Analysis of transcriptional regulation of tetracycline responsive genes in Brugia malayi

Abstract

The Wolbachia endosymbiont of the human filarial parasites is necessary for parasite reproduction, making it an attractive chemotherapeutic target. Previous studies have demonstrated that mRNA levels of several nuclearly encoded genes are altered as a result of exposure to antibiotics that eliminate the endosymbiont, suggesting that they may be involved in maintaining the parasite-endosymbiont relationship. Here, we tested the hypothesis that the increase in mRNA levels of certain nuclearly encoded genes of Brugia malayi in response to tetracycline treatment involved specific regulatory elements present in the promoters of these genes. The promoters of three such genes (BmRPL13, BmRPS4 and BmHSP70) were tested for tetracycline responsiveness utilizing a homologous transient transcription system. Reporter gene expression driven by all three promoters was up-regulated in transfected embryos exposed to tetracycline. Substitution mutagenesis was employed to map the cis-acting elements responsible for this response in the BmHSP70 promoter. Tetracycline responsiveness was found to be distinct from the cis-acting elements involved in regulating the stress response from the BmHSP70 promoter; rather, tetracycline responsiveness was mediated by a TATAA-box like element. This study represents the first demonstration of small molecule-mediated gene regulation of a native B. malayi promoter.

Wolbachiafilarial interactions

“…Many filarial genes show expression changes in response to Wolbachia clearance following tetracycline treatment (Ghedin et al ., ; Strubing et al ., ; Rao et al ., ). Several nuclearly encoded pathway transcripts were increased in response to elimination of Wolbachia in adult B. malayi , including those involved in protein synthesis and the stress response (Ghedin et al ., ), and this has recently been shown to involve specific regulatory elements present in the promoters of these genes (Liu et al ., ). Changes in the expression levels of specific filarial genes such as phosphate permease (Heider et al ., ) and heat shock protein 60 (Pfarr et al ., ) following Wolbachia clearance have also been shown, suggesting that the encoded proteins are involved at some level.…”

The symbiotic role of Wolbachia in Onchocercidae and its impact on filariasis

“…Comparative genomic/transcriptomic/proteomic analyses of filarial species associated or not with Wolbachia, and before and after Wolbachia depletion, have pushed forward our knowledge of the molecular/metabolic basis of the mutualism between Wolbachia and its host, ultimately resulting in the discovery of new drug targets [52,54,[111][112][113][114][115]. Several new approaches directed against Wolbachia are currently under investigation (Table 2) [94,109,[116][117][118][119][120][121][122].…”

Prediction pipeline for discovery of regulatory motifs associated with Brugia malayi molting

“…malayi involved biolistic transfection of isolated embryos [ 23 ] which were developmentally incompetent. While this system has been used to identify the conserved motifs necessary for promoter function in constitutive promoters [ 24 , 51 , 52 , 53 ] and cis acting regulatory regions in some regulated promoters [ 54 , 55 ], these studies were limited to genes expressed in embryos. However, with recent innovations in filarial transgenics, it is now possible to efficiently produce stably transfected developmentally competent infective larvae, which can in turn be used to produce transgenic parasites in which transgenes are stably integrated into the parasite genome [ 44 ].…”