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Inter and Intra-Assemblage Characterizations of Giardia intestinalis: from clinic to genome
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. (Prof. Staffan Svärd laboratory)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The protozoan parasite Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the most common causes of diarrheal disease throughout the world, where an estimated 500 million people are infected annually. Despite efforts in trying to elucidate factors associated with virulence in G. intestinalis little is currently known. The disease outcome is highly variable in Giardia infected individuals, ranging from asymptomatic carriers to severe disease. The reasons behind the differences in disease outcome are vaguely understood and studies trying to link infectivity to different Giardia assemblages or sub-assemblages have rendered conflicting results. Prior to this study, little was known about the prevalence and genetic diversity of different G. intestinalis assemblages across the world.

In this thesis, molecular characterization of clinical G. intestinalis samples from Eastern Africa and Central America, has been performed, enabling a better understanding of the prevalence of different Giardia genotypes in endemic areas (Papers I and II). A correlation between Giardia colonization and the presence of Helicobacter pylori in the human host was established. We found that the currently available genotyping tools provide low resolution when used to characterize assemblage A Giardia. Also, genotyping of assemblage B isolates at these loci is troublesome due to the polymorphic substitutions frequently found in the sequencing chromatograms. This ambiguity was investigated by using micromanipulation to isolate single assemblage B Giardia cells (Paper III). Both cultured trophozoites and cysts from giardiasis patients were analyzed. The data showed that allelic sequence heterozygosity (ASH) does occur at the single cell level, but also that multiple sub-assemblage infections appear to be common in human giardiasis patients.

Furthermore, genome-wide sequencing followed by comparative genomics was performed in order to better characterize differences between and within different Giardia assemblages. The genome of a non-human infecting, assemblage E isolate (Paper IV) was sequenced.  The genomes of two freshly isolated human infecting assemblage AII isolates were also sequenced (Paper V). Subsequent, comparative analyses were performed and included the genomes of two human infecting isolates, WB (AI) and GS/M (B). Several important differences were found between assemblages A, B and E, but also within assemblage A; including unique gene repertoires for each isolate, observed differences in the variable gene families and an overall difference in ASH between the different isolates. Also, a new multi-locus genotyping (MLG) strategy for genotyping of assemblage A Giardia has been established and evaluated on clinical samples from human giardiasis patients.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2012. , p. 85
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 893
Keyword [en]
Giardia, protozoa, parasite infection, diarrhea, genome sequencing, comparative genomics, genotyping, ASH, MLG
National Category
Natural Sciences Medical and Health Sciences
Research subject
Infectious Diseases; Molecular Medicine; Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-167063ISBN: 978-91-554-8259-6 (print)OAI: oai:DiVA.org:uu-167063DiVA: diva2:480741
Public defence
2012-02-23, B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2012-02-02 Created: 2012-01-19 Last updated: 2012-02-15Bibliographically approved
List of papers
1. Dominance of Giardia assemblage B in León, Nicaragua.
Open this publication in new window or tab >>Dominance of Giardia assemblage B in León, Nicaragua.
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2008 (English)In: Acta Tropica, ISSN 0001-706X, E-ISSN 1873-6254, Vol. 106, no 1, p. 44-53Article in journal (Refereed) Published
Abstract [en]

Giardiasis is a major problem in León, Nicaragua, yet despite this no data are available regarding the prevalence of different Giardia genotypes in this area. To address this question, a molecular analysis of Giardia isolates from humans and dogs living in the same area in León, Nicaragua was performed. Giardia isolates from 119 Nicaraguan patients and 8 dogs were successfully genotyped using single and/or nested beta-giardin PCR with subsequent restriction length fragment polymorphism (RFLP) analysis. The analyses of human samples yielded 94 (79%) assemblage B isolates and 25 (21%) assemblage A isolates. Only the non-human-associated assemblages C and D were found in the dog samples. Sixteen isolates with assemblage A pattern, 26 isolates with assemblage B pattern and all dog isolates were further characterized by sequencing the nested beta-giardin PCR product and by molecular analyses of the glutamate dehydrogenase (gdh) gene. Within the study area the assemblage A isolates were highly genetically homogenous, showing only sub-genotypes A2 (n=3) or A3 (n=13) at the beta-giardin locus and AII only at the gdh locus while assemblage B showed a high genetic polymorphism at both loci. Seven different sub-genotypes were identified within 13 of the sequenced assemblage B beta-giardin isolates. The remaining 13 sequenced assemblage B-isolates appeared to contain several different variants of the beta-giardin gene since the chromatograms displayed one to seven double peaks. The gdh sequences showed an even higher polymorphism since only 2 of 26 assemblage B isolates were without double peaks. Two mixed infections between assemblage A and B were found when the gdh gene was analyzed. Polymorphisms were also observed in the dog-associated assemblages C and D, but to a lesser extent than in assemblage B.

Keyword
Nicaragua, Giardia intestinalis, (-Giardin gene, glutamate dehydrogenase gene, genotyping, diarrhea
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-16512 (URN)10.1016/j.actatropica.2008.01.004 (DOI)000255487100007 ()18325480 (PubMedID)
Available from: 2008-05-27 Created: 2008-05-27 Last updated: 2017-12-08Bibliographically approved
2. Multi-locus genotyping of Giardia intestinalis in Ugandan children with and without Helicobacter pylori colonization
Open this publication in new window or tab >>Multi-locus genotyping of Giardia intestinalis in Ugandan children with and without Helicobacter pylori colonization
2012 (English)In: PLoS Neglected Tropical Diseases, ISSN 1935-2735Article in journal (Other academic) Submitted
Abstract [en]

Background

The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts world-wide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, little data is available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.

Methodology/Principal findings

Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. No significant association was found in the studied population with regards to the presence of Giardia and gender, type of toilet, source of drinking-water or type of housing. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on the β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage A, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable and a significant association was found between Giardia assemblage B and H. pylori infection, OR=5.0 (1.9- 16).

Conclusions/Significance

This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that Giardia-infected children have a 3-fold higher risk of being infected by H. pylori. The data also suggests that assemblage B Giardia may be more closely associated with H. pylori infection.

National Category
Medical and Health Sciences Natural Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-167046 (URN)
Available from: 2012-01-19 Created: 2012-01-19 Last updated: 2012-02-15Bibliographically approved
3. Allelic sequence heterozygosity in single Giardia parasites
Open this publication in new window or tab >>Allelic sequence heterozygosity in single Giardia parasites
2012 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, article id 65Article in journal (Refereed) Published
Abstract [en]

Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

Keyword
Giardia, ASH, assemblage B, genotyping, single cell, micromanipulation
National Category
Medical and Health Sciences Natural Sciences
Research subject
Epidemiology; Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-167049 (URN)10.1186/1471-2180-12-65 (DOI)000308649900001 ()22554281 (PubMedID)
Available from: 2012-01-19 Created: 2012-01-19 Last updated: 2017-12-08Bibliographically approved
4. 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, p. 543-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
5. Genomic variation within Giardia intestinalis assemblage A isolates
Open this publication in new window or tab >>Genomic variation within Giardia intestinalis assemblage A isolates
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic patients, to study the genetic diversity within assemblage A and to identify new assemblage A-specific genotyping targets.

Results: Several biological differences between the assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 (AS98) and AII-2 (AS175)) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of an assemblage AI isolate (WB). Our analyses indicate that the divergence between AI and AII is approximately 1%, represented by ~100,000 single nucleotide polymorphisms (SNP). Moreover, SNPs are homogeneously distributed over the chromosomes with an enrichment in regions containing surface antigens and non-coding sequences. The level of allelic sequence heterozygosity (ASH) in the two AII isolates were found to be 0.25-0.35%, which is 25-30-fold higher than in the WB isolate. 37 proteinencoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolatespecific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 7 members, showed high sequence diversity between the assemblage A isolates and they could have important roles in hostparasite interactions. A subset of the variable genes was used to develop new genotyping methods for assemblage A isolates.

Conclusions: Our results show that there is a significant genomic variation in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms. This identified putative virulence genes and generated a new assemblage A-specific genotyping approach.

Keyword
Giardia, comparative genomics, assemblage A, ASH, MLG
National Category
Medical and Health Sciences Natural Sciences
Research subject
Biology with specialization in Microbiology; Molecular Medicine; Infectious Diseases
Identifiers
urn:nbn:se:uu:diva-167052 (URN)
Available from: 2012-01-19 Created: 2012-01-19 Last updated: 2012-02-15

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