Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

Landmann et al. Parasites & Vectors 2012, 5:16 http://www.parasitesandvectors.com/content/5/1/16 RESEARCH Open Access Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi Frédéric Landmann1*†, Jeremy M Foster2†, Barton E Slatko2 and William Sullivan1 Abstract Background: RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans. Results: We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding g-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week. Conclusions: We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis. Keywords: RNAi, nematode, immunostaining, Brugia, filaria Background Filarial nematodes cause debilitating pathologies in tropical areas with > 1 billion people at risk. Current anthelmintic drugs predominantly target larval stages only, and a developing resistance has been indicated [1-3]. Since almost all filarial species that cause disease in humans rely on the bacterial endosymbiont Wolbachia for proper embryogenesis, development and viability, these symbionts have become a major drug target * Correspondence: † Contributed equally 1 Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95604, USA Full list of author information is available at the end of the article for novel anti-filarial strategies [4,5]. It is crucial to characterize the molecular and cellular mechanisms underlying Wolbachia transmission, since their segregation patterns in the embryo determine the localization in adult hypodermal chords [6]. Such knowledge could lead to the molecular identification of new drug targets. Despite the published genomes of Brugia malayi [7], a causative agent of lymphatic filariasis and elephantiasis, and its Wolbachia endosymbiont [8], both the developmental biology of filarial nematodes and the mutualism with Wolbachia are still poorly understood. The free living nematode Caenorhabditis elegans, remains the closest animal model to date. While C. elegans represents a © 2012 Landmann et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Landmann et al. Parasites & Vectors 2012, 5:16 http://www.parasitesandvectors.com/content/5/1/16 valid model for general aspects of nematode biology [9], its estimated > 500 million year separation from B. malayi [10] and free-living lifestyle leave questions related to parasitism unanswered [11]. Furthermore, C. elegans data cannot inform on mutualism with Wolbachia since within the Nematoda this bacterium appears limited to parasitic nematodes within the family Onchocercidae [4]. Therefore inference of gene function or biological processes in parasitic species based on data from C. elegans should be made with caution. While reverse genetic tools such as RNA interference (RNAi) are routinely used in C. elegans research to characterize gene function [12], RNAi experiments in animal parasitic nematodes have proven notoriously challenging [13-18] with few successes reported. A bioinformatic study comparing the RNAi effector protein complements of various animal parasitic nematodes to that of C. elegans found that while quantitative differences exist (with C. elegans having the richest complement), all species were qualitatively similar and should be RNAi-competent [19]. Indeed, there are a few reports of successful RNAi in filarial nematodes [20-26] although several of these studies targeted the same genes. Various explanations for the limited success of RNAi in animal parasitic nematodes have been proposed [15,19,27]. We describe an efficient in vitro RNAi procedure we have developed to successfully copy RNAi-induced phenotypes observed in C. elegans during early embryogenesis in the parasitic nematode B. malayi. We also further optimized our immunofluorescence protocols [6] to permit detailed characterization of the embryonic RNAi phenotypes. These methodologies may be extended to genes expressed in the germ line or in adult tissues. Our results demonstrate that RNAi can be a reliable and effective tool for gene function studies in parasitic species. The enhanced procedures for both RNA delivery and subsequent immunofluorescencebased phenotype analysis open the way to address fundamental questions in parasitic nematode biology, emancipation from the C. elegans model, and investigation of the interaction between the filarial parasite and its Wolbachia symbiont. Methods See Additional file 1 for a detailed step-by-step protocol and Additional files 2, 3, 4 for illustration of key steps in the procedure. Preparation of heterogeneous short interfering RNA (hsiRNA) Total RNA was prepared from adult B. malayi (TRS Labs, Athens, GA, USA) by established methodology http://www.filariasiscenter.org/molecular-resources/protocols and ~ 700 ng RNA used as template for Page 2 of 8 production of cDNA using the ProtoScript M-MuLV First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (New England Biolabs. Ipswich, MA, USA). DNA templates for in vitro transcription were generated by PCR using Crim (...truncated)