The symbiotic role of Wolbachia in Onchocercidae and its impact on filariasis

REVIEW 10.1111/1469-0691.12069 The symbiotic role of Wolbachia in Onchocercidae and its impact on filariasis T. Bouchery, E. Lefoulon, G. Karadjian, A. Nieguitsila and C. Martin UMR 7245, MCAM MNHN CNRS, Muséum National d Histoire Naturelle, Paris, France Abstract Symbiotic associations between eukaryotes and microorganisms are frequently observed in nature, and range along the continuum between parasitism and mutualism. The genus Wolbachia contains well-known intracellular bacteria of arthropods that induce several reproductive phenotypes that benefit the transmission of the bacteria. Interestingly, Wolbachia bacteria have been found in the Onchocercidae, a family of filarial nematodes, including species that cause human filarial diseases, e.g. lymphatic filariasis and onchocerciasis. The endosymbiont is thought to be mutualistic in the Onchocercidae, and to provide essential metabolites to the filariae. Currently, Wolbachia bacteria are targets of antibiotic therapy with tetracyclines, which have profound effects on the development, viability and fertility of filarial parasites. This overview article presents the Onchocercidae and Wolbachia, and then discusses the origin and the nature of the symbiosis. It highlights the contribution of Wolbachia to the survival of the filariae and to the development of pathology. Finally, the infection control implications for filariases are debated. Potential directions for future research are also discussed. Keywords: Antibiotherapy, filariasis, onchocercidae, symbiosis, Wolbachia Article published online: 11 October 2012 Clin Microbiol Infect 2013; 19: 131 140 Corresponding author: C. Martin, UMR 7245, MCAM MNHN CNRS, Muséum National d Histoire Naturelle, 61 rue Buffon, CP52, 75231 Paris Cedex 05, France E-mail: cmartin@mnhn.fr Introduction Among the Nematoda, the order Spirurida encompasses the family Onchocercidae, commonly known as filariae. They infect a broad range of terrestrial vertebrates, and many of them are responsible for medical and veterinary issues in mammals (Table 1) [1 27]. They are all transmitted by haematophagous arthropod vectors that are either pool-feeders (telmophagous) (e.g. black flies for onchocerciasis transmission, or tabanid flies for loasis) or vessel-feeders (solenophagous) (e.g. mosquitoes for lymphatic filariasis transmission, or ticks for canid filariasis) (Table 1). Filarial nematode species can host Wolbachia bacterial endosymbionts. Research on the symbiosis of Wolbachia and Onchocercidae has highlighted the contributions of bacteria to the reproduction of filariae, the inflammatory disease pathogenesis, and the immunomodulation of the vertebrate host, and has led to the consideration of antibiotic therapy as a novel method of treatment. Biology of Filariae and their Endosymbiont Wolbachia The microfilariae (stage 1 or L1) are taken up by the vector during a blood meal. In this intermediate host, they moult and develop until they become stage 3 (infective) larvae (L3). Throughout this development, the larvae migrate from the ingested blood to other tissues, depending on the vector species [28]. During another blood meal of the vector, the infective larvae are passed to the dermis of the vertebrate s skin. Once there, the L3 larvae moult into stage 4 larvae within 2 days for the genera Onchocerca and Dirofilaria, and within approximately 10 days for the other genera [28]. Most of the species migrate through the host s body from the skin to their definitive niche, mainly the lymphatic system, the coelomic cavities, the cardiopulmonary system, and connective tissues (Table 1). The fourth and final moult results in adult male or female worms. Clinical Microbiology and Infection ª2012 European Society of Clinical Microbiology and Infectious Diseases

132 Clinical Microbiology and Infection, Volume 19 Number 2, February 2013 CMI TABLE 1. Prominent filarial species of human and veterinary interest Species Vectors Genus (family) Vertebrate hosts Main tissue localization of adult worms Localization of microfilariae Main pathologies Wolbachia supergroup References Onchocercinae Acanthocheilonema dracunculoides (Cobold, 1870) Hippobosca (Hippoboscidae) Canids Coelomic cavity and subcutaneous tissue Blood Usually asymptomatic NA [1,2] Heterodoxus (Boopiidae) Acanthocheilonema reconditum (Grassi, 1889) Ctenocephalides (Pulicidae) Canids Subcutaneous tissue Blood Usually asymptomatic Absent [1,3] Pulex (Pulicidae) Heterodoxus (Boopiidae) Acanthocheilonema viteae* (Krepkogorskaya, 1933) Ornithodoros (Argasidae) Rodents Subcutaneous tissue Blood Usually asymptomatic Absent [1,4,5] Brugia malayi (Brug, 1927) Mansonia (Culicidae) Humans, a LS, LN, testes Blood Adenopathy D [1,6,7] Anopheles (Culicidae) Lymphoedema Aedes (Culicidae) Brugia pahangi (Buckley & Edeson, 1956) Aedes (Culicidae) Dogs, felids, b, c LS, LN, testes Blood Lymphoedema D [1,6,7] Anopheles (Culicidae) Brugia timori (Partono et al., 1977) Anopheles (Culicidae) Humans LS, LN, testes Blood Lymphoedema D [1,7,8] Aedes (Culicidae) Cercopithifilaria grassii (Noè, 1907) Rhipicephalus (Ixodidae) Dogs Subcutaneous tissue Skin Usually asymptomatic NA [1,9,10] Litomosoides sigmodontis* (Chandler, 1931) Ornithonyssus (Macronyssidae) Rodents Coelomic cavity Blood Usually asymptomatic D [1,11] Mansonella (Mansonella) ozzardi (Manson, 1897) Culicoides (Ceratopogonidae) Humans Coelomic cavity Blood Usually asymptomatic F [1,12] Simulium (Simuliidae) Mansonella (Esslingeria) perstans (Manson, 1891) Culicoides (Ceratopogonidae) Humans and monkeys Coelomic cavity Blood Usually asymptomatic F [1,13] Mansonella (Esslingeria) streptocerca (Macfie & Corson, 1922) Culicoides (Ceratopogonidae) Humans and monkeys Intradermal Skin Dermatitis NA [1,14] Onchocerca cervicalis (Railliet & Henry, 1910) Culicoides (Ceratopogonidae) Equids Nuchal ligament Skin Dermatitis [1,15] Ocular trauma Onchocerca gutturosa (Neumann, 1910) Simulium (Simuliidae) Bovids Nuchal ligament Skin Dermatitis [1,16] Culicoides (Ceratopogonidae) Onchocerca lupi (Rodonaja, 1967) Unknown Canids Skin Ocular trauma [1,17] Onchocerca ochengi (Bwangamoi, 1969) Simulium (Simuliidae) Bovids Intradermal Skin Nodules C [1,11,18,19] Onchocerca volvulus (Leuckart, 1893) Simulium (Simuliidae) Humans Subcutaneous tissue Skin Dermatitis C [1,20] Ocular trauma Wuchereria bancrofti (Cobbold, 1877) Culex (Culicidae) Humans LS, LN, testes Blood Lymphoedema D [1,7,21] Anopheles (Culicidae) Aedes (Culicidae) Dirofilariinae Dirofilaria immitis (Leidy, 1856) Aedes (Culicidae) Canids, felids, d Right ventricle Blood Heart worm disease C [1,22,23] Pulmonary artery Vena cava Dirofilaria repens (Railliet et Henry, 1911) Aedes (Culicidae) Canids, felids, e Subcutaneous tissue Blood Subcutaneous tissue nodules C [1,24] Culex (Culicidae) Connective tissue Loa loa (Cobbold, 1864) Chrysops (Tabanidae) Humans Subcutaneous tissue Blood Calabar swelling Absent [1,25] Connective tissue Ocular trauma Setariinae Setaria equina (Abildgaard, 1789) Aedes (Culicidae) Horses Coelomic cavity Blood Usually asymptomatic Absent [1,26,27] LN, lymph nodes; LS, lymphatic system; NA, data not available. Extra hosts are indicated by a e : a monkeys, cats, dogs, viverrids, pangolins; b cebids, erinaceids, lorisids, sciurids, manids, viverrids; c transmissible to humans; d ferrets, raccoons, sea lions; e raccoons, sea lions.*rodent filariae commonly used as experimental models of filariasis.

CMI Bouchery et al. Wolbachia and filariasis 133 Filarial diseases are often asymptomatic in wild fauna. Clinical manifestations and pathologies can depend on the tissue localization of microfilariae or adult worms, the parasite burden, and the vertebrate host s immune response. In endemic zones, one in six people are affected by human filariases, representing an estimated 150 180 million infested people, of whom 37 million harbour Onchocerca volvulus, the agent of onchocerciasis, also known as river blindness. The latter pathology is responsible for many ocular and cutaneous lesions: chronic onchocercal dermatitis with pruritus, cutaneous lichenification, and depigmentation of the skin ( leopard skin ). In addition, subcutaneous nodules are formed around female worms. Similarly, Onchocerca ochengi females induce the formation of a nodule in the dermis of the bovine host, in which they remain sedentary and in close association with the host s tissue. Lymphatic filariasis is caused by Wuchereria bancrofti and Brugia spp. It is the second largest cause of longterm disability, with one-third of patients suffering from clinical presentations of the infection, namely lymphoedema of the extremities (elephantiasis) and hydrocoele. Many filariases have also been described in domestic dogs, the best known being the heartworm disease caused by Dirofilaria immitis. Live heartworms can cause endarteritis and muscular hypertrophy of arteriole walls, especially in the caudal pulmonary arteries. If left untreated, these alterations lead to pulmonary hypertension and right-sided congestive heart failure [22,29]. In horses, filarial infections can be caused by various agents, one of them being Setaria equina; this is a rather asymptomatic filariasis, although it is potentially responsible for ocular lesions [27]. Many cases of zoonoses caused by mammalian filariae belonging to the two subfamilies Onchocercinae and Dirofilariinae have been identified [30]. Two rare zoonoses have also been described: one caused by Meningonema peruzzii, from the subfamily Splendidofilariinae [31], and another caused by Pelicitus sp., an avian filaria [32]. Many filariae harbour Wolbachia, a Gram-negative intracellular a-proteobacterium (order Rickettsiales). Up to now, Wolbachia bacteria have been identified in two of the eight subfamilies of Onchocercidae, namely the Onchocercinae and the Dirofilariinae (Fig. 1) [33,34]. Among them, Wolbachia bacteria are present in all human filariae except Loa loa [35], and have not yet been analysed in Mansonella streptocerca (Table 1). Wolbachia bacteria are vertically transmitted to the filarial progeny through the female germline. They contaminate numerous classes of arthropods (40% of terrestrial arthropod species are infected [36]), but only a single family of nematodes, the Onchocercidae [37]. Wolbachia bacteria have not been detected in any other nematode groups [38,39], although their presence in Radopholus similis, a plant-parasitic nematode, has been suggested [40]. Wolbachia bacteria are commonly classified in different supergroups, although they are attributed to one species, namely Wolbachia pipientis. These supergroups show an asymmetric distribution within a large host range: supergroups A, B, E, H, I and K are found in arthropods; and supergroups C, D and J are limited to filariae. Interestingly, supergroup F encompasses both arthropod and filarial hosts [37,41 44]. The Evolutionary History of the Symbiosis and the Basis of Mutualism Until recently, it was hypothesized that Wolbachia infection may have been acquired as a single event in the Onchocercidae [34,45], and that there was a congruency between the phylogenies of Wolbachia and the filarial host [45]. Analyses of new onchocercid species have challenged these features, and the relationships between Onchocercidae and Wolbachia appear to be more complex. The presence of Wolbachia in Onchocercidae has been overestimated: from 89.5% in 2003 [39], more extensive analyses have led this estimate to be reduced to 37% [37]. The absence of Wolbachia in these Onchocercidae could be linked to two types of evolutionary event. First, the absence of Wolbachia is likely to be an ancestral condition [33,34,37]. Indeed, the morphologically primitive Onchocercidae, such as the Waltonellinae and Oswaldofilariinae subfamilies, have no Wolbachia (Fig. 1). This suggests that these filariae diversified before the first Wolbachia infection in the Onchocercidae lineage. Second, it was recently shown that an unexpectedly high number of secondary losses occurred in Dirofilariinae and Onchocercinae. These losses are supported by the identification of Wolbachia-like gene sequences in filarial host genomes of species that do not harbour Wolbachia, such as Onchocerca flexuosa and Acanthocheilonema viteae [46]. The hypothesis of acquisition by a single event seems to be contradicted by the identification of supergroup F. Indeed, the existence of this supergroup, which includes Wolbachia from both arthropods and nematodes, reveals that another acquisition event has happened more recently and independently from the one leading to supergroups C and D [37,41]. Up to now, the nature of the symbiosis between Wolbachia and Onchocercidae has been considered to be mutualistic, as both partners seem to benefit from the association [47]. Indeed, many studies have investigated the effects of antibiotic chemotherapy on various Onchocercid species, and have demonstrated that depletion of Wolbachia is associated with stunting, sterilization and death of adult worms [8,48,49]. The evidence for mutualism was strengthened by the analysis of the complete genomes of the Wolbachia strain from the human

134 Clinical Microbiology and Infection, Volume 19 Number 2, February 2013 CMI FIG. 1. The presence of Wolbachia infection mapped on the hypothetical evolution and distribution of Onchocercidae. The eight subfamilies of Onchocercidae are represented on the pie chart ; the sector size of each subfamily is based on the number of onchocercid genera present per subfamily. The host range is shown by a symbol: the crocodile represents clade Reptilia, the frog represents class Amphibia, the bird represents class Aves, and the monkey represents class Mammalia. For each subfamily, X/Y represents the number of genera in which the presence of Wolbachia was analysed/the total number of genera in the subfamily. Underneath X/Y and below the black arrow, the number of genera that harbour Wolbachia (Wb+) is specified. The dendrogram in the centre of the diagram represents the hypothetical evolutionary history of Onchocercidae, based on morphological criteria. Wolbachia was absent from the lineages leading to Oswaldofilariinae, Waltonellinae, Setariinae, and Splendidofilariinae. Wolbachia of supergoup C was acquired on the lineage leading to the Dirofilariinae. Wolbachia was acquired on the lineage leading to the Onchocercinae, diverging into supergroups C, D, J, and F. lymphatic filaria Brugia malayi [50] and the bovine tissular filaria O. ochengi [51]. This revealed that the genome of Wolbachia carries the genes required for haem metabolism and/or riboflavin, whereas its filarial host does not [50 52]. An interaction between Wolbachia and the host s iron metabolism has been recently demonstrated in a parasitoid wasp [53]. Thus, one can argue that Wolbachia could help filarial nematodes to acquire and keep iron, as observed in arthropods. However, how transport, degradation and regulation of haem occurs within filarial parasites remains an open question [54]. In filariae, Wolbachia bacteria are present in all larval stages, although their density varies between individual worms and throughout the developmental stages [55,56]. Usually, Wolbachia bacteria are localized in the hypodermal cells of the lateral cords in filariae [57,58]. Wolbachia bacteria are also found in the ovaries, in the oocytes, and throughout embryonic development in the uterus of females. However, they are never found in the male reproductive system [59,60]. It was recently shown that Wolbachia bacteria of supergroups C and D follow the same pattern of embryonic segregation: through asymmetric mitotic segregation, they reach a subset of hypodermal cells [57]. From this hypodermic localization, Wolbachia bacteria then have to invade the ovaries [59,61]. However, the tissue distribution of Wolbachia bacteria in filariae can be more complex: for example, a supergroup F Wolbachia bacterium has been detected in the intestinal cells but not in the hypodermis of Mansonella (Cutifilaria) perforata [37]. Consequently, this pattern of tissue localization raises the issue of different bacterial segregation during embryogenesis, reflecting different tropisms of the bacteria in their hosts. Furthermore, a nutritional benefit of supergroup F Wolbachia for the host has been documented in the bedbug Cimex lectularius, wherein the antibiotic depletion of

CMI Bouchery et al. Wolbachia and filariasis 135 the symbionts can be counteracted by oral supplementation with B vitamins [62]. The role of Wolbachia in Filarial Biology and Development of Pathology From the entry of the parasite to the establishment of the chronic disease, Wolbachia plays multiple roles, such as exacerbating proinflammatory pathogenesis and immunomodulation of the host, as well as enhancing survival of the filariae. During the early phase of the infection, the inflammation mediated by Wolbachia in the skin favours the vascular migratory pattern of the Litomosoides sigmodontis infective larvae and their subsequent establishment in the pleural cavity of the murine host. For example, CCL17 is involved in the early immune responses limiting filarial parasite invasion into the host [63]. Indeed, genetic deficiency of CCL17 or administration of anti-ccl17 antibodies results in mast cell accumulation and degranulation, enhanced vascular permeability, and increased filarial load. This mechanism is dependent on the presence of Wolbachia endosymbionts in infective L3 larvae and is promoted by Toll-like receptor (TLR)2-dependent signalling [63]. Wolbachia can trigger a proinflammatory response through interaction with monocytes/macrophages, dendritic cells, and neutrophils [64 66]. This role of Wolbachia can be replicated in vitro by exposing innate immune cells to parasite extracts. Indeed, antigen-presenting cells stimulated with whole Wolbachia-containing worm extracts release high quantities of inflammatory cytokines, whereas stimulation with Wolbachia antibiotic-depleted worm extracts or with extracts of Wolbachia-free filariae does not trigger such a response [67 69]. In addition, various pathogen-associated molecular pattern candidates for Wolbachia have been shown to mediate the inflammatory response, such as diacylated lipoproteins, which drive the innate inflammatory response and Th1 polarization [68], Wolbachia surface protein (which is a highly expressed surface protein of Wolbachia), which triggers both inflammation and expression of regulatory markers such as cytotoxic T- lymphocyte antigen 4 on T-cells [70], heat shock protein 60, which induces inflammatory cytokine release by monocytes and their subsequent apoptosis [71,72], and, potentially, GroEL, even though this has not yet been formally proved [51]. The recognition of Wolbachia pathogen-associated molecular patterns has been shown to be dependent on TLR2/TLR6 recognition and signalling [68,69] and, to a lesser extent, on TLR1 recognition. Long-term exposure to Wolbachia proteins drives macrophage/monocyte tolerance both in vitro and in vivo [67]. The role of Wolbachia in filarial biology may be seen as a defensive mutualism. For example, the presence of Wolbachia in O. ochengi contributes to the recruitment of a high level of neutrophils in the vicinity of the filaria in the nodule [73]. In contrast, after depletion of Wolbachia by antibiotic treatment, eosinophils are recruited in the vicinity of O. ochengi and are effective for filarial destruction [74 76]. If the treatment is stopped, the recrudescence of Wolbachia can reconstitute the neutrophilia. Wolbachia thus prevents the degranulation of eosinophils by triggering an ineffective neutrophil response by the host [73]. Increasingly, Wolbachia is being revealed as an important cause of pathology in filarial diseases. Symptoms are correlated with increases in the levels of circulating Wolbachia protein and DNA, and with the detection of antibodies directed against the endosymbiont [77 79]. Such an increase in the detection of Wolbachia in blood of patients may be attributable, in part, to the natural excretion of Wolbachia products by the worms, or the moribund microfilariae or adults that release Wolbachia [61,66,80]. Most of the pathology observed in filariases results from the inflammatory response. In onchocerciasis, the main pathology is caused by the migration of microfilariae to the cornea, where the inflammatory response leads to corneal inflammation of variable severity. This pathology is neutrophil-mediated through Wolbachia TLR2 activation of macrophages and local stromal cells. These cells produce proinflammatory cytokines and CXC chemokines, which mediate neutrophil recruitment [81,82]. In chronically infected individuals, the repeated invasion of microfilariae and their subsequent death perpetuates the inflammatory cell influx, which causes permanent tissue damage, owing to neutrophil degranulation and secretion of cytotoxic products such as nitric oxide, myeloperoxidase, and oxygen radicals [83]. In lymphatic filariasis, the presence of elevated levels of lymphangiogenic factors has been shown to be associated with the severity of the pathology [84,85]. As TLR2 stimulation by extracts of B. malayi has been shown to trigger vascular endothelial growth factor A and angiopoietin-1 production [86], it is possible that Wolbachia is involved in the pathogenesis. This role has been indirectly confirmed by Wolbachia depletion with doxycycline treatment in Bancroftian patients, in whom a reduction in the dilatation of the scrotal lymph vessels was observed [87]. Wolbachia as a Filaricidal Target Current mass drug administration programmes designed to control and eliminate filariasis support the use of diethylcarbamazine or

136 Clinical Microbiology and Infection, Volume 19 Number 2, February 2013 CMI ivermectin, each given in combination or not with albendazole [88]. These agents are potent microfilaricides, but have limited macrofilaricidal effects. As adult filariae are long-lived and fecund for most of their lifetime, patients must be treated for many years for there to be any chance of breaking the transmission cycle [89]. Furthermore, several researchers are raising concerns about the possible development of resistance in the worms [90,91]. In some onchocercian communities, even after several rounds of ivermectin treatment, the average microfiladermia increases instead of decreasing as expected. In the area of mass therapy, both for lymphatic filariasis and for onchocerciasis, children often present with microfilariae in the blood or skin, thus indicating that the transmission is still ongoing. All in all, the need for a new therapeutic approach against filariasis has emerged. For the last decade, therapeutic research has focused on the value of Wolbachia as a filaricidal target. First, the use of antibiotics (tetracycline class) against onchocerciasis in vitro and with animal models has been shown to block transmission by disrupting embryogenesis [92 94]. This sterilization results from Wolbachia depletion, as antibiotic treatment of filariae free of Wolbachia has no influence on embryogenesis or viability of the parasite [66]. Recently, it has been demonstrated that this depletion induces the apoptosis of both the germinal line and the somatic cells in the embryos and in the microfilariae [95]. Anti-Wolbachia therapy with doxycycline (a semisynthetic derivative of the tetracycline family) has been recommended for the treatment of individuals with lymphatic filariasis and onchocerciasis [8,48,58,84,85,95 102] (Table 2). It allows the development of filariae in their vector to be blocked, thus arresting the transmission [103,104]. Furthermore, macrofilaricidal effects against onchocerciasis have been reported in humans 2 years after treatment [97,105]. Communitydirected treatment has been undertaken and been successful [106,107], although mass treatment may still be a long way off. Moreover, the use of antibiotics in areas of coendemicity with L. loa (Wolbachia-free) is safe, and does not reduce the overall efficacy of the treatment [105]. However, doxycycline requires a prolonged treatment regimen (4 6 weeks), and is contraindicated for children under 9 years old and for pregnant/breast-feeding women. No other tetracycline has been used in humans, because doxycycline is the easiest to apply to humans with regard to safety and side effects. However, the most used tetracycline in animals such as cattle is oxytetracycline [73,108]. In addition, rifampicin could be used as an alternative treatment for cases that cannot be treated with doxycycline. Indeed, it was shown that 4 weeks of rifampicin treatment can reduce filarial embryogenesis [109]; however, 1 week of treatment was not efficient [110]. Comparative genomic/transcriptomic/proteomic analyses of filarial species associated or not with Wolbachia, and before and after Wolbachia depletion, have pushed forward our knowledge of the molecular/metabolic basis of the mutualism between Wolbachia and its host, ultimately resulting in the discovery of new drug targets [52,54,111 115]. Several new approaches directed against Wolbachia are currently under investigation (Table 2) [94,109,116 122]. One of the more promising of these is corallopyronin A, a non-competitive inhibitor of the RNA polymerase of Wolbachia [117]. The blockade of RNA transcription gives rise to bacterial death and thus to Wolbachia depletion in Litomosoides sigmodontis. TABLE 2. Old and new therapies against Wolbachia Drugs Target Mechanisms Effects on filariae References Current antibiotic treatment Doxycycline/ 30S ribosomal tetracycline subunit (+50S ribosomal subunit) 1. Blockade of protein synthesis by preventing the binding of aminoacyl-trna to the ribosome 2. Activation of apoptosis of germline and somatic cells of embryos and microfilariae 3. Nitric oxide production (a) Decrease in filaria growth. (b) In vitro female sterilization; (c) Disappearance of Mfs (d) Blockage of first and third moults. (d) Death of adult worms New anti-wb approaches Berberin FtsZ Blockade of bacterial cytokinesis In vitro sterilization of Brugia malayi female [116] In vitro decrease in Wb growth in B. malayi Corallopyronin A RNA polymerase Blockade of RNA synthesis In vivo sterilization of Litomosoides sigmodontis female Decrease in Litomosoides sigmodontis growth Succinyl acetone ALAD Blockade of haem pathway (a) Reduction in motility. (b) Sterility [54] Rapamycin btor Inhibition of btor, which In vitro decrease in Wb growth in B. malayi [119] controls autophagy Globomycin LspA Accumulation of pro-lipoprotein In vitro decrease of Wb growth in B. malayi [120] in the cytoplasmic membrane In vitro reduction in motility of B. malayi Rifampicine Inhibition of RNA polymerase (a) Abnormal and decreased embryogenesis [94,105,110] (b) Reduction of the L3 to L4 moult (C) Decrease in worm growth [8,48,49,58,73,84,85,95,97 102,104,122] ALAD, 5 -aminolevulinic acid dehydratase; btor, B. malayi target of rapamycin; FtsZ, filamenting temperature-sensitive protein Z; LspA, lipoprotein signal peptidase II; Wb, Wolbachia. The target of doxycycline is mainly the bacterial 30S ribosomal subunit and, to a lesser extent, the 50S subunit. [117]

CMI Bouchery et al. Wolbachia and filariasis 137 The size of the filariae was reduced and embryogenesis was stunted, without consequences for mice. The mean level of endobacteria after treatment was even lower than the reference standard obtained with doxycycline treatment. The cytokinesis, polymerase and metabolic pathways of the bacteria have been targeted in vitro in different filariae. For example, filamenting temperature-sensitive protein Z, which is involved in bacterial cytokinesis, can be blocked by berberin treatment. Filamenting temperature-sensitive protein Z is expressed in all stages of B. malayi. This blockade interrupts bacterial division, and therefore growth of the bacterial population. Without bacterial proliferation, embryogenesis of the filaria is interrupted, and microfilarial release is decreased [116]. However, these new approaches have yet to be tested in humans. Conclusion Over the last decade, filarial research has been conducted on the assumption that the symbiotic relationship between Wolbachia and filariae could offer opportunities to control filariases. Antibiotic therapies against Wolbachia now provide a valuable means of clinical control by reducing bacteria-induced inflammation, an effective prevention strategy by breaking the parasitic cycle (e.g. reducing microfilarial production, and preventing their development in their vectors), and a way to eliminate already-established parasites (i.e. by a macrofilaricidal effect). Refinement of the target and/or tools used against the bacteria is already in progress, helped by the recent advances in omics technologies. Acknowledgements We thank A. Smith for English proofreading. Author Contributions Wrote the paper: all authors. Funding This work was supported by European Community grant FP7- HEALTH-2010-243121. A. Nieguitsela was supported by a postdoctoral fellowship from Infectiopole Sud. 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