Amoebiasis is a disease caused by the enteric protozoan parasite Entamoeba histolytica
. Following invasion of human tissue by E. histolytica
, the two major clinical manifestations are hemorrhagic colitis and liver abscesses (18
). For infection to succeed, invading trophozoites must produce an adaptive response that ensures their protection against the host response and survival. Hence, E. histolytica
proteins whose production is triggered or modulated by environmental stress are of great interest, since characterization of these species should help us understand the mechanisms which sustain pathogenesis and could lead to new treatments for amoebiasis.
Microbial pathogens have evolved a number of strategies for protecting themselves from their hosts. One of these is the so-called heat shock response, which is elicited by a sudden increase in ambient temperature (13
) and induces the synthesis of a limited set of proteins (called heat shock proteins [HSPs] or molecular chaperones). Homologues of known HSPs have been identified and partially characterized in E. histolytica
). With the aim of determining gene expression changes during E. histolytica
's adaptive response during infection, we developed an oligonucleotide-based microarray with transcript information randomly obtained from a cultured virulent strain of the pathogen. Array analysis revealed that gene transcription in E. histolytica
exposed to heat shock is dramatically reduced, since 471 of 1,131 unique genes were down regulated, whereas specific HSP-encoding genes were up regulated. In conjunction with real-time PCR results, these genetic information data reveal for the first time a very interesting differential allelic expression of key genes participating in virulence, such as the immunodominant antigen Gal/GalNAc lectin, certain cysteine proteinases, and the so-called 20-kDa antigen.
The aims of this study were (i) to establish a highly discriminating method for monitoring gene expression changes in E. histolytica
and (ii) to determine the mRNA expression profile of E. histolytica
cells growing in a drastically modified environment. We decided to construct an oligonucleotide-based microarray, using information obtained directly from sequence analysis of E. histolytica
transcripts, a strategy that is generally thought to overcome problems due to gene redundancy and the presence of introns. A cDNA library of the virulent E. histolytica
strain HM-1:IMSS and a liver-specific cDNA subtraction library were prepared and sequenced. The bioinformatic analysis of sequenced clones enabled us to define 1,300 bona fide transcripts, all from parasites growing in vitro and enriched with randomly chosen transcripts from parasites growing in the liver. A comparison of our file with the recent publication of the E. histolytica
genome sequence (10
) allowed us to detect and reference 96 transcripts that have not yet been annotated in the genome (referenced as gDNA in our public database at http://genoscript.pasteur.fr
). Additionally, of the 1,131 individual genes in the list, 342 corresponded to coding sequences of unknown function. Finally, E. histolytica
genes known to be involved in virulence or in cytoskeleton activities were also added to the file. Due to the high AT base content of the E. histolytica
genome and in order to achieve a high specificity and limit background noise in the hybridization procedure, we constrained the design of our 70-mer oligonucleotides with the following parameters: a 32% GC content, AT stretches of no longer than 30 nucleotides, and the absence of degenerate bases. All experimental procedures for microarray setting and statistical analysis of data are compiled at http://genoscript.pasteur.fr
and summarized in the supplemental material.
We exposed E. histolytica trophozoites growing exponentially at 37°C to a heat shock by moving them to a temperature of 42°C for 4 hours. Cell viability was monitored by using the trypan blue exclusion test, which indicated that 96% of trophozoites were still alive after the treatment. The 3.4-fold increase of Hsp70 indicated that a heat shock response was obtained under our experimental conditions (data not shown). Given that the array represented a relatively small number of genes and that major changes in gene expression are likely during changes in the parasite's milieu, we chose to compare two normalization methods. The first takes into account the overall mean value of E. histolytica gene expression. In the second method, normalization incorporates the use of external controls by spotting oligonucleotides (spikes) prepared from an unrelated source (Arabidopsis thaliana). In both cases, statistical analysis was performed using a paired Student t test. After drawing locally weighted regression normalization curves, we noted that normalization with spikes fitted the asymmetric data set well, with linear correlation coefficients between biological replicates and between technical replicates being >0.9 and >0.95, respectively; the analysis allowed us to conclude that the method using spikes showed a greater specificity for changes in gene expression observed after heat shock treatment, together with a more asymmetric data set (few induced genes and numerous repressed genes).