Anti-filarial Activity of Antibiotic Therapy Is Due to Extensive Apoptosis after Wolbachia Depletion from Filarial Nematodes

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

New Insights into the Evolution of Wolbachia Infections in Filarial Nematodes Inferred from a Large Range of Screened Species

Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals.We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected.The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further genomic analyses on some of the newly sampled species are welcomed to decipher the open questions

Cytoskeleton defects are revealed in somatic tissues, without apoptotic phenotypes.

<p>Female <i>B. malayi</i> lateral chords from un-treated (A, C) or tetracycline-treated (B, D) jirds. No pyknotic nuclei were detected, and the TUNEL levels were similar in both samples (A, B). (C, D) Apical microtubule network. Scale bar  = 100 µm.</p

Co-evolution between an Endosymbiont and Its Nematode Host: Wolbachia Asymmetric Posterior Localization and AP Polarity Establishment

While bacterial symbionts influence a variety of host cellular responses throughout development, there are no documented instances in which symbionts influence early embryogenesis. Here we demonstrate that Wolbachia, an obligate endosymbiont of the parasitic filarial nematodes, is required for proper anterior-posterior polarity establishment in the filarial nematode B. malayi. Characterization of pre- and post-fertilization events in B. malayi reveals that, unlike C. elegans, the centrosomes are maternally derived and produce a cortical-based microtubule organizing center prior to fertilization. We establish that Wolbachia rely on these cortical microtubules and dynein to concentrate at the posterior cortex. Wolbachia also rely on PAR-1 and PAR-3 polarity cues for normal concentration at the posterior cortex. Finally, we demonstrate that Wolbachia depletion results in distinct anterior-posterior polarity defects. These results provide a striking example of endosymbiont-host co-evolution operating on the core initial developmental event of axis determination

Apoptosis and apoptotic bodies are detected in <i>O. volvulus</i> tissues from human nodules of doxycycline treated patients.

<p>A, B. Cross-sections of adult female worm showing absence of apoptosis and intact embryonic inter-uterine stages (oocytes, pretzel stages, coiled embryonic microfilariae). C-G. Cross-sections of adult female worms depleted of <i>Wolbachia</i> showing extensive apoptosis of germline and early embryonic cells and uterine epithelial cells. Stars label inter-uterine content, black arrowheads label apoptotic germline and early embryonic cells as well as human cells surrounding the worm, white arrowheads point to somatic cells, such as epithelial cells surrounded uteri. Scale bar  = 20 µm.</p

<i>In vivo</i> tetracycline treatment dramatically reduces the <i>Wolbachia</i> population in adult <i>B. malayi</i> females.

<p>Female <i>B. malayi</i> lateral chords from un-treated (A) or tetracycline-treated (B) jirds. Total DNA is revealed with propidium iodide (red), host nuclei are counterstained with an anti acetylated histone H4 (green), therefore the red foci reveal only <i>Wolbachia.</i> The chords are flanked by muscle quadrants stained with phalloidin (green). Scale bar  = 100 µm.</p

Increased expression of <i>ced-</i>3 gene and activation of CED-3 protein in tetracycline treated <i>B. malayi</i>.

<p>A) <i>Ced</i>-3 gene expression level normalized by expression level of <i>gst</i> in BM (<i>B. malayi</i>) and BM-TET (tetracycline treated <i>B. malayi</i>) adult females. B) Western blot detection of CED-3 in microfilaria and 14 day old L4 larvae. Lanes 1-2, microfilariae from untreated control (1) and tetracycline treated (2). Lanes 3-4 lanes, L4 larvae, untreated control (3) and tetracycline treated (4). Activated (cleaved) CED-3 is more abundant in microfilariae and L4 larvae from treated jirds compared to untreated controls. Lane 5, molecular weight markers.</p

<i>Wolbachia</i>-dependent non cell-autonomous apoptosis.

<p>Schematic drawing of a <i>B. malayi</i> female focusing on the reproductive apparatus, showing levels of apoptosis before and after <i>Wolbachia</i> removal. Apoptosis remains a rare event during germline maturation, and is developmentally programmed during embryogenesis. After <i>Wolbachia</i> depletion, cumulative apoptosis is observed in germ cell, embryo and microfilaria. The absence of <i>Wolbachia</i> (from the hypodermal chords and from the few embryonic cells derived from the C blastomere) leads to a massive non cell-autonomous, “bystander” apoptosis, in embryonic cells normally devoid of <i>Wolbachia</i> (green nuclei).</p