Interaction of a Wolbachia WSP-like protein with a nuclear-encoded protein of Brugia malayi

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Abstract

The Brugia malayi endosymbiont Wolbachia has recently been shown to be essential for its host’s survival and development. However, relatively little is known about Wolbachia proteins that interact with the filarial host and which might be important in maintaining the obligate symbiotic relationship. The Wolbachia surface proteins (WSPs) are members of the outer membrane protein family and we hypothesise that they might be involved in the WolbachiaBrugia symbiotic relationship. Notably, immunolocalisation studies of two WSP members, WSP-0432 and WSP-0284 in B. malayi female adult worms showed that the corresponding proteins are not only present on the surface of Wolbachia but also in the host tissues, with WSP-0284 more abundant in the cuticle, hypodermis and the nuclei within the embryos. These results confirmed that WSPs might be secreted by Wolbachia into the worm’s tissue. Our present studies focus on the potential involvement of WSP-0284 in the symbiotic relationship of Wolbachia with its filarial host. We show that WSP-0284 binds specifically to B. malayi crude protein extracts. Furthermore, a fragment of the hypothetical B. malayi protein (Bm1_46455) was found to bind WSP-0284 by panning of a B. malayi cDNA library. The interaction of WSP-0284 and this protein was further confirmed by ELISA and pull-down assays. Localisation by immunoelectron microscopy within Wolbachia cells as well as in the worm’s tissues, cuticle and nuclei within embryos established that both proteins are present in similar locations within the parasite and the bacteria. Identifying such specific interactions between B. malayi and Wolbachia proteins should lead to a better understanding of the molecular basis of the filarial nematode and Wolbachia symbiosis.

Highlights

Brugia malayi harbors the obligate endosymbiont Wolbachia. ► Wolbachia surface protein WSP-0284 is located not only on the surface of bacteria. ► WSP- 0284 is released within the worm tissue and binds to the B. malayi Bm-G2 protein. ► Bm-G2 partially corresponding to the Bm1_46455 protein is localised similarly like WSP-0284 within parasite and bacteria. ► Bm-G2/WSP-0284 protein complex is potentially involved in this symbiotic relationship.

Introduction

Filariasis is a serious public health problem worldwide. It is endemic in 83 countries and territories and results in significant economic loss by causing considerable morbidity (Molyneux, 2003). Some 120 million people are infected with the lymphatic filarial nematodes Wuchereria bancrofti, Brugia malayi and Brugia timori (WHO, 2004) with 1.3 billion people at risk. Lymphatic filariasis (LF) provokes acute dermatolymphagioadenitis and lymphedema, often leading to elephantiasis as a result of damage and dysfunction of the lymphatics (Taylor et al., 2010). The related filarial nematode Onchocerca volvulus affects 37 million people (Molyneux, 2003) and causes onchocerciasis (river blindness) as well as skin lesions due to inflammation induced by dead microfilaria. The ocular lesions (keratitis, retinal lesions) are caused by microfilaria that migrate to the cornea and penetrate the ciliary body. This may lead to atrophy of the optic nerve and consequent blindness (Taylor et al., 2010).

Currently, the most widely used strategy for the control of filariasis as a public health problem employs mass drug administration of albendazole in combination with ivermectin (IVM) or diethylcarbamazine (DEC) for LF, and IVM alone for onchocerciasis. One of the limitations of this approach is that the drugs are not macrofilaricidal (i.e. they do not kill the adult worms), and thus are not efficient tools for complete elimination of the worms (Nandha et al., 2007, Ottesen et al., 2008).

Most of the human filarial nematode species, including Brugia spp., W. bancrofti and O. volvulus, harbour the obligate intracellular endosymbiont Wolbachia (Taylor et al., 2005a). Wolbachia bacteria were first identified in insects where they are parasitic and associated with reproductive manipulation (Werren et al., 2008). In filaria, Wolbachia is an obligate mutualistic symbiont that plays an essential role in oogenesis and embryogenesis in adult worms and during larval development (Smith and Rajan, 2000, Pfarr and Hoerauf, 2007, Ghedin et al., 2008, Ghedin et al., 2009). Elimination of Wolbachia by antibiotic treatment leads to infertility of the female worms, inhibition of larval moulting, and atrophy and death of adult worms (macrofilaricidal effect) (Hoerauf et al., 2000, Casiraghi et al., 2002, Taylor et al., 2005b). This evidence prompted the study of Wolbachia as a target for anti-filarial nematode chemotherapy. Multiple in vitro and in vivo studies, including several clinical trials in humans using Wolbachia targeting antibiotics, reported anti-filarial effects confirming the essential role Wolbachia plays in worm survival and thus vulnerability for elimination (Bazzocchi et al., 2008, Hoerauf et al., 2008, Specht et al., 2008, Specht and Wanji, 2009, Supali et al., 2008, Mand et al., 2009). The endosymbiont was also found to contribute to LF and onchocerciasis pathogenesis and thus morbidity (Taylor et al., 2000, Taylor, 2003). In particular, a member of the Wolbachia surface protein (WSP) family was shown to induce an inflammatory response associated with the pathogenesis of onchocerciasis through the activation of an innate immune response (Brattig et al., 2004) and to have anti-apoptotic activity by delaying the apoptosis in human polymorphonuclear cells that are essential for the initiation and execution of the innate immune response against bacterial pathogens (Bazzocchi et al., 2007).

The molecular mechanisms involved in the interaction of the filarial endosymbionts and their hosts remain largely unexplored. With the availability of genomic data of B. malayi and its Wolbachia endosymbiont (wBm) (Foster et al., 2005, Ghedin et al., 2007), it has become possible to study the underlying mechanisms of this symbiotic system, and it has pointed to some specific pathways and filarial and Wolbachia proteins that might be essential in maintaining the WolbachiaBrugia symbiotic relationship. In particular, it was postulated that surface proteins such as the WSP family proteins of the endosymbiont are the most likely to be involved in the WolbachiaBrugia symbiotic interaction (Ghedin et al., 2008). Three WSP-like outer surface proteins (wBm0100, wBm0284 and wBm0432) have been identified in the Wolbachia genome (Foster et al., 2005), and these are similar to the outer membrane protein (OMP) family of bacteria known to be involved in bacteria–host interactions (Baldo et al., 2010). In arthropods, a WSP-like protein is thought to be a key player for establishment and persistence of the symbiosis but little is known about the role of this protein and its possible interaction partners in the arthropods and/or nematodes (Baldo et al., 2010).

The WSP-like proteins are highly conserved in Wolbachia from filarial nematodes and are also reported to have a heterogeneous pattern of amino acid diversity characteristic of other OMPs (Braig et al., 1998, Baldo et al., 2005, Baldo et al., 2010, Serbus et al., 2008). Moreover, through analysis of the B. malayi secretome, a number of Wolbachia OMPs were found to be secreted or released by the worm (Bennuru et al., 2009). In the work described here, we have examined the role that members of the WSP family might play in the host–endosymbiont relationship, focusing in particular on one member of the family, WSP-0284 (GI: 3266802, locus tag: wBm0284).

Section snippets

Cloning, expression and purification of the Wolbachia surface protein WSP-0284

The cDNA corresponding to the WSP gene wBm0284 was amplified from positions 73 to 846 of the predicted open reading frame (ORF) by PCR from female B. malayi random-primed cDNA using gene-specific primer set: 5′ CACCGAAACAGAAGGATTCTACTT 3′ and 5′ ACAACATGTTTAAACCTTGC 3′. This region consisted of the entire predicted ORF minus the predicted signal sequence. The 787 bp PCR product was first cloned into pENTR™/D-TOPO®, entry cloning for the Gateway® System (Invitrogen, Carlsbad, CA, USA). The first

The Wolbachia surface proteins are located on the bacterial cell surface and are also found within filarial tissues

To analyse and compare the ultrastructural localisation of WSPs in adult B. malayi female worms, we used antibodies raised against two WSP members: WSP-0432 and WSP-0284. WSPs, which contain both trans-membrane domains and a standard signal peptide for secretion, were expected to be found on the surface of the Wolbachia of the filarial worms examined, as previously observed for the WSP corresponding to wBm0432 (Kramer et al., 2003, McGarry et al., 2003, McGarry et al., 2004, Brattig et al., 2004

Acknowledgements

We thank the National Institute of Allergy and Infectious Diseases (NIAID, USA)/National Institutes of Health (NIH, USA) Filariasis Research Reagent Repository Center (FR3) for supplying B. malayi-infected jirds and the adult B. malayi worms. We would also like to thank Dr. Mark Taylor and Dr. Louise Ford, Liverpool School of Tropical Medicine, Liverpool, UK for provision of the anti-WSP-0432 antibody. We also thank Dr. William Harnett and Dr. Katrina Houston from the University of Strathclyde,

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