Characterization of the Giardia lamblia Transcriptome

Marine Biological Laboratory, Woods Hole MA

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Abstract

This is a proposal to determine patterns of genome-wide gene expression throughout the life cycle of the important environmentally transmitted human pathogen Giardia lamblia. Giardiasis is a major contributor to the enormous worldwide burden of human diarrheal diseases, yet the basic biology of this parasite is not well understood. No giardial virulence factor is known and this protist's ability to survive in diverse, hostile environments may be a key to its pathophysiology, as Giardia must be able to respond to large and small changes in its external environment for appropriate timing of crucial events in its life cycle. Examination of genome-wide gene expression patterns will provide a coherent picture of activation and inactivation of biological pathways throughout Giardia's life cycle. As the genome sequence of Giardia will soon be known and because we are able to reproduce Giardia's life cycle in vitro, we propose to utilize Serial Analysis of Gene Expression (SAGE) to monitor genome-wide levels of messenger RNA (mRNA) expression throughout Giardia's life cycle. We will perform SAGE for Giardia lamblia by generating approximately 12,2000 15 bp nucleotide sequence tags from the mRNA of 14 stages of its life cycle, modeled in vitro. We will detect up- and down-regulation of genes related to giardial infection (excystation), pathogenicity (trophozoites), transmission (encystation), and survival in the environment (cysts). We will additionally predict which genes and biochemical pathways are constitutively expressed in Giardia lamblia. We will confirm mRNA expression levels of genes with the most variable expression levels predicted by SAGE using semi- quantitative Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR). This research will provide a comprehensive understanding of changes in giardial gene expression in response to important host physiological signals and will serve as a valuable model for study of other parasites and complex eukaryotes, such as yeast and animals.