Endosymbiotic Wolbachia of parasitic filarial nematodes as drug targets

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1 Review Article Indian J Med Res 122, September 2005, pp Endosymbiotic Wolbachia of parasitic filarial nematodes as drug targets Ramakrishna U. Rao Department of Internal Medicine, Infectious Diseases Division,Washington University School of Medicine, St. Louis, MO, USA Received October 20, 2004 The parasitic nematodes Wuchereria bancrofti, Brugia malayi and B. timori cause a dreadful disease in humans known as lymphatic filariasis, which afflicts more than 120 million people worldwide. As per recent epidemiologic estimates on prevalence of W. bancrofti and B. malayi, about 428 million people are at risk, with 28 million microfilaria carriers and 21 million clinical cases spread out in 13 States and 5 Union Territories of India. The Indian subcontinent that comprises Bangladesh, India, Maldives, Nepal and Sri Lanka harbours 50 per cent of the world s lymphatic filarial disease burden. Recently, an endobacterium of Wolbachia species that belongs to the family Rickettsiaceae was found in all life cycle stages of these nematodes and the transmission is exclusively vertical through the embryonic stages of the female worms. People with filariasis have been exposed to these Wolbachia bacteria or their proteins by the natural killing of parasites. Wolbachia have also been identified occasionally in body fluids of infected patients. Evidence suggests that these Wolbachia are mutualistic symbionts and can be cured from the nematodes by several antibiotics having antirickettsial properties. Treatment of nematodes with tetracyclines affect Wolbachia and they get cleared from worm tissues; and this elimination causes reproductive abnormalities in worms and affect worm s embryogenesis, resulting in sterility. Although it is impractical, prolonged treatment with doxycycline significantly reduces the numbers of microfilaria in circulation, which is an important strategy to control transmission of filariasis by mosquito vectors. In this review, the current knowledge of Wolbachia as a drug target and potential ways to reduce the infection through anti-wolbachia treatments is discussed. Key words Antibiotics - Brugia malayi - diethylcarbamazine - doxycycline - embryogenesis - endobacteria - filariasis - Onchocerca volvulus - rickettsia - Wolbachia - Wuchereria bancrofti Biology of filarial Wolbachia Wolbachia of filarial nematodes (Fig.) are the obligate intracellular alpha-proteobacteria and have some resemblances with insect Wolbachia. They were first found in hypodermal tissues of lateral chords, uterine wall and in embryos of filarial nematodes (Figs 199 A, B), embedded as single or multiple organisms in host-derived vacuoles. They attain different shapes (oval, round or rod- shaped) and are µm in size. The body is covered with a double membrane enclosing the cytoplasm rich in dense ribosomes (Fig. C). In late 1970s, two groups first identified these endobacteria in filarial worms and speculated that

Filarial worm -M (~ 4-5.5 cm) Filarial worm -Y (~ 1.3-2.

microscopy showing (arrow) Wolbachia endobacteria (C).

2 Filarial worm -M (~ cm) Filarial worm -Y (~ cm) Filarial Wolbachia (~ µm) Fig. Adult Brugia malayi filarial nematode worms (A & B) and a section of female worm s hypodermis under transmission electron microscopy showing (arrow) Wolbachia endobacteria (C). Magnifications, 10x (A, B). Scale bars, 1 cm (A, B) and 1µm (C). 200 INDIAN J MED RES, SEPTEMBER 2005

3 antibiotics could be used to treat filarial infections 1-3. All life cycle stages of filarial worms are infected with these bacteria, but the intensity of the infections varies between the life cycle stages 4,5, and appears that they have their own developmental life cycle within the worms which is yet to be clearly defined. Several filarial nematodes have been shown to contain these bacteria 6,7 and, interestingly, only a few species (for example: Loa loa, Acanthocheilonema viteae, Setaria equina and Onchocerca flexuosa) do not carry these bacteria 7-9. It is common in several other bacterial infections, that they can crossover from one host to another. Studies suggest that Wolbachia in filarial nematodes have coexisted for several million years and have not crossed over from their intermediate hosts (mosquitos for example) recently 10,11. However, loss of Wolbachia across the nematode family was reported during their evolution 7. Identification of new molecules in drug discovery research against filarial nematodes was boosted by the observation that Wolbachia can be used as a drug target and thus hold great promise towards therapeutic options available for filariasis treatment. Wolbachia as a target for therapy in animal models We and others 12,13 have shown that antibiotics active against Rickettsiaceae, particularly the tetracyclines, rifampicin and chloramphenicol, were effective in reducing the filarial larval molt (from L3 to L4) and their development in vitro. In contrast, effect of tetracycline analogues lacking antirickettsial properties also affected larval molting indicating that the drug might have other pharmacological effects on worms 14. In Brugia infected animals, tetracycline was prophylactic and affected the molting of infective larvae 15-17, and caused distortion of male/female sexratios 15. Sex-ratio bias by Wolbachia has a positive influence on insect population. Accordingly, Wolbachia may produce sex-ratio distortion during nematode development as well. This scenario would have profound implications in filarial biology as more females survive to produce millions of microfilariae and the role of males is restricted to reproduction. Antibiotics also affect adult filarial worms in vitro by reducing their ability to produce microfilariae and their viability 18. Several reports have shown effects of antibiotics on filarial nematodes in experimental RAO: WOLBACHIA AS TARGETS FOR THERAPY 201 animal models 16,17, More importantly members of tetracycline family (tetracycline, oxytetracycline, doxycycline, and minocycline) were found to be effective against worms. These antibiotics also affect Wolbachia after treatment Modes of action of these antibiotics are generally on bacterial RNA polymerases, protein synthesis, and other processes, and these agents may affect similar pathways in both worms and their Wolbachia. In several nematode worm infections these antibiotics have multiple effects on worm growth and development; worm fertility (particularly female worm embryogenesis) and worm survival, with evidence suggesting that prolonged treatment can be detrimental to worms 19,21. Moreover, when microfilaraemic animals were treated, their microfilarial numbers were considerably reduced in the circulation 19. In contrast, in animals infected with aposymbiotic A. viteae worms, which do not carry these bacteria, similar long-term treatment had no effect on worm biology and development 19, suggesting that these bacteria play a very important role in the growth and reproduction of the filarial worms that harbour them. The combination studies with rifampicin in animal models have been found promising to achieve acceptable short-term regimen plans with doxycycline 24. Interestingly, in addition to anti-wolbachia properties 18-23, tetracyclines markedly affected the normal embryogenesis profiles by causing damage and degeneration of intrauterine embryos 18-23,25. Polymerase chain reaction (PCR) assay also confirmed the clearance of Wolbachia DNA after prolonged therapy 20,23. The reduction or clearance of bacterialspecific hsp60 and Wolbachia surface protein (WSP) as determined by immunohistochemical staining indicated the absence or clearance of Wolbachia in treated worms 20,26. Wolbachia as a target of therapy against pathogenic human filarial infections The availability of safe drug doxycycline has encouraged clinical investigators to test their hypothesis that elimination of Wolbachia is beneficial in reducing the human filarial infections. The first clinical trials were done in people having onchocerciasis infections. A 6 wk course of daily doxycycline treatment (100 mg/day) depleted Wolbachia in worms, and caused extensive

4 202 INDIAN J MED RES, SEPTEMBER 2005 degeneration of embryos by 4 months posttreatment 27. The worms became sterile after the loss of Wolbachia, and infected individuals also had significantly fewer or no microfiladermia 27. The combination therapy with doxycycline and ivermectin also remarkably reduced microfiladermia following reductions in Wolbachia in worms 28,29. Similar effects were observed in W. bancrofti- infected patients after multiple doses of doxycycline (200 mg/day for 6 wk) 30. In this study, patients were treated with doxycycline followed by a single dose of ivermectin. Doxycycline treatment alone reduced Wolbachia numbers (96%) after 4 months of treatment, followed by 99 per cent reductions in number of microfilariae by one year of treatment. It would be interesting to see whether Wolbachia can repopulate in these worms after cessation of antibiotic therapy. Additional studies are needed to effectively measure macrofilaricidal activity of these drugs in such clinical studies. Interestingly, doxycycline treatment showed no effect on Loa loa (free of Wolbachia) infections in humans 31. Despite this demonstrated efficacy, multi-dose antibiotic therapy and their mass treatment regimens remain impractical especially in children and pregnant women 32. Therefore, the efficacy of short-term antibiotic treatments along with antifilarial drug combinations such as diethylcarbamazine (DEC) and albendazole in various endemic countries remains to be tested. Other than doxycycline treatment studies in selective populations carrying onchocerciasis, loaisis, or lymphatic filariasis 27-31, no clinical trials with this potent antibiotic has been reported in other endemic areas. Therefore, it is still premature to have a consensus regarding the effective universal dosage and duration of treatment for either microfilaria clearance or adult worm sterility. Moreover, the results of treatment may be affected by the immunological status of the host, age, host susceptibility and total worm burden. The filarial Wolbachia genome sequencing has been recently completed 33,34 and several new targets necessary for the bacteria are being identified. These might lead to investigate a new class of anti-wolbachia drugs that benefit filarial chemotherapy research (B. Slatko & J. Foster, personal communication). Perspective Human filariasis continued to be a major public health problem in parts of Indian subcontinent and other tropical areas of the world as a vector borne communicable disease The current antifilarial therapies are restricted to DEC or ivermectin in combination with albendazole Identification of new parasite molecules, biological targets (for example Wolbachia) and lead compounds against them are under way to minimize or control filariasis. The results emerged from experimental animal models and limited human studies are very promising; and targeting Wolbachia might be a strategic new approach to treat filariasis. This concept has been extensively reviewed recently More potent prophylactic antibiotic drugs or antibacterial agents in eliminating Wolbachia followed by parasites may be identified within pharmaceutical research platforms. For example, tigecycline (Wyeth Pharmaceuticals), an injectable class of tetracycline derivative that inhibits bacterial protein synthesis and cell growth which is under clinical trials can be tested against experimental filarial infections 46. Another promising approach, exploiting novel drug delivery such as liposomes, is based on the positive modulation of pharmacokinetics of drugs Incorporation of potent antibiotics into liposomes can consequently increase bioavailability and prolonged drug circulation time, usually allows for lowering of the dosage, and hence diminish adverse toxicities associated with therapy Antibiotics also have anti-inflammatory properties and therefore, it is possible to reduce posttreatment reactions (for example, ocular lesions in onchocerciasis and Mazzotti reactions) by administering these before standard antifilarial therapy. Targeting inclusion bodies of several pathogenic protozoans has always been an area of interest to develop novel therapeutics Similarly, the evidence presented so far with filarial Wolbachia strongly suggests that these worm-specific endobacteria emerge as one of the targets for reducing worm burden, fertility and transmission. Because of drug resistance and possible toxicity, general antibiotic use is not an option for widespread use. More research is needed to explore new biochemical pathways in Wolbachia life cycle that

5 are important for parasites to survive and new enzymes involved in Wolbachia growth and development; and once identified, their inhibitors might be the silver bullets to use in filarial therapy. References 1. McLaren DJ, Worms M, Laurence B, Simpson M. Microorganisms in filarial larvae (Nematoda). Tran R Soc Trop Med Hyg 1975; 69 : Kozek W. Transovarially-transmitted intracellular microorganisms in adult and larval stages of Brugia malayi. J Parasitol 1977; 63 : Kozek W, Marroquin H. Intracytoplasmic bacteria in Onchocerca volvulus. Am J Trop Med Hyg 1977; 26 : McGarry HF, Egerton GL, Taylor MJ. Population dynamics of Wolbachia bacterial endosymbionts in Brugia malayi. Mol Biochem Parasitol 2004; 135 : Fenn K, Blaxter M. Quantification of Wolbachia bacteria in Brugia malayi through the nematode lifecycle. Mol Biochem Parasitol 2004; 137 : Taylor MJ, Hoerauf A. Wolbachia bacteria of filarial nematodes. Parasitol Today 1999; 15 : Casiraghi M, Bain O, Guerrero R, Martin C, Pocacqua V, Gardner SL, et al. Mapping the presence of Wolbachia pipientis on the phylogeny of filarial nematodes: evidence for symbiont loss during evolution. Int J Parasitol 2004; 34 : Büttner DW, Wanji S, Bazzocchi C, Bain O, Fischer P. Obligatory symbiotic Wolbachia endobacteria are absent from Loa loa. Filaria J 2003; 9 : Chirgwin SR, Porthouse KH, Nowling JM, Klei TR. The filarial endosymbiont Wolbachia sp. is absent from Setaria equina. J Parasitol 2002; 88 : Bandi C, Anderson TJC, Genchi C, Blaxter ML. Phylogeny of Wolbachia in filarial nematodes. Proc R Soc London B Biol Sci 1998; 265 : Casiraghi M, Anderson TJC, Bandi C, Bazzocchi C, Genchi C. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology 2001; 122 : Smith HL, Rajan TV. Tetracycline inhibits development of the infective-stage larvae of filarial nematodes in vitro. Exp Parasitol 2000; 95 : Rao R, Weil GJ. In vitro effects of antibiotics on Brugia malayi worm survival and reproduction. J Parasitol 2002; 88 : Rajan TV. Relationship of anti-microbial activity of tetracyclines to their ability to block the L3 to L4 molt of RAO: WOLBACHIA AS TARGETS FOR THERAPY 203 the human filarial parasite Brugia malayi. Am J Trop Med Hyg 2004; 71 : Casiraghi M, McCall JW, Simoncini L, Kramer LH, Sacchi L, Genchi C, et al. Tetracycline treatment and sex-ratio distortion: a role for Wolbachia in the moulting of filarial nematodes? Int J Parasitol 2002; 32 : Bosshardt SC, McCall JW, Coleman SU, Jones KL, Petit TA, Klei TR. Prophylactic activity of tetracycline against Brugia pahangi infection in jirds (Meriones unguiculatus). J Parasitol 1993; 79 : McCall JW, Jun JJ, Bandi C. Wolbachia and the antifilarial properties of tetracycline. An untold story. Italian J Zool 1999; 66 : Rao R, Moussa H, Weil GJ. Brugia malayi: Effects of antibacterial agents on larval viability and development in vitro. Exp Parasitol 2002; 101 : Hoerauf A, Nissen-Pàehle, K, Schmetz C, Henkle-Dührsen, K, Blaxter, M L, Büttner D W, et al. Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. J Clin Invest 1999; 103 : Hoerauf A, Volkmann L, Nissen-Pàehle, K, Schmetz C, Autenrieth I, Büttner DW, et al. Targetting of Wolbachia endobacteria in Litomosoides sigmodontis: comparison of tetracyclines with chloramphenicol, macrolides and ciprofloxin. Trop Med Int Health 2000; 5 : Langworthy NG, Renz A, Meckenstedr U, Henkle-Dührsen K, Bronsvoort M, Tanya VN, et al. Macrofilaricidal activity of tetracycline against the filarial nematode, Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship. Proc R Soc London Series B 2000; 267 : Townson S, Hutton D, Siemienska J, Hollick L, Scanlon, T, Tagboto SK, et al. Antibiotics and Wolbachia in filarial nematodes: antifilarial activity of rifampicin, oxytetracycline and chloramphenicol against Onchocerca gutturosa, Onchocerca lienalis and Brugia pahangi. Ann Trop Med Parasitol 2000; 94 : Bandi C, McCall J W, Genchi C, Corona S, Venco L, Sacchi L. Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia. Int J Parasitol 1999; 29 : Volkmann L, Fischer K, Taylor M, Hoerauf A. Antibiotic therapy in murine filariasis (Litomosoides carinii): Comparative effects of doxycycline and rifampicin on Wolbachia and filarial viability. Trop Med Int Health 2003; 8 : Sacchi L, Corona S, Kramer L, Calvi L, Casiraghi M, Franceschi A. Ultrastructural evidence of the degenerative events occurring during embryogenesis of the filarial nematode Brugia pahangi after tetracycline treatment. Parasitologia 2003; 45 :

6 204 INDIAN J MED RES, SEPTEMBER Kramer LH, Passeri B, Corona S, Simoncini L, Casiraghi M. Immunohistochemical/immunogold detection and distribution of the endosymbiont Wolbachia of Dirofilaria immitis and Brugia pahangi using a polyclonal antiserum raised against WSP (Wolbachia surface protein). Parasitol Res 2003; 89 : Hoerauf A, Volkmann L, Hamelmann C, Adjei O, Autenrieth IB, Fliescher B, et al. Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis. Lancet 2000; 355 : Hoerauf A, Mand S, Adjei O, Fleischer B, Büttner DW. Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfiladermia after ivermectin treatment. Lancet 2001; 357 : Hoerauf A, Mand S, Volkmann L, Büttner M, Marfo- Debrekyei Y, Taylor M, et al. Doxycycline in the treatment of human onchocerciasis: Kinetics of Wolbachia endobacteria reduction and of inhibition of embryogenesis in female Onchocerca worms. Microbes Infect 2003; 5 : Hoerauf A, Mand S, Fischer K, Kruppa T, Marfo-Debrekyei Y, Debrah AY, et al. Doxycycline as a novel strategy against bancroftian filariasis-depletion of Wolbachia endosymbionts from Wuchereria bancrofti and stop of microfilaria production. Med Microbiol Immunol 2003; 192 : Brouqui P, Fournier PE, Raoult D. Doxycycline and eradication of microfilaremia in patients with loiasis. Emerg Infect Dis 2001; 7 : Walgate R. Could antibiotics cure river blindness? Bull World Health Organ 2002; 80 : accessed on October Foster J, Ganatra M, Kamal I, Ware J, Makarova K, Ivanova N, et al. The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode. PLoS Biol 2005; 3 : e Devaney E, Yazdanbakhsh M. Prospects and challenges in lymphatic filariasis. Parasite Immunol 2001; 23 : Freeing the World of LF: A call to action. 3rd meeting of the Global Alliance to Eliminate Lymphatic Filariasis. March 2004; Molyneux DH, Bradley M, Hoerauf A, Kyelem D, Taylor MJ. Mass drug treatment for lymphatic filariasis and onchocerciasis. Trends Parasitol 2003; 19 : Ottesen EA, Ismail MM, Horton J. The role of albendazole in programmes to eliminate lymphatic filariasis. Parasitol Today 1999; 15 : Ottesen EA. Major progress toward eliminating lymphatic filariasis. N Engl J Med 2002; 347 : Melrose WD. Chemotherapy for lymphatic filariasis: progress but not perfection. Expert Rev Anti Infect Ther 2003; 1 : Sabesan S, Ravi R, Das PK. Elimination of lymphatic filariasis in India. Lancet Infect Dis 2005; 5 : Rajendran R, Sunish IP, Mani TR, Munirathinam A, Abdullah SM, Arunachalam N, et al. Impact of two annual single-dose mass drug administrations with diethylcarbamazine alone or in combination with albendazole on Wuchereria bancrofti microfilaraemia and antigenaemia in south India. Trans R Soc Trop Med Hyg 2004; 98 : Taylor MJ, Hoerauf A. A new approach to the treatment of filariasis. Curr Opin Infect Dis 2001; 14 : Hoerauf A, Adjei O, Büttner DW. Antibiotics for the treatment of onchocerciasis and other filarial infections. Curr Opin Investig Drugs 2002; 3 : Rao R. Wolbachia in worms: endosymbiont of parasitic filarial nematodes. Recent Res Develop Ex Med TransWorld Research Network Publications (Kerala, India). 2004; 1 : Muralidharan G, Micalizzi M, Speth J, Raible D, Troy S. Pharmacokinetics of tigecycline after single and multiple doses in healthy subjects. Antimicrob Agents Chemother 2005; 49 : Sangare L, Morisset R, Omri A, Ravaoarinoro M. Incorporation rates, stabilities, cytotoxicities and release of liposomal tetracycline and doxycycline in human serum. J Antimicrob Chemother 1998; 42 : Sangare L, Morisset R, Ravaoarinoro M. In vitro antichlamydial activities of free and liposomal tetracycline and doxycycline. J Med Microbiol 1999; 48 : Bakker-Woudenberg IA, Schiffelers RM, Storm G, Becker MJ, Guo L. Long-circulating sterically stabilized liposomes in the treatment of infections. Methods Enzymol 2005; 391 : McFadden GI, Roos DS. Apicomplexan plastids as drug targets. Trends Microbiol 1999; 7 : Wilson RJ. Parasite plastids: approaching the endgame. Biol Rev Camb Philos Soc 2005; 80 : Reprint requests: Dr R.U. Rao, Department of Internal Medicine, Infectious Diseases Division, Washington University School of Medicine, 660 S Euclid Ave, St. Louis, MO 63110, USA rrao@wustl.edu

Canine and feline heartworm infections remain a

Canine and feline heartworm infections remain a Article 3 CREDITS The Emerging Role of Wolbachia Species in Heartworm Disease Kristen Frank, DVM R. Dennis Heald, DVM, DACVIM Gulf Coast Veterinary Specialists Houston, Texas Abstract: Heartworm disease