Characterization of innate immunity genes in the parasitic nematode Brugia malayi

Symbiosis (2016) 68:145–155 DOI 10.1007/s13199-015-0374-7 Characterization of innate immunity genes in the parasitic nematode Brugia malayi Silvia Libro 1 & Barton E. Slatko 1 & Jeremy M. Foster 1 Received: 5 October 2015 / Accepted: 24 December 2015 / Published online: 5 January 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract The filarial nematode Brugia malayi is one of the causative agents of lymphatic filariasis, a neglected tropical disease that affects 120 million people worldwide. The limited effectiveness of available anthelmintics and the absence of a vaccine have prompted extensive research on the interaction between Brugia and its obligate bacterial endosymbiont, Wolbachia. Recent studies suggest that Wolbachia is able to manipulate its nematode host immunity but relatively little is known about the immune system of filarial nematodes. Therefore, elucidation of the mechanisms underlying the immune system of B. malayi may be useful for understanding how the symbiotic relationship is maintained and help in the identification of new drug targets. In order to characterize the main genetic pathways involved in B. malayi immunity, we exposed adult female worms to two bacterial lysates (Escherichia coli and Bacillus amyloliquefaciens), dsRNA and dsDNA. We performed transcriptome sequencing of worms exposed to each immune elicitor at two different timepoints. Gene expression analysis of untreated and immune-challenged worms was performed to characterize gene expression patterns associated with each type of immune stimulation. Our results indicate that different immune Presented at the 8th Congress of the International Symbiosis Society, July 12–18, 2015, Lisbon, Portugal Electronic supplementary material The online version of this article (doi:10.1007/s13199-015-0374-7) contains supplementary material, which is available to authorized users. * Silvia Libro 1 Genome Biology Division, New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA elicitors produced distinct expression patterns in B. malayi, with changes in the expression of orthologs of wellcharacterized C. elegans immune pathways such as insulin, TGF-β, and p38 MAPK pathways, as well as C-type lectins and several stress-response genes. Keywords Nematode . Brugia . Transcriptomics . Wolbachia . Immunity 1 Introduction The filarial nematode Brugia malayi is one of the causative agents of lymphatic filariasis (elephantiasis), a neglected tropical disease that affects 120 million people in endemic tropical areas. The disease is transmitted to the human host via infected mosquitoes which allow third-stage filarial larvae (L3) to enter the host’s bloodstream. Larvae reach maturity in the lymphatic system and reproduce, generating millions of microfilariae that migrate to the capillaries from where they can be ingested by new mosquitos. In the mosquito, microfilariae develop into first-stage larvae, second-stage larvae and then infective thirdstage larvae, and the cycle repeats. Current treatments such as albendazole and ivermectin rely on mass drug administration (MDA) programs - but they predominantly target larval stages, necessitating a treatment course of up to 10–15 years (Molyneux et al. 2003). Due to this and other limitations of MDA, extensive effort has been devoted to research for alternative treatments. In particular, several projects are currently focusing on understanding the biology of the relationship between B. malayi and its obligate endosymbiont, the alpha-proteobacterium Wolbachia. Wolbachia is present in intracytoplasmic vacuoles in several filarial nematodes and is required for worm fertility and survival (Foster et al. 2013; Taylor et al. 2005) making it a 146 promising therapeutic target for filariasis control. One example of anti-filarial strategies exploiting this obligate mutualism is the use of antibiotics against Wolbachia, such as doxycycline, to cause premature death and permanent sterilization of adult worms. While the mechanisms of the symbiotic relationship are not fully understood, recent studies suggest that apoptosis and autophagy- two conserved cellular pathways essential for homeostasis and innate immunity- are involved in the regulation of Wolbachia titer in filarial nematodes (Landmann et al. 2011; Voronin et al. 2012), suggesting that Wolbachia is able to modulate and evade the host immune system in order to survive. In particular, these studies report extensive apoptosis in germline and somatic cells of embryos, microfilariae, and fourth-stage larvae following antibiotic-mediated depletion of Wolbachia (Landmann et al. 2011) and show that Wolbachia can be recognized and eliminated by autophagy in filarial nematodes and insects (Voronin et al. 2012). Similar to other mutualistic bacterial symbioses, these data are indicative of a cross-talk between Wolbachia and its nematode host immune system. Therefore, determining the mechanisms underlying B. malayi immunity can provide critical information to understand how the symbiosis with Wolbachia is maintained. So far, few studies have focused on the B. malayi immune system and our knowledge is mostly based on sequence similarity with the free-living nematode Caenorhabditis elegans (Engelmann and Pujol 2010; Irazoqui et al. 2010). We initiated experiments aimed at the identification of candidate immune-related genes in the filarial nematode B. malayi. Live adult females were exposed to four different immune elicitors (dsRNA, dsDNA, Gram-positive and Gramnegative bacterial lysates) for 24 and 36 h. The expression profiles of treated and un-treated worms were compared and the identity of differentially expressed (DE) transcripts was determined based on current genome annotations. Our results indicate that different immune elicitors produced distinct expression patterns in B. malayi, with changes in the expression of orthologs of well-characterized C. elegans immune pathways such as insulin, transforming growth factor beta (TGF-β), and p38 MAPK pathways, as well as C-type lectins and several stress-response genes. S. Libro et al. and that the genome of B. malayi encodes several components of the pathway (Dalzell et al. 2011). 2.1 Preparation of dsRNA The template for the production of dsRNA was LITMUS 28i (New England Biolabs), a double-stranded cloning/in vitro transcription phagemid vector that has opposing T7 promoters. cDNA was obtained by PCR amplification of a 172 bp region of the vector using Q5® High-Fidelity 2X Master Mix (Cat. #M0492, New England Biolabs) and custom designed primers (IDT): 5-TAA TAC GAC TCA CTA TAG GGC AGA T-3 for the forward primer and 5-TAA TAC GAC TCA CTA TAG GCC TTG ACT AG-3 for the reverse primer. The PCR product was verified by 1 % agarose gel electrophoresis and then purified with the QIAquick PCR Purification Kit (Cat. #28104, Qiagen) following the manufacturer’s protocol but with one extra wash with PE buffer. PCR produ (...truncated)