Infection by Entamoeba histolytica, the causative agent of amoebiasis, is a global health problem, which affects 500 million people worldwide (Stanley, 2003). Most commonly, this pathogen causes haemorrhagic dysentery and liver abscesses. During tissue invasion, E. histolytica adapts to changing oxygen tensions as it goes from the anaerobic colonic lumen to an oxygen-rich environment in the colonic tissue (Stanley, 2003). Additionally, the parasite must cope with cytotoxic reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are produced and released by activated phagocytes that are attracted to the site of infection (MacMicking et al., 1997; Bogdan et al., 2000; Stanley, 2003). Therefore, a significant contribution to E. histolytica's pathogenic potential is likely to be due to its ability to cope with oxidative and nitrosative stresses generated during tissue invasion.
The cellular components targeted by ROS and RNS include proteins (metal cofactors, thiolate side-chains, tyrosine and methionine residues), nucleic acids and lipids (Halliwell and Gutteridge, 2007). Common defence strategies against oxidative and nitrosative stresses include detoxification enzymes and repair systems that enable cells to resist RNS and ROS (Justino et al., 2005; Vandenbroucke et al., 2008). Several microbial transcription factors and regulons, which are involved in the response to both oxidative and nitrosative stresses as well as in the transition from anaerobic metabolism to aerobic metabolism, have redox-sensitive active sites that are modified and/or damaged by both ROS and RNS. Not surprisingly, given the overlap in the types of damage caused by ROS and RNS, common mechanisms exist to deal with these stressors. These include the Crp-Fnr superfamily of transcriptional regulators, which respond both to nitrosative and oxidative stresses (Korner et al., 2003), as other transcriptional regulators do, e.g. in Escherichia coli: NsrR, OxyR, SoxRS, MetR, ferric uptake regulator and NorR, regulating a wide range of cellular processes (Spiro, 2006). A survey of the genomes of the parasitic protists E. histolytica (Loftus et al., 2005), Giardia lamblia (Morrison et al., 2007) and Trichomonas vaginalis (Carlton et al., 2007) revealed the absence of homologues of any of the above-mentioned transcriptional regulators. In contrast, genes coding for detoxification systems for ROS and RNS are present in the genomes of these anaerobic protists. Some of these genes may have been acquired by lateral gene transfer from prokaryotes (Andersson et al., 2003; 2006). The E. histolytica genome has four genes encoding flavodiiron proteins (FDPs), enzymes endowed with oxygen and/or nitric oxide reductase activity that are widespread in prokaryotes (Saraiva et al., 2004; Kurtz, 2007), and have been studied in the protozoa T. vaginalis (Sarti et al., 2004) and G. lamblia (Di Matteo et al., 2008). E. histolytica's genome also contains genes encoding other enzymes involved in the detoxification of ROS, including peroxiredoxin, rubrerythrin, hybrid-cluster protein and superoxide dismutase (SOD). Peroxiredoxin constitutes a major defence against oxidative stress as it is induced by a high-oxygen environment (Akbar et al., 2004) and trichostatin A (Isakov et al., 2008), and contributes to E. histolytica's virulence (Davis et al., 2006; Sen et al., 2007). Although peroxiredoxin and SOD are ubiquitous in all domains of life, FDPs, rubrerythrin and hybrid-cluster proteins have thus far been identified only in prokaryotes and in these anaerobic protists.
Whole-genome expression profiling has been used to assess the effects of oxidative and nitrosative stresses in diverse eukaryotes and prokaryotes (Thum and Bauersachs, 2007; Vandenbroucke et al., 2008). A recent meta-analysis of microarray data performed to assess the common denominators in the oxidative stress response across different domains of life revealed that there are both strong species-specific responses and common strategies for diverse organisms to cope with this challenge (Vandenbroucke et al., 2008). Microarray technology has been used in Entamoeba to investigate a wide variety of biological questions, including virulence (MacFarlane and Singh, 2006; Davis et al., 2007), host colonic and hepatic invasion (Gilchrist et al., 2006; Santi-Rocca et al., 2008) and development (Ehrenkaufer et al., 2007). In order to determine the molecular mechanisms by which E. histolytica trophozoites respond when challenged with oxidative and nitrosative stresses, we used whole-genome expression profiling using a short oligonucleotide microarray containing 9435 of the annotated 9938 amebic genes. Our results demonstrated a significant transcriptional response of E. histolytica HM-1:IMSS, a canonical virulent strain, to H2O2 (286 genes regulated), NO (1036 genes regulated) and a significant overlap among the genes responsive to both conditions (164 genes). To further identify which components of these response mechanisms may be correlated with E. histolytica's virulence potential, the response to oxidative stress was assessed for a non-pathogenic strain, E. histolytica Rahman. In contrast to the observations for the virulent HM-1:IMSS strain, the Rahman strain had fewer transcriptional changes and the overall fold-changes for the regulated genes were significantly lower. Overall, our results provide insights into the molecular network regulating adaptation to oxidative and nitrosative stresses in E. histolytica and suggest that one important difference between virulent and non-virulent amebae is their ability to deal with the stresses encountered during host invasion.