Genome and transcriptome studies of the protozoan parasites Trypanosoma cruzi and Giardia intestinalis

Trypanosoma cruzi and Giardia intestinalis are two human pathogens and protozoan parasites responsible for the diseases Chagas disease and giardiasis, respectively. Both diseases cause suffering and illness in several million individuals. The former disease occurs primarily in South America and Central America, and the latter disease occurs worldwide. Current therapeutics are toxic and lack efficacy, and potential vaccines are far from the market. Increased knowledge about the biology of these parasites is essential for drug and vaccine development, and new diagnostic tests. In this thesis, high-throughput sequencing was applied together with extensive bioinformatic analyses to yield insights into the biology and evolution of Trypanosoma cruzi and Giardia intestinalis. Bioinformatics analysis of DNA and RNA sequences was performed to identify features that may be of importance for parasite biology and functional characterization. This thesis is based on five papers (i-v). Paper i and ii describe comparative genome studies of three distinct genotypes of Giardia intestinalis (A, B and E). The genome-wide divergence between A and B was 23% and 13% between A and E. 4557 groups of three-way orthologs were defined across the three genomes. 5 to 38 genotype-specific genes were identified, along with genomic rearrangements. Genes encoding surface antigens, vsps, had undergone extensive diversification in the three genotypes. Several bacterial gene transfers were identified, one of which encoded an acetyltransferase protein in the E genotype. Paper iii describes a genome comparison of the human infecting Trypanosoma cruzi with the bat-restricted subspecies Trypanosoma cruzi marinkellei. The human infecting parasite had an 11% larger genome, and was found to have expanded repertoires of sequences related to surface antigens. The two parasites had a shared ‘core’ gene complement. One recent horizontal gene transfer was identified in T. c. marinkellei, representing a eukaryoteto-eukaryote transfer from a photosynthesizing organism. Paper iv describes the repertoire of small non-coding RNAs in Trypanosoma cruzi epimastigotes. Sequenced small RNAs were in the size range 16 to 61 nucleotides, and the majority were derived from transfer RNAs and other non-coding RNAs. 92 novel transcribed loci were identified in the genome, 79 of which were without similarity to known RNA classes. One population of small RNAs were derived from protein-coding genes. Paper v describes transcriptome analysis using paired-end RNA-Seq of three distinct genotypes of Giardia intestinalis (A, B and E). Gene expression profiles recapitulated the known phylogeny of the examined genotypes, and 61 to 176 genes were differentially expressed. 49,027 distinct polyadenylation sites were mapped and compared, and the median 3′UTR length was 80 nucleotides (A). One 36-nt novel intron was identified and the previously reported introns (5) were confirmed. List of Publications This thesis is based on the following articles and manuscripts that will be referred to in the text by their roman numerals. i Franzén O, Jerlström-Hultqvist J, Castro E, Sherwood E, Ankarklev J, Reiner DS, Palm D, Andersson JO, Andersson B, Svärd SG. Draft Genome Sequencing of Giardia intestinalis Assemblage B Isolate GS: Is Human Giardiasis Caused by Two Different Species?. PLoS Pathogens, 2009, Aug;5(8):e1000560. ii Jerlström-Hultqvist J, Franzén O, Ankarklev J, Xu F, Nohýnková E, Andersson JO, Svärd SG, Andersson B. Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate. BMC Genomics, 2010, 11:543. iii Franzén O, Talavera-López C, Ochaya S, Butler CE, Messenger LA, Lewis MD, Llewellyn MS, Marinkelle CJ, Tyler KM, Miles MA, Andersson B. Comparative genomic analysis of human infective Trypanosoma cruzi lineages with the bat-restricted subspecies T. cruzi marinkellei . BMC Genomics. Accepted (in press). iv Franzén O †, Arner E †, Ferella M, Nilsson D, Respuela P, Carninci P, Hayashizaki Y, Åslund L, Andersson B, Daub CO. The Short NonCoding Transcriptome of the Protozoan Parasite Trypanosoma cruzi . PLoS Neglected Tropical Diseases, 2011, 5(8): e1283. † Equal contribution. v Franzén O ‡, Jerlström-Hultqvist J ‡, Einarsson E, Ankarklev J, Ferella M, Andersson B, Svärd SG. Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNA-Seq. Submitted manuscript. ‡ Equal contribution.