Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties

Abstract

BackgroundThe availability of two genetically very similar cell lines (A and B) derived from the laboratory isolate Entamoeba histolytica HM-1:IMSS, which differ in their virulence properties, provides a powerful tool for identifying pathogenicity factors of the causative agent of human amoebiasis. Cell line A is incapable inducing liver abscesses in gerbils, whereas interaction with cell line B leads to considerable abscess formation. Phenotypic characterization of both cell lines revealed that trophozoites from the pathogenic cell line B have a larger cell size, an increased growth rate in vitro, an increased cysteine peptidase activity and higher resistance to nitric oxide stress. To find proteins that may serve as virulence factors, the proteomes of both cell lines were previously studied, resulting in the identification of a limited number of differentially synthesized proteins. This study aims to identify additional genes, serving as virulence factors, or virulence markers.ResultsTo obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR. Out of 6242 genes represented on the array, 87 are differentially transcribed (≥two-fold) in the two cell lines. Approximately 50% code for hypothetical proteins. Interestingly, only 19 genes show a five-fold or higher differential expression. These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A. The aig1-like GTPasesare of special interest because the majority of them show higher levels of transcription in the pathogenic cell line B. Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach.ConclusionsIn this study we have identified a defined set of genes that are differentially transcribed between the non-pathogenic cell line A and the pathogenic cell line B of E. histolytica. The identification of transcription profiles unique for amoebic cell lines with pathogenic phenotypes may help to elucidate the transcriptional framework of E. histolytica pathogenicity and serve as a basis for identifying transcriptional markers and virulence factors.

Chemical and genetic validation of thiamine utilization as an antimalarial drug target

“…Synthesis of cDNA was accomplished with 200 U Super-ScriptIII Reverse Transcriptase from 1 mg RNA using 0.5 mM dNTP, Random Primer (250 mg), 5 mM dithiothreitol and 40 U RNaseOUT in 20 ml for 2 h at 42°C. SuperScriptIII Reverse Transcriptase was omitted as negative control for the subsequent real-time quantitative PCR using 2.5 RealMasterMix/20 Â SYBR Green kit as previously described 49 Table S2), and 50 ng cDNA prepared on the day of the experiment with the following amplification conditions: 95°C for 2 min, followed by 35 cycles at 95°C for 15 s, 49°C for 20 s and 68°C for 20 s, and a melting step ramping from 67-95°C. In each experiment from separate preparations of cDNA, each sample was analysed in duplicate.…”

Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes

“…The amplified PCR products or synthetic genes were digested with XmaI and XhoI and ligated into XmaI/XhoI-digested pEhExHA [37], to produce plasmids HA-U1A, HA-RabX13, HA-RabC1, HA-nNS1, HA-gRabX13, and HA-gRabX13-MS2, respectively. The AIG17 construct was described elsewhere [38]. The transformants expressing the above plasmids were established by liposome-mediated transfection of the wild-type HM1:IMSS Cl6 strain as previously described [39].…”

Control of Entamoeba histolytica Adherence Involves Metallosurface Protease 1, an M8 Family Surface Metalloprotease with Homology to Leishmanolysin

“…We propose the name E. histolytica metallosurface protease 1 (EhMSP-1) for the E. histolytica-specific protein and EhMSP-2 for the common homologue (NCBI GeneID numbers 3409717 and 3406949, respectively). EhMSP-2 was one of 87 genes with significant differential expression (Ն2-fold) in a recent microarray analysis comparing gene expression in virulent and avirulent E. histolytica trophozoite strains derived from the same genetic background (mRNA levels were more than 20-fold higher in the avirulent strain) (5). On the basis of these data and the fundamental contributions of leishmanolysin and its orthologues to Leishmania and Trypanosoma virulence, we decided to characterize the E. histolytica surface metalloproteases, beginning with the pathogen-specific family member EhMSP-1.…”