Sashendra Aponso (5489673)
Page 1 of 9
Symbiosis in Parasites: Wolbachia bacteria of Filariae
Filarial nematodes are long threadlike roundworms that belong to the phylum Nematoda.
Some species of filariae infect humans, living parasitically in the human lymphatic system,
subcutaneously or in deep connective tissues. (Markel & Voge 274-320)
Among the most important diseases caused by Filariae is lymphatic filariasis. Transmitted by
mosquitoes, the filariae that cause lymphatic filariasis are Wuchereria bancrofti, Brugia malayi
and B. timori. The presence of Filariae in the lymph system leads to lymphedema, resulting in
swelling of the limbs (elephantiasis) and hydrocele in the scrotum. Although lymphatic filariasis
is endemic in around 83 countries, the high population levels of the tropical countries it is found
and the disability of sufferers to get on with their occupation as before mean that it has a
significant disease burden (See figure 1). In 1994 it was estimated that approximately 106.2
million people suffered from bancroftian filariasis. (World Health Organization Website)
Figure 1: Global Distribution of Lymphatic Filariasis.
Guerrant et al 2006, Tropical Infectious Diseases 2nd Ed
The widespread nature of filarial disease calls for the development of appropriate public
health interventions and therapeutic strategies. When speculating potential therapeutics for
filarial diseases, it is important to have a thorough understanding of the nature of filarial
nematodes since such an understanding reveals more therapeutic targets.
One such area that demands further investigation and has tremendous potential for
therapeutics is the symbiotic relationship between filarial nematodes and Wolbachia bacteria. In
this paper I will first examine the nature of the symbiosis that exists between Wolbachia and
Aponso
Page 2 of 9
filarial nematodes. In doing so I will identify potential opportunities for therapeutic intervention
and proceed to discuss how this symbiosis has been exploited for therapeutic purposes.
Wolbachia Symbiosis in Filarial Nematodes
Often in nature, organisms form close associations with each other. Symbiosis refers to the
close association between organisms of two different species. The association may be long term
and either or both organisms may benefit from the relationship. Depending on the relative
location of the organisms, the symbiosis may be ectosymbiosis (organism A lives on organism
B) or endosymbiosis (organism A lives within organism B). While filarial nematodes had been
previously studied, it was the advent of electron microscopy that allowed researchers to discover
the endosymbiotic relationship between filarial nematodes and Wolbachia bacteria that live
inside the nematodes.
In the 1970’s, researchers McLaren, Vincent, Kozek et al discovered intracellular bacteria
residing in filarial nematodes. (Taylor and Hoerauf 1999) As Wolbachia are also found in certain
insect species where they existed symbiotically, McLaren and his colleagues hypothesized that
the bacteria were related to insect-symbiont Wolbachia bacteria. More importantly, they
speculated that Wolbachia in nematodes contributed to the pathogenesis of filarial disease, in
which case they would be a novel target for drug therapy. (Kozek and Figueroa 1977)
Subsequent research led to the discovery of Wolbachia in several filarial species including
Wuchereria bancrofti, Onchocerca volvulus & Brugia malayi, all of which affect humans.
Electron microscopy studies have shown that within the nematode, Wolbachia bacteria are
confined to the hypodermal cells of the lateral cord (the layer immediately below the cuticle) and
reproductive tissues of the female worm (ovaries, oocytes, developing embryonic stages in the
uterus)
The latter location suggests the high probability of vertical transmission from mother to daughter
nematode via egg cell cytoplasm (Kozek et al. 1977). The bacteria were found throughout all
stages of the life-cycle (Kozek 1977, McGarry et al 2004). Wolbachia bacteria can exist
individually or in groups and often live in a host-derived vacuole inside the host tissue. See
figure 2 below.
Figure 2: Clusters of Wolbachia within a filarial nematode (a) and
scattered within the nematode’s lateral
cord (b). Taylor et al, 1999
2
Aponso
Page 3 of 9
Figure 3: Wolbachia (red) in O. volvulus.
A
B
Taylor et al. 2005. Image courtesy D.W.
Buttner.
Although clusters of Wolbachia are found
in nematode tissue, there is no evidence of a
pathological effect on the nematode itself. One
observation, however, was that heavily infected
eggs appeared to have arrested development (1)
and that these bacteria are not present in male
reproductive tissue (Taylor & Hoerauf, 1999,
Sacchi et al, 2002, Kozek, 2005). Figure 3 (left)
shows Wolbachia stained in red, present in the
hypodermal tissue (labeled A) and within
embryos in a nematode uterus. (Taylor et al 2005)
With regards as to how the bacteria entered the
nematodes to begin with, it is speculated that
Wolbachia which initially infected only insects,
spread from arthropods to nematodes at a certain
point in time. This however, is still to be
determined. (Foster et al. 2005)
Having
explored
the
physical
microenvironment of Wolbachia in filarial
nematodes, we now turn our attention to the
nature of the symbiosis. Genomic analysis of the
Wolbachia symbiont of B. Malayi (wBm) has
given us information regarding the nature of the
symbiosis and what each party contributes and
gains from the association. (Foster et al, 2005)
Bacterial Contribution to the Symbiosis
1) Supply of metabolites- Wolbachia may supply the nematode with essential metabolites.
Filarial nematodes (W. bancrofti & B. Malayi) lack the genes required for riboflavin
biosynthesis. (Ghedin et al. 2004) wBm on the other hand, contains all the enzymes
required for producing riboflavin and flavin adenine dinucleotide. (FAD) In this light,
Wolbachia appears to be a source of these important coenzymes for the host nematode.
(Foster et al. 2005) wBm has almost all the components required for Isoprenoid (a lipid
subfamily) synthesis. The wBm genome has all but one gene for the biosynthesis of
isoprenoids. It is possible that the missing gene-product (1-deoxy-D-xylulose-5-phosphate)
is supplied by the host. Studies of B. Malayi suggest that Heme may be provided by wBm
and play an important role in filarial reproduction and development. (Narita et al 1999)
Heme functions as a prosthetic group of cytochromes and Wolbachia contain genes for all
but one of the components of the heme biosynthesis pathway. Foster et al (2005) speculate
that the function of the missing gene may be complemented by the function of another
gene, thus producing heme that the nematode host can use.
2) Supply of nucleotides- wBm has complete biosynthesis pathways for de novo biosynthesis
of purines and pyrimidines. Most other endosymbionts and parasites like Buchnera and
3
Aponso
Page 4 of 9
Rickettsia lack this. This suggests that the bacteria produce nucleotides not only for internal
consumption but for supplementation of the nucleotide pool of the host when needed (egduring oogenesis and embryogenesis, when the requirement for DNA synthesis is very
high) (Kuhn et al 1978)
The Nematode’s Contribution to the Symbiosis
1) Supply of amino acids- Wolbachia has limited biosynthetic capabilities. It is capable of
synthesizing only one amino acid (meso-diaminopimelate) which is a major constituent
of peptidoglycan. (Foster et al 2005) Since it requires several other amino acids, which it
cannot otherwise obtain or synthesize for itself, it can be concluded that it obtains these
amino acids from the nematode. The proposed mechanism of obtaining amino acids is
through the proteolysis of host proteins. This is strongly supported by the fact that the
wBm genome is shown to encode a variety of different proteases.
2) Supply of biosynthesis precursors- as with most endosymbionts, Wolbachia lacks
complete pathways for de novo biosynthesis of vitamins and cofactors such as Coenzyme
A, NAD, biotin, lipoic acid, ubiquinon, folate and pyridoxal phosphate. Interestingly. In
Wolbachia retains genes for the final steps in a few of these pathways. Foster et al (2005)
suggest that having these incomplete biosynthesis pathways makes Wolbachia dependent
on the supply of the required precursors from its nematode host.
3) Provision of a growth environment- apart from the amino acids, nematode reproductive
tissues and hypodermal tissues serve as an environment in which Wolbachia is able to
grow and reproduce, propagating itself. Researchers also suggest that the environment
inside the worm is abundant in compounds that serve as growth substrates for Wolbachia.
Of particular interest are pyruvate and TCA cycle intermediates as these are produced as
byproducts of nematode respiration. nMRI studies of B. Malayi nematodes have shown
that phosphophenolpyruvate is a major energy reservoir. (Shukla-Dave et al 1999)
The following schematic diagram (figure 4) summarizes the mutualistic nature of the symbiosis.
Figure 4
Metabolites, riboflavin,
FAD, heme, nucleotides.
Amino acids, growth factors
required for bacterial growth
4
Aponso
Page 5 of 9
Exploiting the Symbiosis for Therapeutic Purposes
The close symbiosis of filarial nematodes and Wolbachia bacteria opens up a new avenue
of treatment possibilities wherein we exploit the dependence of the nematode on the worm, for
the purposes of eliminating it. Review of recent literature on anti-Wolbachia treatments has
shown that these therapeutics act in two ways, as will be discussed in the following sections.
1) They have been shown to cause sterility in Filariae
2) They appear to be strong macrofilaricides, killing Wolbachia and as a result, causing
death of adult worms.
A) Sterilizing Effect of Anti-Wolbachia Therapeutics
The presence of Wolbachia in the reproductive tissues of female filarial nematodes has
given researchers clues as to a possible contribution by the bacteria towards nematode
reproduction. Clinical trials have used doxycycline as the drug of choice in eliminating
Wolbachia in the nematodes of patients. In lymphatic filariasis patients infected with W.
bancrofti, a 6 week regimen of Doxycycline (200mg/day) was shown to cause a >95% reduction
in Wolbachia levels. Bacteria level was assessed from blood microfilariae and compared to pretreatment levels. (Hoerauf et al, 2003)
Trials of Doxycycline against O. volvulus nematodes have resulted in the block of
embryogenesis. This was seen in long term reduction of skin microfilaria. Most importantly, the
effects of blocking embryogenesis via anti-Wolbachia treatment were shown to persist for up to
two years after initiating the treatment. (Hoerauf et al. 2000, 2003) This was especially
significant when compared to ivermectin controls since doxycycline treatment was seen to bring
about a dramatic decline in microfilarial load and a subsequent period of amicrofilaremia
(absence of microfilaria) that was sustained for approximately 2 years. The hypothesis proposed
by Hoerauf and colleagues is that the block in embryogenesis in the adult female worm occurs
when Wolbachia are below a particular threshold or are entirely absent. (Taylor et al, 2005)
While human clinical trials investigating the sterilizing effect of anti-Wolbachia
therapeutics have shown clearly that embryogenesis is disrupted in adult female nematodes,
studies in animal models have allowed us to get a better understanding of the exact effects of
how killing the bacteria interferes with the nematode’s reproductive function.
While Doxycycline has been the drug of choice for human clinical trials, animal studies
have employed the antibiotic tetracycline. As Hoerauf and colleagues demonstrated with regards
to doxycycline, Bosshardt et al (1993) demonstrated that administering tetracycline to jirds that
were infected with B. pahangi resulted in the inhibition of third-stage larvae developing into
adult worms and the development of microfilaremia (presence of microfilaria).
L. sigmodontis (a nematode) is commonly used as a mouse model of human filarial
diseases. A 1999 study by Hoerauf et al examined bacterial and nematode responses to
tetracycline treatment. Immunohistology of bacterial hsp60 was used to visualize the bacteria,
enabling researchers to trace changes in bacterial density in relation to worm tissue development.
Immunohistology and electron microscopy revealed that after 28 days of tetracycline treatment,
the Wolbachia bacteria were completely eliminated. However the worm ovaries in which
5
Aponso
Page 6 of 9
Wolbachia had been eliminated were degenerated, as seen using microscopy. The treatment was
found to cause a block of filarial establishment, decreasing worm fertility (as seen by the
degenerated ovaries) and causing a 90% decrease in amicrofilaremia. See figures 5 and 6 below.
Figure 5- Tetracycline therapy in a mouse model of filarial infection
Mice carrying L. sigmodontis
nematode (infected with
Wolbachia).
Mice carrying A. vitaea
nematode (not infected
with Wolbachia)
Tetracycline treatment (28 days)
Tetracycline treatment
(28 days)
Bacteria eliminated
No effect on worm
development and
fertility.
Adult worms rendered infertile.
Block of worm establishment &
growth. Ovary degeneration of
nematodes. 90% reduction in
fertility.
Figure 6:
(g) Cross-section of an adult L sigmodontis
female depicting parts of the uterus with bacteria
in morulae (mo) and microfilariae (mf)
(h) Longitudinal sections of an adult L
sigmodontis female from a tetracycline-treated
mouse. No staining is seen in the lateral
hypodermal cords (lhc) and in the oocytes (oc) in
the ovary (ov). The oocytes are clearly
degenerated.
Image from Taylor & Hoerauf 1999. Para. Today
6
Aponso
Page 7 of 9
While the previous experiment showed reproductive degeneration upon Wolbachia
elimination, research by Bandi et al (1999) has shown that treatment with tetracycline brings
about inhibition of embryogenesis when there is loss of bacteria from all but the caudal end of
the nematode’s reproductive tract. Dose-reduction experiments have been conducted to
determine the minimum dose required to destroy the bacteria. These have revealed that it is
impossible to destroy the bacteria without rendering the nematodes unviable in the process.
(Bandi et al 1999) This brings us to the second feature of anti-Wolbachia therapeutics; their
macrofilaricidal properties.
Macrofilaricidal Effect of anti-Wolbachia therapeutics:
While we have seen above how anti-Wolbachia treatments render worms infertile, Taylor
and colleagues showed that doxycycline also acts to directly kill the adult worm. (Stolk et al
2005) This adult-killing (macrofilaricidal) property of doxycycline is important since it expands
the potential stages of the filarial life cycle that we are able to target and disrupt. Drugs such as
ivermectin are largely microfilaricidal, so the macrofilaricidal properties of novel anti-Wolbachia
treatments present the possibility of being used in combination with previously used
microfilaricidal treatments, for sustained filarial elimination.
When performing clinical trials of anti-Wolbachia treatment in humans, Taylor and his
colleagues noticed a dramatic reduction in the number of worm nests (in the patient’s scrotum)
and filarial antigen levels in the blood within 14 months of treatment. Using ultrasound to detect
an 80% difference in the mean number of worms in controls versus doxycycline-treated
individuals, the researchers concluded that approximately 80% of adult worms had been
eliminated. (Taylor et al. 2000) The following table indicates the macrofilaricidal effect of
Doxycycline in comparison to conventional microfilaricidal drugs.
(Image from Stolk et al 2005, The Lancet)
7
Aponso
Page 8 of 9
The high efficacy of Doxycycline as a macrofilaricide and its use as a novel therapeutic
are significant since:
1) Novel therapeutics are required in the anticipation of filariae developing drug resistance
to the mass treatment programs that have been implemented globally to eliminate filarial
diseases such as lymphatic filariasis. (eg- DEC administration via table salt)
2) The severe side effects of DEC on Onchocerca-infected individuals means DEC it cannot
be used in certain African countries. Ivermectin is contraindicated in Loa Loa endemic
countries. Thus, a novel therapeutic that has no such side-effects would be invaluable.
Taylor and colleagues (2005) add that although the current 8 week regimen of Doxycycline
(200mg daily) is not suitable for mass administration, it would be interesting to see the regimen
applied to mass treatment. This would possibly call for refining the model to determine the
optimum drug requirement that can be administered, minimizing overall cost but still
maintaining efficacy.
As we have seen, Wolbachia bacteria and filariae have a close symbiotic relationship.
Having understood the nature of the relationship, we are able to exploit the association between
these organisms to eliminate filariae from the human host. The sterilization of female nematodes
and death of adult filariae brought about by antibiotic treatments such as doxycycline (in
humans) and tetracycline (in animal models) demonstrates the efficacy of anti-Wolbachia
treatments as therapeutics. While previous anti-filarial therapies have been primarily
microfilaricidal, the macrofilaricidal properties of anti-Wolbachia therapeutics mean that they
may be developed to be used in conjunction with or to replace existing anti-filarial treatments.
The novel therapeutic target offered by taking advantage of the symbiosis has greatly broadened
the treatment of filarial diseases and shows tremendous promise in reducing the worldwide
burden of these diseases.
8
Aponso
Page 9 of 9
Bibliography:
 Taylor, M.J, and A Hoerauf. "Wolbachia Bacteria of Filarial Nematodes." Parasitology
Today. 15.11 (1999): 437-442.
 Kozek, W.J. and Figueroa, M. (1977) Intracytoplasmic bacteria in Onchocerca volvulus.
Am. J. Trop. Med. Hyg. 26, 663–678
 Kozek, W.J. (1977) Transovarially-transmitted intracellular microorganisms in adult and
larval stages of Brugia malayi. J. Parasitol. 63, 992–1000
 McGarry, H.F., Egerton, G. and Taylor, M.J. (2004). Population dynamics of Wolbachia
bacterial endosymbionts in Brugia malayi. Molecular and Biochemical Parasitology 135,
57–67.
 Sacchi, L., Corona, S., Casiraghi, M. and Bandi, C. (2002). Does fertilizationin the
filarial nematode Dirofilaria immitis occur through endocytosis of spermatozoa?
Parasitology 124, 87–95.
 Kozek, W. J. (2005). What is new in the Wolbachia/Dirofilaria interaction? Veterinary
Parasitology (in press).
 Foster, Jeremy. "The Wolbachia Genome of Brugia malayi: Endosymbiont Evolution
within a Human Pathogenic Nematode." PLoS Biology. 3.4 (2005): 599-614.
 Ghedin ES, Wang S, Foster JM, Slatko BE (2004) First sequenced genome of a parasitic
nematode. Trends Parasitol 20: 151–153. Narita S, Taketani S, Inokuchi H (1999)
Oxidation of protoporphyrinogen IX in Escherichia coli is mediated by the aerobic
coproporphyrinogen oxidase. Mol Gen Genet 261: 1012–1020.
 Kuhn O, Tobler H (1978) Quantitative analysis of RNA, glycogen and nucleotides from
different developmental stages of Ascaris lumbricoides(var. suum). Biochim Biophys
Acta 521: 251–66. Shukla-Dave A, Degaonkar M, Roy R, Murthy PK, Murthy PS, et al.
(1999) Metabolite mapping of human filarial parasite, Brugia malayi, with nuclear
magnetic resonance. Magn Reson Imaging 17: 1503–1509.
 Hoerauf, A., Mand, S., Adjei, O., Fleisher, B. and Bu¨ ttner, D.W. (2001). Depletion of
Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia
after ivermectin treatment. Lancet 357,1415–1416
 Taylor, Mark, and Claudio Bandi. "Wolbachia Bacterial Endosymbionts of Filarial
Nematodes." Advances in Parasitology 60. (2005): 246-274.
 Stolk, Wilma . "Anti-Wolbachia Treatment for Lymphatic Filariasis." Lancet 365.
(2005): n. pag. Web. 11 Feb 2010. <www.thelancer.com>.
 Saridaki, Aggeliki, and Kostas Bourtzis. "Wolbachi: more than just a bug in insects
genitals." Current Opinion in Microbiology. 13. (2010): 67-72.
 15 Bosshardt, S.C. et al. (1993) Prophylactic activity of tetracycline against Brugia
pahangi infection in jirds (Meriones unguiculatus). J. Parasitol. 79, 775–777
 Hoerauf, A. et al. (1999) Tetracycline therapy targets intracellular bacteria in the filarial
nematode Litomosoides sigmodontis and results in filarial infertility. J. Clin. Invest. 103,
11–18
 Bandi, C. et al. (1999) Effects of tetracycline on the filarial worms Brugia pahangi and
Dirofilaria immitis and their bacterial
 Taylor MJ, Bandi C, Hoerauf AM, Lazdins J. Wolbachia bacteria of filarial nematodes: a
target for control? Parasitol Today 2000; 16: 179–80.
9