George Tzertzinis

Determination of Wolbachia Genome Size by Pulsed-Field Gel Electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.

Molecular Evidence for a Functional Ecdysone Signaling System in Brugia malayi

Background Filarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle). Methods and Findings We report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR). Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor) homolog (Bma-RXR) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone. Conclusions Our study identifies and characterizes the two components (Bma-EcR and Bma-RXR) necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together confirm that an ecdysone signaling system operates in B. malayi and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.

Detection of Serum IgG Antibodies Specific for Wolbachia Surface Protein in Rhesus Monkeys Infected with Brugia malayi

The mechanism of lymphedema development in individuals with lymphatic filariasis is presently poorly understood. To investigate whether Wolbachia, symbiotic bacteria living within filarial nematodes, may be involved in disease progression, Wolbachia-specific immune responses were assayed in a group of Brugia malayi-infected rhesus monkeys. Serum IgG antibodies specific for a major Wolbachia surface protein (WSP) were detected in 2 of 12 infected monkeys. It is interesting that both of these monkeys developed lymphedema after becoming amicrofilaremic. WSP-specific antibody responses were temporally associated with increases in antifilarial IgG1 antibodies as well as lymphedema development. These findings suggest that Wolbachia may be important in understanding disease caused by filarial worms.

EcR-B1 and Usp nuclear hormone receptors regulate expression of the VM32E eggshell gene during Drosophila oogenesis

Ecdysone signaling plays key roles in Drosophila oogenesis, as its activity is required at multiple steps during egg chamber maturation. Recently, its involvement has been reported on eggshell production by controlling chorion gene transcription and amplification. Here, we present evidence that ecdysone signaling also controls the expression of the eggshell gene VM32E, whose product is a component of vitelline membrane and endochorion layers. Specifically blocking the function of the different Ecdysone receptor (EcR) isoforms we demonstrate that EcR-B1 is responsible for ecdysone-mediated VM32E transcriptional regulation. Moreover, we show that the EcR partner Ultraspiracle (Usp) is also necessary for VM32E expression. By analyzing the activity of specific VM32E regulatory regions in usp(2) clones we identify the promoter region mediating ecdysone-dependent VM32E expression. By in vitro binding assay and site-directed mutagenesis we demonstrate that this region contains a Usp binding site necessary for VM32E regulation. Our results further support the crucial role of ecdysone signaling in controlling transcription of eggshell structural genes and suggest that the heterodimeric complex EcR-B1/Usp mediates the ecdysone-dependent VM32E transcriptional activation in the main body follicle cells.

An EcR homolog from the filarial parasite, Dirofilaria immitis requires a ligand-activated partner for transactivation

Filarial parasites are responsible for several serious human diseases with symptoms such as lymphoedema, elephantiasis, and blindness. An understanding of how these parasites pass through developmental checkpoints may suggest potential targets for intervention. A useful model system for the study of the human parasites is the closely related nematode, D. immitis, the causative agent of dog heartworm disease. Ecdysteroids have been identified in filarial nematodes and have been shown to have a biological affect both on molting and microfilarial production. The ecdysteroid, 20-hydroxyecdysone and its receptor, EcR, have a well-characterized developmental role in insects, where it is involved in the control of molting and metamorphosis. We have identified a D. immitis nuclear receptor, DiEcR that shows strong sequence similarity to the insect EcR and shares many of its biochemical properties, including ligand-dependent activation of transcription. However, unlike most insect EcRs, DiEcR requires a ligand-activated RXR partner to exhibit ligand-dependent transcriptional activation of a reporter gene in tissue culture.

Determination of silencing potency of synthetic and RNase III-generated siRNA using a secreted luciferase assay

RNA interference (RNAi) is an established tool for functional genomics studies that is also showing great potential for medical applications. Currently, one of the main goals in RNAi technology is the design and discovery of potent small interfering RNAs (siRNAs). Using a secreted luciferase from Gaussia princeps (GLuc), we developed a reporter assay, which allows for rapid potency assessment of siRNAs, by measuring luminescence activity in cell culture supernatants. The method was applied in microtiter plate format and validated by comparison to quantitative reverse transcription PCR (RT-PCR) and Western blot analysis. This reporter assay was used to evaluate in HeLa cells the potency of different siRNA mixtures generated by RNase III, or several synthetic siRNAs, all directed against human p53. The results show that all four siRNA mixtures generated by RNase III induce 50%-75% decrease of the reporter activity at less than 10 nM transfected concentration. In contrast, only one out of the five commercially available synthetic siRNAs showed comparable potency. These results suggest that one advantage of using enzymatic complex siRNA mixtures for RNAi is that, unlike single synthetic siRNAs, selecting a target region is not important to ensure potency.

Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections.

Background A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. Methodology/ Principal Findings Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. Conclusions The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites.

Substrate Cooperativity in Marine Luciferases

Marine luciferases are increasingly used as reporters to study gene regulation. These luciferases have utility in bioluminescent assay development, although little has been reported on their catalytic properties in response to substrate concentration. Here, we report that the two marine luciferases from the copepods, Gaussia princeps (GLuc) and Metridia longa (MLuc) were found, surprisingly, to produce light in a cooperative manner with respect to their luciferin substrate concentration; as the substrate concentration was decreased 10 fold the rate of light production decreased 1000 fold. This positive cooperative effect is likely a result of allostery between the two proposed catalytic domains found in Gaussia and Metridia. In contrast, the marine luciferases from Renilla reniformis (RLuc) and Cypridina noctiluca (CLuc) demonstrate a linear relationship between the concentration of their respective luciferin and the rate of light produced. The consequences of these enzyme responses are discussed.

Identification of genes containing ecdysone response elements in the genome of Brugia malayi.

Recent studies have demonstrated that filarial parasites contain a functional homologue of the insect ecdysone receptor (EcR). As a first step in deciphering the physiological role that ecdysteroids play in filarial parasites, adult female parasites cultured in the presence and absence of 20-OH ecdysone were metabolically labeled. Gel electrophoretic analysis of proteins extracted from the cultured parasites revealed changes in the level of expression of several proteins, indicating that adult female parasites contained an ecdysone-responsive gene network. A bioinformatic analysis was then conducted to identify putative ecdysone response elements (EcREs) in the Brugia malayi genome. A total of 18 genes were identified that contained putative EcREs located in the 4 kbp upstream from the start of their open reading frames. The most common functional classifications of the encoded proteins were factors involved in transcription and metabolism. These genes revealed a number of different developmental patterns of transcription. The promoter of one EcRE-containing gene was cloned into a luciferase reporter vector and transfected into B. malayi embryos. Reporter gene expression from embryos transfected with this construct was up-regulated by 20-OH ecdysone. Deletion and substitution mutations in the canonical EcRE resulted in a loss of the ecdysone response. These results demonstrate the presence of functional EcREs in the B. malayi genome.

Controlled expression of functional miR-122 with a ligand inducible expression system.

BackgroundTo study the biological function of miRNAs, and to achieve sustained or conditional gene silencing with siRNAs, systems that allow controlled expression of these small RNAs are desirable. Methods for cell delivery of siRNAs include transient transfection of synthetic siRNAs and expression of siRNAs in the form of short hairpins using constitutive RNA polymerase III promoters. Systems employing constitutive RNA polymerase II promoters have been used to express miRNAs. However, for many experimental systems these methods do not offer sufficient control over expression.ResultsWe present an inducible mammalian expression system that allows for the conditional expression of short hairpin RNAs that are processed in vivo to generate miRNAs or siRNAs. Using modified nuclear receptors in a two hybrid format and a synthetic ligand, the Rheoswitch system allows rapid and reversible induction of mRNA expression. We evaluated the systems properties using miR-122 as a model miRNA. A short hairpin encoding miR-122 cloned into the expression vector was correctly processed to yield mature miRNA upon induction with ligand and the amount of miRNA produced was commensurate with the concentration of ligand. miR-122 produced in this way was capable of silencing both endogenous target genes and appropriately designed reporter genes. Stable cell lines were obtained, resulting in heritable, consistent and reversible expression of miR-122, a significant advantage over transient transfection. Based on these results, obtained with a microRNA we adapted the method to produce a desired siRNA by designing short hairpins that can be accurately and efficiently processed.ConclusionWe established an Inducible expression system with a miR-122 backbone that can be used for functional studies of miRNAs and their targets, in heterologous cells that do not normally express the miRNA. Additionally we demonstrate the feasibility of using the miR-122 backbone to express shRNA with a desired siRNA guide strand for inducible RNAi silencing.