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Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq
Karolinska Institutet.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
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2013 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 9, no 3, e1003000Article in journal (Refereed) Published
Abstract [en]

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 ofthe 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.

Place, publisher, year, edition, pages
2013. Vol. 9, no 3, e1003000
Keyword [en]
Giardia intestinalis, transcriptome, RNA-seq, poly(A), intestinal parasite
National Category
Microbiology Infectious Medicine
Research subject
Biology with specialization in Microbiology; Biology with specialization in Evolutionary Organismal Biology
Identifiers
URN: urn:nbn:se:uu:diva-182829DOI: 10.1371/journal.pcbi.1003000ISI: 000316864200066OAI: oai:DiVA.org:uu-182829DiVA: diva2:560968
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Hidden Diversity Revealed: Genomic, Transcriptomic and Functional Studies of Diplomonads
Open this publication in new window or tab >>Hidden Diversity Revealed: Genomic, Transcriptomic and Functional Studies of Diplomonads
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The diplomonads are a diverse group of eukaryotic microbes found in oxygen limited environments such as the intestine of animals were they may cause severe disease. Among them, the prominent human parasite Giardia intestinalis non-invasively colonizes the small intestine of humans and animals where it induces the gastrointestinal disease giardiasis. Two of the eight genetic groups of G. intestinalis, assemblage A and B, are known to infect humans and have zoonotic potential. At the start of project, genome scale data from assemblage B-H was either sparse or entirely missing.

In this thesis, genome sequencing was performed on the assemblage B isolate GS (Paper I) and the P15 isolate (Paper III) of the hoofed-animals specific assemblage E to investigate the underlying components of phenotypic diversity in Giardia. Comparisons to assemblage A isolate WB revealed large genomic differences; entirely different repertoires of surface antigens, genome rearrangements and isolate specific coding sequences of potential bacterial origin. We established that genomic differences are also manifested at the transcriptome level (Paper VIII). In a follow up analysis (Paper IV) we concluded that the Giardia assemblages are largely reproductively isolated. The large genomic differences observed between Giardia isolates can explain the phenotypic diversity of giardiasis.

The adaptation of diplomonads was further studied in Spironucleus barkhanus (Paper II), a fish commensal of grayling, that is closely related to the fish pathogen Spironucleus salmonicida, causative agent of systemic spironucleosis in salmonid fish. We identified substantial genomic differences in the form of divergent genome size, primary sequence divergence and evidence of allelic sequence heterozygosity, a feature not seen in S. salmonicida.

We devised a transfection system for S. salmonicida (Paper VI) and applied it to the study of the mitochondrial remnant organelle (Paper VII). Our analyses showed that S. salmonicida harbor a hydrogenosome, an organelle with more metabolic capabilities than the mitosome of Giardia. Phylogenetic reconstructions of key hydrogenosomal enzymes showed an ancient origin, indicating a common origin to the hydrogenosome in parabasilids and diplomonads.

In conclusion, the thesis has provided important insights into the adaptation of diplomonads in the present and the distant past, revealing hidden diversity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 104 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 990
Keyword
Giardia intestinalis, Spironucleus salmonicida, Spironucleus barkhanus, intestinal parasite, hydrogenosome, mitosome, lateral gene transfer, horizontal gene transfer, diplomonad, metamonad, sexual recombination, transfection, protein complex purification
National Category
Microbiology Evolutionary Biology Infectious Medicine
Research subject
Biology with specialization in Evolutionary Organismal Biology; Biology with specialization in Microbiology; Biology with specialization in Molecular Biology; Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-182831 (URN)978-91-554-8520-7 (ISBN)
Public defence
2012-12-14, B22, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:00 (English)
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Supervisors
Available from: 2012-11-22 Created: 2012-10-16 Last updated: 2013-02-11Bibliographically approved

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