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Hidden Diversity Revealed: Genomic, Transcriptomic and Functional Studies of Diplomonads
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. (Svärd Group)
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 [en]
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: urn:nbn:se:uu:diva-182831ISBN: 978-91-554-8520-7 (print)OAI: oai:DiVA.org:uu-182831DiVA: diva2:562514
Public defence
2012-12-14, B22, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2012-11-22 Created: 2012-10-16 Last updated: 2013-02-11Bibliographically approved
List of papers
1. Draft genome sequencing of Giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species?
Open this publication in new window or tab >>Draft genome sequencing of Giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species?
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2009 (English)In: PLoS Pathogens, ISSN 1553-7366, Vol. 5, no 8, e1000560- p.Article in journal (Refereed) Published
Abstract [en]

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16 x coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-127462 (URN)10.1371/journal.ppat.1000560 (DOI)000270804500015 ()19696920 (PubMedID)
Available from: 2010-07-15 Created: 2010-07-13 Last updated: 2013-02-11Bibliographically approved
2. Large genomic differences between the morphologically indistinguishable diplomonads Spironucleus barkhanus and Spironucleus salmonicida.
Open this publication in new window or tab >>Large genomic differences between the morphologically indistinguishable diplomonads Spironucleus barkhanus and Spironucleus salmonicida.
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2010 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, 258- p.Article in journal (Refereed) Published
Abstract [en]

Background

Microbial eukaryotes show large variations in genome structure and content between lineages, indicating extensive flexibility over evolutionary timescales. Here we address the tempo and mode of such changes within diplomonads, flagellated protists with two nuclei found in oxygen-poor environments. Approximately 5,000 expressed sequence tag (EST) sequences were generated from the fish commensal Spironucleus barkhanus and compared to sequences from the morphologically indistinguishable fish parasite Spironucleus salmonicida, and other diplomonads. The ESTs were complemented with sequence variation studies in selected genes and genome size determinations.

Results

Many genes detected in S. barkhanus and S. salmonicida are absent in the human parasite Giardia intestinalis, the most intensively studied diplomonad. For example, these fish diplomonads show an extended metabolic repertoire and are able to incorporate selenocysteine into proteins. The codon usage is altered in S. barkhanus compared to S. salmonicida. Sequence variations were found between individual S. barkhanus ESTs for many, but not all, protein coding genes. Conversely, no allelic variation was found in a previous genome survey of S. salmonicida. This difference was confirmed by sequencing of genomic DNA. Up to five alleles were identified for the cloned S. barkhanus genes, and at least nineteen highly expressed S. barkhanus genes are represented by more than four alleles in the EST dataset. This could be explained by the presence of a non-clonal S. barkhanus population in the culture, by a ploidy above four, or by duplications of parts of the genome. Indeed, genome size estimations using flow cytometry indicated similar haploid genome sizes in S. salmonicida and G. intestinalis (similar to 12 Mb), whereas the S. barkhanus genome is larger (similar to 18 Mb).

Conclusions

This study indicates extensive divergent genome evolution within diplomonads. Genomic traits such as codon usage, frequency of allelic sequence variation, and genome size have changed considerably between S. barkhanus and S. salmonicida. These observations suggest that large genomic differences may accumulate in morphologically indistinguishable eukaryotic microbes.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-136422 (URN)10.1186/1471-2164/11/258 (DOI)000277992000002 ()20409319 (PubMedID)
Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
3. Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate.
Open this publication in new window or tab >>Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate.
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2010 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, 543- p.Article in journal (Refereed) Published
Abstract [en]

Background

Giardia intestinalis is a protozoan parasite that causes diarrhea in a wide range of mammalian species. To further understand the genetic diversity between the Giardia intestinalis species, we have performed genome sequencing and analysis of a wild-type Giardia intestinalis sample from the assemblage E group, isolated from a pig.

Results

We identified 5012 protein coding genes, the majority of which are conserved compared to the previously sequenced genomes of the WB and GS strains in terms of microsynteny and sequence identity. Despite this, there is an unexpectedly large number of chromosomal rearrangements and several smaller structural changes that are present in all chromosomes. Novel members of the VSP, NEK Kinase and HCMP gene families were identified, which may reveal possible mechanisms for host specificity and new avenues for antigenic variation. We used comparative genomics of the three diverse Giardia intestinalis isolates P15, GS and WB to define a core proteome for this species complex and to identify lineage-specific genes. Extensive analyses of polymorphisms in the core proteome of Giardia revealed differential rates of divergence among cellular processes.

Conclusions

Our results indicate that despite a well conserved core of genes there is significant genome variation between Giardia isolates, both in terms of gene content, gene polymorphisms, structural chromosomal variations and surface molecule repertoires. This study improves the annotation of the Giardia genomes and enables the identification of functionally important variation.

National Category
Biological Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-133827 (URN)10.1186/1471-2164-11-543 (DOI)000283123400001 ()20929575 (PubMedID)
Available from: 2010-11-19 Created: 2010-11-16 Last updated: 2017-12-12Bibliographically approved
4. Genome-Wide Analyses of Recombination Suggest That Giardia intestinalis Assemblages Represent Different Species
Open this publication in new window or tab >>Genome-Wide Analyses of Recombination Suggest That Giardia intestinalis Assemblages Represent Different Species
2012 (English)In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 29, no 10, 2895-2898 p.Article in journal (Refereed) Published
Abstract [en]

Giardia intestinalis is a major cause of waterborne enteric disease in humans. The species is divided into eight assemblages suggested to represent separate Giardia species based on host specificities and the genetic divergence of marker genes. We have investigated whether genome-wide recombination occurs between assemblages using the three available G. intestinalis genomes. First, the relative nonsynonymous substitution rates of the homologs were compared for 4,009 positional homologs. The vast majority of these comparisons indicate genetic isolation without interassemblage recombinations. Only a region of 6 kbp suggests genetic exchange between assemblages A and E, followed by gene conversion events. Second, recombination-detecting software fails to identify within-gene recombination between the different assemblages for most of the homologs. Our results indicate very low frequency of recombination between the syntenic core genes, suggesting that G. intestinalis assemblages are genetically isolated lineages and thus should be viewed as separated Giardia species.

National Category
Natural Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-175947 (URN)10.1093/molbev/mss107 (DOI)000309927900003 ()22474166 (PubMedID)
Available from: 2012-06-14 Created: 2012-06-14 Last updated: 2017-12-07Bibliographically approved
5. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome
Open this publication in new window or tab >>Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome
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2012 (English)In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 11, no 7, 864-873 p.Article in journal (Refereed) Published
Abstract [en]

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide-glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG-tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-181943 (URN)10.1128/EC.00092-12 (DOI)000307191000004 ()
Available from: 2012-10-02 Created: 2012-10-02 Last updated: 2017-12-07Bibliographically approved
6. Stable transfection of the diplomonad parasite Spironucleus salmonicida
Open this publication in new window or tab >>Stable transfection of the diplomonad parasite Spironucleus salmonicida
2012 (English)In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 11, no 11, 1353-1361 p.Article in journal (Refereed) Published
Abstract [en]

Eukaryotic microbes are highly diverse and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has mainly been studied due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad casuing systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3xHA, 2xOLLAS, 3xMYC) and purification of proteins (2xStrepII-FLAG or SBP-GST) under the control of native or constitutive promoters. Three selectable markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr) or neomycin phosphotransferase (nptII) were successfully applied for generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope-tagged six proteins and confirmed their expression by Western Blot. Next, we demonstrated the utility of these vectors by recording the sub-cellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we describe the creation of a S. salmonicida double transfectant suitable for co-localization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific as well as general diplomonad traits, benefiting research on both Giardia and Spironucleus.

National Category
Microbiology Cell Biology
Research subject
Biology with specialization in Microbiology; Biology with specialization in Molecular Cell Biology
Identifiers
urn:nbn:se:uu:diva-182827 (URN)10.1128/EC.00179-12 (DOI)000310566400006 ()22983987 (PubMedID)
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2017-12-07Bibliographically approved
7. Spironucleus mitochondrial remnants suggest that hydrogenosomes are ancient organelles
Open this publication in new window or tab >>Spironucleus mitochondrial remnants suggest that hydrogenosomes are ancient organelles
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(English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490Article in journal (Refereed) Submitted
Keyword
diplomonad, Spironucleus salmonicida, [FeFe] hydrogenase, hydrogenosome, mitosome, intestinal parasite, Giardia intestinalis
National Category
Microbiology Evolutionary Biology
Research subject
Biology with specialization in Evolutionary Organismal Biology; Biology with specialization in Microbiology; Biology with specialization in Molecular Evolution; Biology with specialization in Molecular Cell Biology
Identifiers
urn:nbn:se:uu:diva-182828 (URN)
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2017-12-07Bibliographically approved
8. Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq
Open this publication in new window or tab >>Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq
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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.

Keyword
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:nbn:se:uu:diva-182829 (URN)10.1371/journal.pcbi.1003000 (DOI)000316864200066 ()
Available from: 2012-10-16 Created: 2012-10-16 Last updated: 2017-12-07Bibliographically approved

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