Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNA-Seq

Abstract

Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most of the genome was transcribed in trophozoites grown in vitro, but at vastly different levels. RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3′ UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology.

Author Summary

Giardia is a single cell intestinal parasite and a common cause of diarrhea in humans and animals. Giardia is an unusual eukaryote by possessing two nuclei, a highly reduced genome and simple transcriptional apparatus. We have characterized the transcriptome of Giardia at single nucleotide resolution, which allowed the calculation of digital gene expression values for the complete set of genes. We performed a comparison of gene expression divergence across three genotypes. Most of the genes were transcribed, and the data were used to refine and correct gene models. Several gene expression differences were identified between the genotypes. A non-transcribed cluster of genes was detected on chromosome 5, likely representing a silenced region. The data also allowed mapping of transcript termini, which provided the first global view of 3′ untranslated regions in this parasite. This study also gives the first genome-wide evidence of transcription of allelic variants in Giardia. In this study, we provide novel insights into the transcriptome of an important human pathogen and model eukaryote. The findings reported here likely relate to the lifestyle of this parasite and its adaptation to parasitism. The data provide starting points for functional investigation of Giardia's biology and diplomonads generally.