title: Molecular characterisation of virulence in Entamoeba histolytica
Creators: name: Preativatanyou, Kanok
Publisher: name: University of Liverpool Repository
Year: 2016

description: Entamoeba histolytica is a parasitic protozoan that infects the human digestive tract. Infection results from ingestion of cyst-contaminated food or water. To date, E. histolytica infection remains a major worldwide public health problem in worldwide endemic areas. The spectrum of clinical manifestations ranges from asymptomatic carrier to mucous and bloody diarrhea or even extraintestinal amoebiasis, usually amoebic liver abscess. Several molecular studies have been carried out to reveal novel aspects of E. histolytica infection. However, the studies focused on genomic-wide analysis comparing between E. histolytica strains are still limited. Thus, the aims of this project are to comprehensively study the comparative analysis of the whole and small RNA transcriptomes amongst nonvirulent and virulent strains of laboratory cultured E. histolytica trophozoites as well as to integrate such transcriptomic findings with the genomic data for advanced understanding of the molecular pathogenesis and virulence in amoebiasis. In this study, genome-wide transcriptome analysis using Illumina RNA-Seq technology can illustrate significant expression differences between nonvirulent and virulent E. histolytica strains. Differential gene expression analysis between nonvirulent Rahman strain and other three virulent strains (i.e. PVBM08B, HM-1:IMSS and IULA:1092:1) reveals that transcripts involved in host cell killing and mucosal invasion, nucleic acid interaction and response to oxidative stress are notably upregulated in the virulent trophozoites. InterProScan results show the upregulation of genes encoding proteolysis-related domains and the co-upregulation of cytoskeleton and actin-modulating domains in the virulent strains. Also, process ontologies related to protein degradation, cellular biosynthesis, DNA metabolism, repair and recombination, mitotic cell division, actin dynamics and response to stress are highly enriched as a core metabolism in the virulent strains, indicating the rapid growing and active metabolic state are the main drivers of virulence. However, the striking underrepresentation of ontologies involved in signaling and regulatory processes was observed in the virulent parasites. It could be inferred that reduced regulation of sensing and correctly responding to the environmental stimuli potentially enable the parasites to become virulent and subsequently cause the invasive infection. Also, NanoString validation reveals the spectrum of virulence-associated gene expression among these four strains, reflecting their different degrees of virulence. Gene copy number variation (CNV) is widespread among the genomes of the E. histolytica strains, reflecting genomic plasticity and variability in gene family content. Herein, this present data show that patterns of CNV contribute to differential expression profiles, therefore it can be extrapolated that differences in gene copy number between genomes could contribute to the variation in phenotypic attributes, including virulence, among E. histolytica strains. Also, genome plasticity can also be seen in Trypanosomes and Leishmania, suggesting that CNV is a potentially important mechanism in generating genetic diversity and regulating gene expression levels in almost exclusively asexual parasite group. For small RNA transcriptomics, the size-fractionated sRNA sequencing data demonstrate the inverse relationship between antisense sRNA abundance and target gene expression levels, strongly suggesting the sRNA-mediated regulation. Differential sRNA regulation in virulence-associated gene expression was found among strains, indicating that sRNA-mediated post-transcriptional regulation may be important in shaping the parasite virulence. In addition, this study identified the novel putative miRNA from the sRNA sequencing data using the biogenesis-based bioinformatic analysis and qPCR validation, implying that miRNA potentially play a regulatory role in E. histolytica. In summary, it can be inferred that genomic plasticity and sRNA-mediated regulation are important mechanisms of virulence modulation in E. histolytica.