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Eukaryotic Cell, May 2006, p. 871-875, Vol. 5, No. 5
1535-9778/06/$08.00+0 doi:10.1128/EC.5.5.871-875.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Unité Biologie Cellulaire du Parasitisme, INSERM 786, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France,1 Pasteur Genopole Île-de-France, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France,2 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel3
Received 8 December 2005/ Accepted 21 February 2006
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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 (1, 11). 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).
Overview of gene expression modulated by heat shock in Entamoeba histolytica. To obtain an overview of our microarray results, we classified genes according to their genomic annotations and then tabulated the numbers of differentially regulated genes in selected functional categories. The most striking finding was our observation of massive gene down regulation after heat shock. Of the 1,131 unique genes probed by the microarray (most of which were picked randomly from cDNA sequences), almost 471 (42%) were significantly repressed during heat treatment. One hundred thirty-five (29%) of the 471 down-regulated genes potentially encode proteins of unknown function. Using genome annotation and BLAST, we identified genes involved in growth-related processes, DNA metabolism and repair, fatty acid metabolism, RNA dynamics, signaling, cytoskeletal activity, vesicular trafficking, metabolic processes, and virulence. An interesting group of genes stood out due to its involvement in protein translation (principally the synthesis of ribosomal proteins). The repression of genes encoding ribosomal proteins has been observed during stress in Saccharomyces cerevisiae (20). A smaller number of genes were up regulated by heat shock; these included the usual chaperones as well as a small set of hypothetical proteins. A comparison of the gene categories present on the microarray with the gene categories modulated by heat shock (Fig. 1) prompted us to conclude that gene down regulation is rather indiscriminate and may correspond to a general response for adaptation to new environmental conditions. These observations support the notion that signals leading to gene down regulation during the heat shock response converge on common pathways resulting from an imbalance in cell homeostasis.
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The BRF subunit of transcription factor IIIB (TFIIIB) from E. histolytica was overexpressed threefold during heat shock. TFIIIB recruits RNA polymerase III (Pol III) to initiate transcription of short, untranslated RNA molecules. The RNA Pol III also transcribes short interspersed nuclear elements (SINEs), which contain Pol III promoters. The E. histolytica genome contains three classes of long interspersed nuclear elements (LINEs) and SINEs (2), accounting for about 6% of the total nuclear DNA. Indeed, SINE transcription accounts for 25% of all RNA transcription in this parasite, and the transcription of these elements may be modulated during stress.
Specific virulence-related genes are modulated by heat shock. Heat shock treatment of the parasite induced the transcription of genes previously described as cysteine proteases 4 and 6 (CP4 and CP6), which have never previously been found to be expressed during parasite growth (4). CP6 was especially up regulated by heat shock, indicating a particular activity of this protease during stress due to its potential role in the degradation of damaged proteins. In contrast, CP1, CP2, CP8, and CP17 were all down regulated (Table 2).
One of the most striking findings generated by the microarray analysis was the observation of differential expression of alleles for the heavy-chain subunit of the Gal/GalNAc lectin, which is an abundant surface protein complex involved in contact-dependent killing and phagocytosis of target cells by E. histolytica (14). The Gal/GalNAc lectin is composed of two subunits, a light (Lgl), glycosylphosphatidylinositol-anchored, 31/35-kDa subunit and a heavy (Hgl), 170-kDa subunit. The complex is associated with an intermediate, 120-kDa subunit (Igl). Each lectin subunit is represented in the E. histolytica genome by multiple alleles; however, the functional distinctions between these alleles have not yet been established. Six genes with 53 to 85% identity encode Lgl isoforms, and at least two genes encoding specific Hgl isoforms (Hgl-2 and Hgl-3) have been isolated and well studied (15, 19). Furthermore, another Hgl isoform and an incomplete sequence have been described from the genome (10). We found that the Hgl-2 allele and one Lgl allele (which we suggest calling Lgl-6) were involved in the heat shock response, whereas all other lectin family components were not. In contrast, the Hgl-3 allele and all other Lgl and Igl subunits underwent gene repression during heat shock. This was also true for other factors that have been associated with the Gal/GalNAc lectin, such as peroxiredoxin (the principal thiol-containing surface antigen in E. histolytica). Peroxiredoxin is important during the transition from the anaerobic environment of the large intestine to the aerobic environment in human tissue (5) and has been found to associate with the Gal/GalNAc lectin in two-hybrid and cellular experiments (8). Our findings suggest that there is a need to assess Hgl-3's specific activity during oxygen peaks. Quantitative reverse transcription-PCR confirmed that transcription of the Hgl-2 allele (16.t0014) and the Lgl-6 allele (168.t0024) increased roughly sixfold during heat shock, whereas all other Hgl, Igl, and Lgl alleles were down regulated (see the supplemental material). Finally, one of the previously described 20-kDa antigen (202.t00007) (21)-encoding genes was found to be up regulated, whereas the second (132.t00016) was down regulated.
In conclusion, this work is a first step toward defining E. histolytica's overall stress-triggered expression program and should help us to understand this parasite's remarkable ability to survive the host response during tissue infection.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Pasteur-Weizmann Research Council and the French Ministry of National Education (via the PRFMMIP program).
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FOOTNOTES |
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Supplemental material for this article may be found at http://ec.asm.org/.
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