previous studies have shown that this concentration of siRNA sufficiently silences gene expression (66)(67)(68)(69). We placed the dialysis tubes in a beaker with 500 mL of culture medium at 37°C in 5% CO 2 . ...
Lymphatic filariasis is a debilitating disease that afflicts over 70 million people worldwide. It is caused by the parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. Despite substantial success, efforts to eliminate LF will likely require more time and resources than predicted. Identifying new drug and vaccine targets in adult filariae could help elimination efforts. This study's aim was to evaluate intestinal proteins in adult Brugia malayi worms as possible therapeutic targets. Using short interfering RNA (siRNA), we successfully targeted four candidate gene transcripts: Bma-Serpin, Bma-ShTK, Bma-Reprolysin, and Bma-LAD-2. Of those, Bma-LAD-2, an immunoglobulin superfamily cell adhesion molecule (IgSF CAM), was determined to be essential for adult worm survival. We observed a 70.42% knockdown in Bma-LAD-2 transcript levels 1 day post-siRNA incubation and an 87.02% reduction in protein expression 2 days post-siRNA incubation. This inhibition of Bma-LAD-2 expression resulted in an 80% decrease in worm motility over 6 days, a 93.43% reduction in microfilaria release (Mf) by day 6 post-siRNA incubation, and a dramatic decrease in (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Transmission electron microscopy revealed the loss of microvilli and unraveling of mitochondrial cristae in the intestinal epithelium of Bma-LAD-2 siRNA-treated worms. Strikingly, Bma-LAD-2 siRNA-treated worms exhibited an almost complete loss of pseudocoelomic fluid. A luciferase immunoprecipitation system assay did not detect anti-Bma-LAD-2 IgE in the serum of 30 LF patients, indicating that LF exposure does not result in IgE sensitization to this antigen. These results indicate that Bma-LAD-2 is an essential protein for adult Brugia malayi and may be an effective therapeutic target. IMPORTANCE Brugia malayi is a parasitic nematode that can cause lymphatic filariasis, a debilitating disease prevalent in tropical and subtropical countries. Significant progress has been made toward eliminating the disease. However, complete eradication may require new therapeutics such as drugs or a vaccine that kill adult filariae. In this study, we identified an immunoglobulin superfamily cell adhesion molecule (Bma-LAD-2) as a potential drug and vaccine candidate. When we knocked down Bma-LAD-2 expression, we observed a decrease in worm motility, fecundity, and metabolism. We also visualized the loss of microvilli, destruction of the mitochondria in the intestinal epithelium, and loss of pseudocoelomic fluid contents after Bma-LAD-2 siRNA treatment. Finally, we demonstrated that serum from filaria-infected patients does not contain preexisting IgE to Bma-LAD-2, which indicates that this antigen would be safe to administer as a vaccine in populations where the disease is endemic.
... With the advent of CRISPR-Cas9 in genomic splicing (25), the ability to knock out this gene is now a possibility (26)(27)(28). However, for the filarial nematodes, RNAi remains the most useful method of gene silencing (12,13,15,(29)(30)(31)(32). ...
A molecule we termed Brugia malayi IL-5 receptor (IL-5R) binding protein (BmIL5Rbp; also known as Bm8757) was identified from B. malayi filarial worms and found to inhibit human interleukin-5 (IL-5) binding to its human receptor competitively. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference (RNAi) was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in its excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33%, reduced the surface protein expression by ~50%, and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof of principle for the local inhibition of the human IL-5R. These findings provide evidence that a parasite-encoded IL-5R antagonist may locally inhibit a vital host innate immune activation of IL-5 on eosinophils.
... Among them are those based on RNA interference (RNAi) and microRNA knock-down. These systems arise as highly powerful tools for the study of the cellular and molecular basis of development and hostparasite interaction in helminth parasites (Pérez et al. 2019;Misra et al. 2017;McCoy et al. 2015;Britton et al. 2015;Spiliotis et al. 2010;Dalzell et al. 2011;Da'dara and Skelly 2015). One of the major hurdles for the use of these technologies has been the delivery of oligonucleotides. ...
Echinococcus multilocularis is the etiological agent of alveolar echinococcosis (AE), a serious parasitic disease in the Northern Hemisphere. The E. multilocularis primary cell cultivation system, together with E. multilocularis genome data and a range of pioneering molecular-based tools have advanced the research on this and other cestodes. RNA interference (RNAi) and microRNA knock-down have recently contributed to the study of the cellular and molecular basis of tapeworm development and host-parasite interaction. These, as well as other techniques, normally involve an electroporation step for the delivery of RNA, DNA, peptides, and small molecules into cells. Using transcriptome data and bioinformatic analyses, we herein report a genome-wide comparison between primary cells of E. multilocularis and primary cells under electropo-rated conditions after 48 h of culture. We observed that ~ 15% of genes showed a significant variation in expression level, including highly upregulated genes in electroporated cells, putatively involved in detoxification and membrane remodeling. Furthermore, we found genes related to carbohydrate metabolism, proteolysis, calcium ion binding and microtubule processing significantly altered, which could explain the cellular dispersion and the reduced formation of cellular aggregates observed during the first 48 h after electroporation.
... In parasitic nematodes, RNAi is possible in some life cycle stages of some species in which some parasitic stages can be maintained (but not expanded) in the laboratory. In O. volvulus, RNAi has been attempted using L3 worms [197,198], but it has been seldom used-certainly not as much as in the case in Brugia malayi (N), where many authors have reported its successful use [199][200][201][202][203][204]. For the purposes of drug screening in Onchocerca volvulus, the bovine parasite O. ochengi has been used as a model [205][206][207][208][209]. Comparative genomics revealed Onchocerca ochengi as the closest relative to Onchocerca volvulus [191], and the microfilariae that can be maintained a few hours in culture can be harvested from slaughterhouse biological samples in larger numbers. ...
Nematodes constitute a very successful phylum, especially in terms of parasitism. Inside their mammalian hosts, parasitic nematodes mainly dwell in the digestive tract (geohelminths) or in the vascular system (filariae). One of their main characteristics is their long sojourn inside the body where they are accessible to the immune system. Several strategies are used by parasites in order to counteract the immune attacks. One of them is the expression of molecules interfering with the function of the immune system. Excretory-secretory products (ESPs) pertain to this category. This is, however, not their only biological function, as they seem also involved in other mechanisms such as pathogenicity or parasitic cycle (molting, for example). We will mainly focus on filariae ESPs with an emphasis on data available regarding Onchocerca volvulus, but we will also refer to a few relevant/illustrative examples related to other worm categories when necessary (geohelminth nematodes, trematodes or cestodes). We first present Onchocerca volvulus, mainly focusing on the aspects of this organism that seem relevant when it comes to ESPs: life cycle, manifestations of the sickness, immunosuppression, diagnosis and treatment. We then elaborate on the function and use of ESPs in these aspects.
... The first and most widely used strategy is confirmation of the important functions of macromolecules. The commonly used techniques include homology analysis (Hung and Weng, 2016;Flo et al., 2017), molecular docking (Esteves and Paulino, 2013;Vadloori et al., 2018), differential gene expression analysis (DeMarco and Verjovski-Almeida, 2009;Parkinson et al., 2012;Saurabh et al., 2014), and RNA interference (RNAi; Misra et al., 2017;Mousavi et al., 2019), among other techniques. The second strategy involves drug target identification based on active compounds. ...
Echinococcosis is an important parasitic disease that threats human health and animal husbandry worldwide. However, the low cure rate of clinical drugs for this disease is a challenge. Hence, novel compounds and specific drug targets are urgently needed. In this study, we identified drug targets of amino alcohols with effects on Echinococcus species. The drug targets were predicted with the idTarget web server. Corresponding three-dimensional structures of the drug targets were built after sequence BLAST analysis and homology modeling. After further screening by molecular docking, the activities of the candidate targets were validated in vitro. We ultimately identified glycogen phosphorylase as a potential drug target for amino alcohols. There are two genes coding glycogen phosphorylase in Echinococcus granulosus (EgGp1 and EgGp2). EgGp1 was abundant in E. granulosus PSCs, while EgGp2 was abundant in the cysts. These proteins were located at suckers and somas of E. granulosus PSCs and near the rostellum of cysts developed from PSCs. The effective compounds docked into a pocket consisting of E124, K543 and K654 and affected (either inhibited or enhanced) the activity of E. granulosus glycogen phosphorylase. In this study, we designed a method to predict drug targets for echinococcosis treatment based on inverse docking. The candidate targets found by this method can contribute not only to understanding of the modes of action of amino alcohols but also to modeling-aided drug design based on targets.
... Silencing of the TsASP2 gene also impeded other T. spiralis developmental stages, as adult worms recovered from mice infected with siRNA-treated larvae were shorter than those recovered from the control and PBS groups. Specific gene silencing by RNAi has been widely applied for gene function identification in other parasites [62,63], and the results of the present study indicated that TsASP2 played a crucial role in T. spiralis invasion of IEC, and silencing of the TsASP2 gene significantly reduced larval infectivity and development in mice. ...
Background
T. spiralis aspartic protease has been identified in excretion/secretion (ES) proteins, but its roles in larval invasion are unclear. The aim of this study was to characterize T. spiralis aspartic protease-2 (TsASP2) and assess its roles in T. spiralis invasion into intestinal epithelial cells (IECs) using RNAi.
Methodology/Principal findings
Recombinant TsASP2 (rTsASP2) was expressed and purified. The native TsASP2 of 43 kDa was recognized by anti-rTsASP2 serum in all worm stages except newborn larvae (NBL), and qPCR indicated that TsASP2 transcription was highest at the stage of intestinal infective larvae (IIL). IFA results confirmed that TsASP2 was located in the hindgut, midgut and muscle cells of muscle larvae (ML) and IIL and intrauterine embryos of the female adult worm (AW), but not in NBL. rTsASP2 cleaved several host proteins (human hemoglobin (Hb), mouse Hb, collagen and IgM). The proteolytic activity of rTsASP2 was host-specific, as it hydrolyzed mouse Hb more efficiently than human Hb. The enzymatic activity of rTsASP2 was significantly inhibited by pepstatin A. The expression levels of TsASP2 mRNA and protein were significantly suppressed by RNAi with 5 μM TsASP2-specific siRNA. Native aspartic protease activity in ML crude proteins was reduced to 54.82% after transfection with siRNA. Larval invasion of IECs was promoted by rTsASP2 and inhibited by anti-rTsASP2 serum and siRNA. Furthermore, cell monolayer damage due to larval invasion was obviously alleviated when siRNA-treated larvae were used. The adult worm burden, length of adult worms and female fecundity were clearly reduced in mice challenged using siRNA-treated ML relative to the PBS group,
Conclusions
rTsASP2 possesses the enzymatic activity of native aspartic protease and facilitates T. spiralis invasion of host IECs.
... Since this first experiment, RNAi has been successfully applied to a number of mammalian-parasitic nematode species, including Ascaris suum, B. malayi, Onchocerca volvulus, the ruminant parasite Haemonchus contortus and the filarial parasite of livestock Setaria digitata (Hagen et al., 2012;Luck et al., 2016;Maule et al., 2011;McCoy et al., 2015;Misra et al., 2017;Samarasinghe et al., 2011;Somarathne et al., 2018;Verma et al., 2017). However, results from RNAi experiments are often variable in terms of both the extent of gene knockdown and the resulting mutant phenotype (Britton et al., 2016;Hagen et al., 2012;Maule et al., 2011;McCoy et al., 2015). ...
... While most studies have attempted to induce RNAi using dsRNA, several recent studies have instead used small interfering RNA (siRNA) for inducing RNAi (Misra et al., 2017;Somarathne et al., 2018). In C. elegans, long dsRNA is processed by the dicer complex into siRNA. ...
... siRNA was used in B. malayi to knock down the potential drug target UCP-galactopyranose mutase, which catalyzes the production of the cell surface component galactofuranose. Galactofuranose is an essential component of cell surface glycoproteins in several nematodes, but is absent in mammals (Misra et al., 2017). UCP-galactopyranose mutase knockdown in B. malayi adults resulted in severe movement defects and impaired embryogenesis, and knockdown in L3 larvae resulted in severely reduced infectivity. ...
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.
