Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi
Introduction
Human filarial parasites cause diseases that inflict significant morbidity upon a large proportion of the poorest people on the planet (World Health Organization (WHO), 2010). Lymphatic filariasis (caused by infection with Brugia malayi, Brugia timori or Wuchereria bancrofti) and onchocerciasis (caused by infection with Onchocerca volvulus) together result in the loss of 5.7 million disability adjusted life years (Mathers et al., 2007). As a result, these diseases have been identified by the international community as two of the five Neglected Tropical Diseases (NTDs) worldwide, and both have been targeted for elimination by the international community in the London Declaration on Neglected Tropical Diseases (Turner et al., 2014).
Elimination programs targeting both onchocerciasis and filariasis have been implemented at the national and international levels. All rely primarily upon a strategy of mass drug distribution to interrupt transmission and thereby eventually locally eliminate the parasite (Cupp et al., 2011). However, these programs rely upon a small arsenal of drugs that must be given over a long period of time (i.e., years). This leaves the programs vulnerable to failure in the face of developing resistance. Furthermore, the prolonged treatment courses necessary for effective elimination present substantial logistical difficulties resulting from the need to maintain high drug coverage rates over a long period of time (i.e., years). The current drug regimens used by these programs face limitations in deployment in many areas. For example, diethylcarbamazine (DEC), a drug commonly used to treat lymphatic filariasis, produces severe ocular and systemic complications when given to individuals infected with O. volvulus (Awadzi, 2003). This precludes the use of DEC in much of Africa, where lymphatic filariasis and onchocerciasis are co-endemic. Similarly, treatment of onchocerciasis using ivermectin is complicated in areas that are co-endemic for the eye-worm Loa loa, as severe adverse events have been documented to occur in individuals treated with ivermectin that are heavily infected with L. loa (Twum-Danso, 2003). For these reasons, there is an urgent need to develop alternative therapeutic interventions to augment the efforts of the elimination programs.
Ecdysteroids have long been known to play a central role in controlling the development of various invertebrates. They have been best characterised in insects. These hormones exhibit their effects through the activity of ecdysteroid receptors, which act as master transcriptional regulators (Koelle et al., 1991, Baehrecke, 1996). In insects, juvenile hormone and ecdysone regulate both egg development and the molting cycle. As juvenile hormone levels decrease, there is a surge in ecdysone levels leading to molting (Riddiford, 1993). This effect is mediated through a heterodimer of the ecdysone receptor (EcR) and the retinoid X receptor (RXR), two members of the nuclear hormone receptor family of transcriptional regulators (Koelle et al., 1991, Thomas et al., 1993, Yao et al., 1993). The fact that molting and the receptors controlling this process are not found in vertebrates makes this process an attractive potential chemotherapeutic target.
A homolog of the EcR has been identified and shown to be active in B. malayi (Tzertzinis et al., 2010). However, its physiological role in controlling the developmental processes in this parasite remains unclear (Mendis et al., 1983, Tzertzinis et al., 2010). In an attempt to decipher the physiological role of the B. malayi ecdysone receptor (BmaEcR), we have conducted transcriptomic, proteomic and phenotypic studies of the effect on 20-hydroxyecdysone (20E) on gravid adult female B. malayi worms.
Section snippets
Phenotypic studies of the effect of 20E on fecund adult female worms
Gravid adult female parasites were obtained from the Filariasis Research Reagent Resource Center (FR3) at the University of Georgia, USA. A total of five worms per well were cultured in a 6-well plate using 3 ml of CF-RPMI media (RPMI 1640 supplemented with 25 mM HEPES buffer, 2 mM glutamine, 100 U/ml of streptomycin, 100 μg/ml of penicillin, 0.25 μg/ml of amphotericin B, and 10% heat-inactivated FBS). Experiments were designed to consist of two replicates of parasites treated with 20E and two
Results
To aid in delineating the role played by the EcR in the filaria, fecund adult female parasites were cultured in vitro in the presence and absence of 20E as described in Section 2.1. Five biological replicates of the experiment were performed, all of which gave similar results. A representative experiment is shown in Fig. 1. Parasites cultured in the presence of 20E exhibited a significant increase (P < 0.001) in the production and expulsion of both microfilariae and eggs/embryos compared with
Discussion
The data presented above suggest that ecdysone may play an important role in the development and birth of microfilaria in B. malayi. Worms cultured in the presence of 20E exhibited an increase in expulsion of microfilariae, pre-microfilariae and eggs/embryos into the culture medium for 2 days. While expulsion of immature stages is commonly seen in adult female worms cultured in vitro (c.f. Fig. 1) as well as in adult females in i.p. infected gerbils (A. Moorhead, unpublished data), the number of
Acknowledgements
We gratefully acknowledge the Filariasis Research Reagent Resource Center (FR3) at the University of Georgia, USA, under a contract from the National Institute of Allergy and Infectious Diseases, USA for providing the parasite material. We are thankful to Ms. Monica Lazaro for providing technical assistance in RNA preparation. This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, USA to TRU (project R21AI103302) and from internal New England Biolabs,
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