Entamoeba histolytica EhPgp5 transcriptional activation depends on putative emetine response elements
Introduction
Drug resistance has emerged as a problem in the treatment and control of many infectious diseases produced by protozoan parasites (Cowman et al., 1994). Due to the health impact of these diseases, especially in developing countries, the interest in the identification of mechanisms involved in drug resistance, and the design of new antiparasitic drugs has been growing (Ullman, 1995). In Entamoeba histolytica the multidrug resistance (MDR) phenotype shows similar characteristics to the MDR phenotypes identified in mammalian cells, and other parasites such as Plasmodium falciparum and Leishmania (Cortes-Selva et al., 2004, Rajagopal and Simon, 2003, Rubio and Cowman, 1996). The MDR phenotype in amoeba is a consequence of an increased expression of P-glycoprotein, due mainly to the overexpression of EhPgp1 and EhPgp5 genes (Descoteaux et al., 1995). The EhPgp1 gene is constitutively expressed in drug resistant trophozoites of clone C2, while the EhPgp5 gene is overexpressed in C2 trophozoites grown in the presence of emetine. The differential expression pattern of both genes suggest that exist a finely regulated mechanisms mainly controlled at transcriptional level.
However, recent work reported that the EhPgp5 mRNA stability is also increased in the trophozoites growing at high emetine concentrations, indicating that, although the transcriptional regulation seems to be the major control point, other mechanisms such as the mRNA stability or the DNA amplification are also regulating the MDR phenotype (López-Camarillo et al., 2003, López et al., 1997).
The EhPgp5 gene is only transcribed in the presence of emetine in drug resistant trophozoites (clone C2) and the transcript level increases according to the emetine concentration in the culture medium (Descoteaux et al., 1995). These findings show that the EhPgp5 gene presents an emetine inducible gene expression pattern. Transcriptional analysis of the EhPgp5 promoter demonstrated that minimal promoter activity was kept by the −235 bp upstream the transcription initiation site. The results also showed that this activity was higher in C2(40) (trophozoites growing for several months in the presence of 40 μM of emetine) than in C2 trophozoites, suggesting that drug responsive elements are located within this region (Pérez et al., 1998).
In this paper, we detected functional drug responsive elements between −111 to −170 bp of the EhPgp5 transcription start site. Experiments currently in progress will allow us to identify the precise sequence of these elements as well as the biochemical characterization of transcriptional factors interacting with it.
Section snippets
Entamoeba histolytica cultures
Trophozoites of emetine sensitive (A) and resistant (C2) clones were axenically cultured in TYI-S-33 medium supplemented with 15% fetal bovine serum (Biofluids), 6% vitamins (JRH, Biosciences), and antibiotics (Lakeside) (Diamond et al., 1978). Exponential trophozoites cultures were harvested by ice-chilling and centrifugation at 150g during 5 min at 4 °C.
Plasmid constructions
Three serial deletions of the minimal promoter were done by PCR DNA amplification of different fragments (360, 140, and 79 bp) using the
EhPgp5 gene promoter activity is induce by emetine in the wild type clone A and in the resistant clone C2
The multidrug resistance phenotype in E. histolytica is produced by the overexpression of the EhPgp genes. In contrast to the constitutively expressed EhPgp1 gene, the EhPgp5 gene expression is induced by the presence of emetine in the medium only in the drug resistant clone C2 (Bañuelos et al., 2002, Descoteaux et al., 1995). Transcriptional analysis of the EhPgp5 gene demonstrated that in both drug sensitive and drug resistant clones, the promoter sequence is similar and the core region is
Acknowledgments
We are grateful to Alfredo Padilla for her valuable help with the artwork. This work received financial support from CONACyT and CGPI-IPN, México.
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