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

Pyknotic nuclei and morphology defects observed in the germline and intrauterine microfilariae.

<p>Germline cells-containing ovaries (A, B) and microfilariae obtained from uteri (C, D) were stained for DNA (propidium iodide, red) and actin (phalloidin, green). Worms dissected from un-treated animals (A, C), and from animals treated with tetracycline (B, D). In (A), propidium iodide reveals germline nuclei surrounded by <i>Wolbachia</i> (appearing as small red foci), while <i>Wolbachia</i> are absent in (B). In (B), arrowheads point towards some pyknotic nuclei, with a condensed chromatin appearing brighter with propidium iodide stain, while stars indicate germ cell nuclei with a normal morphology. Note that depending on the area observed in the ovary, wild type nuclei can slightly vary in volume, as shown in images (A) and (B). 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

Doxycycline treatment leads to apoptosis <i>in vitro.</i>

<p>(A, C) control worms and treated worms (B, D) were TUNEL assayed (green) and stained for DNA (PI in red). (A, B) Germ cells in mitotic proliferation in the ovaries. (C) Proximal uteri filled with developing embryos. (D) Apoptotic oocytes and early embryos (arrows) in a distal uterus, surrounded by sperm cells (arrowheads). Scale bar  = 15 µm.</p

Percentage of <i>B. malayi</i> microfilariae showing different levels of apoptotic positive nuclei.

<p>Percentage of <i>B. malayi</i> microfilariae showing different levels of apoptotic positive nuclei.</p