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Title: Anti-filarial Activity of Antibiotic Therapy Is Due to Extensive Apoptosis after Wolbachia Depletion from Filarial Nematodes}
Article:
Abstract:
Filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility and viability and thus has great promise as a novel approach for treating filarial diseases. However, little is known concerning the basis for this mutualistic relationship. Here we demonstrate using whole mount confocal microscopy that an immediate response to Wolbachia depletion is extensive apoptosis in the adult germline, and in the somatic cells of the embryos, microfilariae and fourth-stage larvae( L4). Surprisingly, apoptosis occurs in the majority of embryonic cells that had not been infected prior to antibiotic treatment. In addition, no apoptosis occurs in the hypodermal chords, which are populated with large numbers of Wolbachia, although disruption of the hypodermal cytoskeleton occurs following their depletion. Thus, the induction of apoptosis upon Wolbachia depletion is non-cell autonomous and suggests the involvement of factors originating from Wolbachia in the hypodermal chords. The pattern of apoptosis correlates closely with the nematode tissues and processes initially perturbed following depletion of Wolbachia, embryogenesis and long-term sterilization, which are sustained for several months until the premature death of the adult worms. Our observations provide a cellular mechanism to account for the sustained reductions in microfilarial loads and interruption of transmission that occurs prior to macrofilaricidal activity following antibiotic therapy of filarial nematodes.
Introduction:
The majority of filarial nematodes host Wolbachia bacteria in a mutualistic symbiotic association. In adult worms, the endosymbionts are situated in the hypodermal lateral chord cells, located within host-derived vacuoles. In females, Wolbachia are also found in the ovaries, oocytes and developing embryos within the uteri[1]–[4]. The mutualistic association of Wolbachia in filarial nematodes has been exploited as a novel approach to the treatment of lymphatic filariasis caused by Wuchereria bancrofti and Brugia malayi and onchocerciasis caused by Onchocerca volvulus[5]. The use of tetracyclines or rifamycins to deplete Wolbachia leads to an arrested development of larval and embryonic stages resulting in permanent sterilization of adult female worms[6]. The adult parasites die prematurely after 1–2 years following depletion of Wolbachia, compared to their typical lifespan of 10–14 years, delivering for the first time a safe and potent macrofilaricidal treatment for filariasis[5]. Although the effects of Wolbachia depletion on the development, fertility and viability of filarial nematodes has been documented( reviewed in[7]), the reason why depletion of Wolbachia leads to these anti-filarial outcomes is unknown. Here we have used whole mount confocal microscopy to observe the consequences of Wolbachia depletion on host nematode cellular and nuclear structure. Our observations reveal an extensive and profound development of apoptosis in germline cells and embryos following antibiotic depletion of Wolbachia, which occurs soon after bacterial depletion in B. malayi and is sustained for at least 21 months in O. volvulus. We find extensive apoptosis even in cells that had not been infected with Wolbachia prior to antibiotic treatment. Nuclear structure in most somatic tissues remains intact, although disruption of cytoskeleton arrangement occurs in the lateral chord cells, where the vast majority of the bacteria reside.
Discussion:
Our observations show that an extensive apoptosis of adult germline cells, embryos and somatic cells of microfilariae occurs following the depletion of Wolbachia from filarial nematodes. The development of apoptosis occurs soon after the depletion of bacteria with tetracycline in experimental infections of B. malayi in animals and in vitro. These observations were confirmed by evidence of activation of Cell death protein-3 in treated adult females, microfilaria and L4 larvae. Furthermore, apoptosis is observed in the germline cells and uterine tissues of O. volvulus at least 21 months following antibiotic treatment of people with onchocerciasis. These observations are consistent with the known anti-filarial effects of Wolbachia depletion on the rapid and sustained blockage of embryogenesis, the decline of microfilarial loads and the interruption of transmission to vectors and the arrested development of larvae to adults in the mammalian host[7],[14]–[16]. Previous studies showing that antibiotic treatment of the Wolbachia-free filarial nematode, Acanthocheilonema viteae has no effect on the viability or biological processes of this species[17], supports our conclusion that the observed apoptosis is due to the loss of Wolbachia rather than a direct effect of tetracycline treatment. The lack of apoptosis in lateral chord cells and other somatic tissues suggests the event is not a global consequence of Wolbachia depletion, which is consistent with the long and gradual decline in the viability of adult worms. In the case of onchocerciasis, permanent sterilization of adult females is a therapeutically attractive outcome, as this blocks the release of the skin dwelling microfilariae, the developmental stage that gives rise to skin and eye disease[3],[7],[14]. In lymphatic filariasis, where the adult stage is responsible for disease pathogenesis of damage to the lymphatics, the removal of adult worms is required. In both cases the permanent sterilization of adult worms will deliver important benefits for the interruption of parasite transmission via blood feeding insect vectors[5],[18]. Regulation of host cell apoptosis is common to many intracellular bacteria[19]–[21]. In many cases the bacterial pathogens commandeer conserved apoptotic regulating cascades. This requires the pathogen to reside within or be directly associated with the affected cell. A unique feature of Wolbachia’s anti-apoptotic effect is that there is no correlation between Wolbachia-populated cells and cells that undergo apoptosis upon Wolbachia depletion( Figure 9). For example, Wolbachia-laden hypodermal chords do not undergo apoptosis upon Wolbachia depletion. In contrast, while only a few cells in the early embryo( the C linage,[12]) possess Wolbachia, the majority of cells in the embryo undergo apoptosis. Similarly, while only the middle third of intrauterine ‘stretched’ microfilariae are Wolbachia-infected, apoptosis occurs throughout the entire organism upon Wolbachia depletion( Figure 9). These data clearly demonstrate that the effects of Wolbachia-depletion on apoptosis are non-cell autonomous. The non-cell autonomous behaviour implies that Wolbachia-infected cells suppress apoptosis in distant non-neighbouring uninfected cells. The source of the Wolbachia-infected cells, which are capable of this action-at-distance apoptosis suppression and when in development this occurs remains unclear. Given the abundant Wolbachia populations in the female lateral chords, it is likely that these are primarily responsible for apoptotic rates in the oocytes, embryos and intrauterine ‘stretched’ microfilariae. This view is supported by the anatomy of the female in which the lateral chords reside in close proximity to the ovaries and uteri[1],[12] and the greater needs of embryo requirements compared to spermatozoa. Further evidence to support the predominant role of depleted chord cell Wolbachia populations in inducing apoptosis in intrauterine developmental stages comes from our observations on released microfilariae. Although microfilariae recovered from tetracycline treated animals did show an increase in the proportion of parasites with medium and high levels of apoptosis, this was not as extensive as we observed with intrauterine ‘stretched’ microfilariae. We were unable, however, to distinguish between microfilariae that were released prior to or after treatment and further experiments using isolated microfilariae only infections are planned to confirm this observation. Our results are, however, consistent with previous observations on the effect of Wolbachia depletion on microfilarial viability and transmission. The majority of microfilariae depleted of Wolbachia retain their motility and viability in the mammalian host but their ability to develop in the vector is compromised[15]. Our studies raise the issue of the mechanism by which Wolbachia suppresses apoptosis. Studies of the influence of other intracellular bacteria on host cell apoptosis demonstrate this may be achieved either by direct targeting of the apoptotic signaling cascade or indirectly by influencing host metabolism[19],[22],[23]. An example of the former occurs in Chlamydia, which produces a protein that targets the host pro-apoptotic protein Bim for proteasome-based destruction[24]. Anaplasma phagocytophilium, a close relative of Wolbachia, inhibits host cell apoptosis through targeting multiple apoptotic pathways[21] and releases a protein, Ats1, via the type IV secretion system that reduces the sensitivity of mitochondria to apoptotic signals[25]. Potential molecular candidates from Wolbachia, which have been shown to inhibit apoptosis, include Wolbachia surface protein( WSP)[26] or lipoproteins such as wBmPAL, which drive innate and adaptive inflammatory immunity associated with disease pathogenesis[27] and can inhibit apoptosis of human neutrophils[28]( Taylor et al. unpublished observation). Bacterial pathogens subvert apoptosis to enhance their ability to replicate and survive in host cells, whereas, Wolbachia, which has a mutualistic rather than a parasitic or pathogenic association with its host nematode, clearly benefits both itself and it’s host by preventing apoptosis during larval, germline and embryonic development to ensure its transmission to the next generation. The only other example of a mutualistic association with Wolbachia and arthropods is the parasitic wasp Asobara tabida in which depletion of Wolbachia results in increased apoptosis in the nurse cells of the female germline[29], suggesting these mutualistic Wolbachia associations may share common pathways. Wolbachia’s effect on apoptosis may be through more indirect mechanisms such as influencing the provisioning of essential metabolites, a process likely to be compromised by the hypodermal cytoskeletal defects following Wolbachia depletion. In this context it is notable that the nematode biological process most sensitive to Wolbachia depletion, embryogenesis and larval development, are processes with high metabolic demands for tissue development, growth and organogenesis. Examples of metabolite provision dependent on Wolbachia under nutritional stress have been described in Drosophila showing that Wolbachia up-regulates bacterioferritin, an iron chelator, and may buffer the host from dramatic changes in iron concentration in the environment[30],[31]. Thus, Wolbachia may provide protection from oxidative stress produced by high internal concentrations of iron. Support for a similar protective mechanism operating in filarial nematodes comes from genomic studies demonstrating that B. malayi lacks the heme biosynthetic pathway while the Wolbachia genome retains most of the entire heme and riboflavin pathways[32]. Heme and riboflavin are efficient iron chelators. Thus the abundance of Wolbachia in the chords has a tremendous iron buffering potential. Together our observations provide a cellular mechanism to explain some of the anti-filarial effects, which occur following antibiotic depletion of Wolbachia from filarial nematodes. The pattern of apoptosis we observe closely correlates with the outcomes previously observed in antibiotic therapy in laboratory models of filarial nematodes and in human field trials with doxycycline[5]. The first event, which occurs soon after bacterial depletion from adult female worms, is the blockage of embryogenesis and cessation of microfilarial production, which is consistent with the extensive and profound apoptosis observed in uterine embryonic stages of B. malayi. This results in long-term sterilization of adult female worms and the slow decline in microfilarial levels, until patients reach a sustained amicrofilarial state, with benefits to disease reduction in onchocerciasis and interruption of transmission in both onchocerciasis and lymphatic filariasis. This state of sterilization persists for at least 21 months, which is reflected in the retention of widespread apoptotic cells in adult female germline and uterine cells of O. volvulus obtained from patients treated with doxycycline, which suggests this is a permanent status. The increase in the proportion of released microfilariae with medium to high levels of apoptotic cells, but less extensive than that observed in intrauterine ‘stretched’ microfilarial stages, is consistent with the observation that following Wolbachia depletion, a proportion of microfilariae retain their motility and viability, yet their capacity to develop within the vector host is reduced. A similar pattern of activation of apoptosis in Wolbachia-depleted L4 larvae suggests that this process also accounts for the arrested development of L4 larvae to adult worms. Finally, we also observed that apoptosis is not widely induced in the nuclei of somatic tissues and the syncytial chord cells of adult worms, where the majority of the bacterial population exist, which is again consistent with the long-term macrofilaricidal effects seen in both lymphatic filariasis and onchocerciasis, which only manifest after 12 months or 18–27 months respectively. Further studies to define the process by which Wolbachia regulates host nematode apoptosis may provide alternative targets to screen for drugs or biomarkers of anti-Wolbachia activity, which could yield alternative and improved therapeutic options for the control and elimination of lymphatic filariasis and onchocerciasis.
Filarial nematodes are a group of pathogenic helminth parasites that include those responsible for the diseases , lymphatic filariasis and onchocerciasis . These nematodes have evolved a mutualistic association with the intracellular bacterial symbiont , Wolbachia , providing a novel approach to their treatment and control using antibiotics . Why depletion of Wolbachia leads to anti-filarial activity remains unknown . Here we show that depletion of the bacteria leads to a rapid and sustained induction of cell death through apoptosis of reproductive cells , developing embryos , microfilariae and developing larvae . Apoptosis surprisingly affects embryonic cells other than those infected with the bacteria , leading us to conclude that the Wolbachia populations in the adult hypodermis , rather than the few bacteria present in the developing embryos , are essential to avoid apoptosis . Our observation provides a mode of action to account for some of the key anti-filarial activities of antibiotic treatment of filarial nematodes and identifies one of the biological processes that form the basis of this mutualistic symbiosis .