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

Brugia malayi Gene Expression in Response to the Targeting of the Wolbachia Endosymbiont by Tetracycline Treatment

Filarial parasites afflict hundreds of millions of individuals worldwide, and cause significant public health problems in many of the poorest countries in the world. Most of the human filarial parasite species, including Brugia malayi, harbor endosymbiotic bacteria of the genus Wolbachia. Elimination of the endosymbiont leads to sterilization of the adult female worm. The need exists for the development of new chemotherapeutic approaches that can practically exploit the vulnerability of the filaria to the loss of the Wolbachia. In this study we performed ultrastructural and microarray analyses of female worms collected from infected jirds treated with tetracycline. Results suggest that the endosymbiotic bacteria were specifically affected by the antibiotic. Furthermore, in response to the targeting of the endosymbiont, the parasites modulated expression of their genes. When exposed to tetracycline, the parasites over-expressed genes involved in protein synthesis. Expression of genes involved in cuticle biosynthesis and energy metabolism was, on the other hand, limited

Normal Mouse Intestinal Epithelial Cells as a Model for the in vitro Invasion of Trichinella spiralis Infective Larvae

It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine; however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. Although the previous observations indicated that invasion also occurs in vitro when the infective larvae are inoculated onto cultures of intestinal epithelial cells (e.g., human colonic carcinoma cell line Caco-2, HCT-8), a normal readily manipulated in vitro model has not been established because of difficulties in the culture of primary intestinal epithelial cells (IECs). In this study, we described a normal intestinal epithelial model in which T. spiralis infective larvae were shown to invade the monolayers of normal mouse IECs in vitro. The IECs derived from intestinal crypts of fetal mouse small intestine had the ability to proliferate continuously and express specific cytokeratins as well as intestinal functional cell markers. Furthermore, they were susceptible to invasion by T. spiralis. When inoculated onto the IEC monolayer, infective larvae penetrated cells and migrated through them, leaving trails of damaged cells heavily loaded with T. spiralis larval excretory-secretory (ES) antigens which were recognized by rabbit immune sera on immunofluorescence test. The normal intestinal epithelial model of invasion mimicking the natural environment in vivo will help us to further investigate the process as well as the mechanisms by which T. spiralis establishes its intestinal niche

Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues

Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host

The Wolbachia endosymbiont as an anti-filarial nematode target

Human disease caused by parasitic filarial nematodes is a major cause of global morbidity. The parasites are transmitted by arthropod intermediate hosts and are responsible for lymphatic filariasis (elephantiasis) or onchocerciasis (river blindness). Within these filarial parasites are intracellular alpha-proteobacteria, Wolbachia, that were first observed almost 30 years ago. The obligate endosymbiont has been recognized as a target for anti-filarial nematode chemotherapy as evidenced by the loss of worm fertility and viability upon antibiotic treatment in an extensive series of human trials. While current treatments with doxycycline and rifampicin are not practical for widespread use due to the length of required treatments and contraindications, anti-Wolbachia targeting nevertheless appears a promising alternative for filariasis control in situations where current programmatic strategies fail or are unable to be delivered and it provides a superior efficacy for individual therapy. The mechanisms that underlie the symbiotic relationship between Wolbachia and its nematode hosts remain elusive. Comparative genomics, bioinfomatic and experimental analyses have identified a number of potential interactions, which may be drug targets. One candidate is de novo heme biosynthesis, due to its absence in the genome sequence of the host nematode, Brugia malayi, but presence in Wolbachia and its potential roles in worm biology. We describe this and several additional candidate targets, as well as our approaches for understanding the nature of the host-symbiont relationship

Endosymbiont DNA in Endobacteria-Free Filarial Nematodes Indicates Ancient Horizontal Genetic Transfer

Background: Wolbachia are among the most abundant symbiotic microbes on earth; they are present in about 66% of all insect species, some spiders, mites and crustaceans, and most filarial nematode species. Infected filarial nematodes, including many pathogens of medical and veterinary importance, depend on Wolbachia for proper development and survival. The mechanisms behind this interdependence are not understood. Interestingly, a minority of filarial species examined to date are naturally Wolbachia-free. Methodology/PrincipalFindings:We used 454 pyrosequencing to survey the genomes of two distantly related Wolbachia- free filarial species, Acanthocheilonema viteae and Onchocerca flexuosa. This screen identified 49 Wolbachia-like DNA sequences in A. viteae and 114 in O. flexuosa. qRT-PCR reactions detected expression of 30 Wolbachia-like sequences in A. viteae and 56 in O. flexuosa. Approximately half of these appear to be transcribed from pseudogenes. In situ hybridization showed that two of these pseudogene transcripts were specifically expressed in developing embryos and testes of both species. Conclusions/Significance: These results strongly suggest that the last common ancestor of extant filarial nematodes was infected with Wolbachia and that this former endosymbiont contributed to their genome evolution. Horizontally transferred Wolbachia DNA may explain the ability of some filarial species to live and reproduce without the endosymbiont while other species cannot