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Studies of Giardia-host interactions: role of cysteine-rich surface proteins.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Giardia intestinalis is a eukaryotic parasite that colonizes the small intestine of humans and animals causing the diarrheal disease known as giardiasis. This parasite is not invasive and does not internalize into host cells but it rather attaches to the brush border surface of the small intestine disrupting the epithelial barrier. Giardia causes around 280 million symptomatic infections in humans every year, while it can also cause chronic and asymptomatic infections. Giardiasis is a multifactorial disease but only few factors that directly contribute in the pathogenesis and virulence of the disease have been identified. G. intestinalis has eight genetic groups, but only two of them (A and B) are known to infect humans.

In this thesis, whole genome sequencing was performed for two human assemblage A isolates (AS175 and AS98) and were compared to assemblage A isolate WB genome (Paper I). Genome-wide variations were identified among the three isolates including isolate-specific coding sequences and high level of nucleotide diversity of multi-gene families such as VSPs and HCMPs.

We further used an in vitro model for parasite interaction with host intestinal epithelial cells (IECs) to study the interplay between Giardia and the human host. We have identified the major Giardia excretory-secretory products (ESPs) released by two Giardia isolates (WB and GS) when they interact with the Caco-2 IECs (Paper II). Wide changes in the transcriptome (Paper III) and the proteome (Paper IV) of the parasite (WB isolate) and the host IECs have been studied giving us a further understanding of the parasite-host interactions. An understudied gene family (HCMPs) was studied and further characterized during interactions in both RNA and protein level (Paper III, IV).

In conclusion, the thesis has provided a further understanding of Giardia-host interactions in vitro and the molecular mechanisms involved.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 85
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1816
Keywords [en]
Giardia, intestinal parasite, parasite infection, host-parasite interaction, virulence factors, HCMPs, cysteine-rich proteins, secretome, proteome
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-382071ISBN: 978-91-513-0670-4 (print)OAI: oai:DiVA.org:uu-382071DiVA, id: diva2:1305906
Public defence
2019-06-14, A1:111a, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-05-24 Created: 2019-04-19 Last updated: 2019-06-18
List of papers
1. Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates
Open this publication in new window or tab >>Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates
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2015 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 16, article id 697Article in journal (Refereed) Published
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. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. Results: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by similar to 100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding 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 isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/ Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. Conclusions: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.

National Category
Genetics Microbiology
Identifiers
urn:nbn:se:uu:diva-264039 (URN)10.1186/s12864-015-1893-6 (DOI)000361093400009 ()26370391 (PubMedID)
Funder
Swedish Research Council FormasSwedish Research Council
Available from: 2015-10-06 Created: 2015-10-05 Last updated: 2019-04-19Bibliographically approved
2. Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells
Open this publication in new window or tab >>Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells
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2017 (English)In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 11, no 12, article id e0006120Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

Giardia intestinalis is a non-invasive protozoan parasite that causes giardiasis in humans, the most common form of parasite-induced diarrhea. Disease mechanisms are not completely defined and very few virulence factors are known.

METHODOLOGY:

To identify putative virulence factors and elucidate mechanistic pathways leading to disease, we have used proteomics to identify the major excretory-secretory products (ESPs) when Giardia trophozoites of WB and GS isolates (assemblages A and B, respectively) interact with intestinal epithelial cells (IECs) in vitro.

FINDINGS:

The main parts of the IEC and parasite secretomes are constitutively released proteins, the majority of which are associated with metabolism but several proteins are released in response to their interaction (87 and 41 WB and GS proteins, respectively, 76 and 45 human proteins in response to the respective isolates). In parasitized IECs, the secretome profile indicated effects on the cell actin cytoskeleton and the induction of immune responses whereas that of Giardia showed anti-oxidation, proteolysis (protease-associated) and induction of encystation responses. The Giardia secretome also contained immunodominant and glycosylated proteins as well as new candidate virulence factors and assemblage-specific differences were identified. A minor part of Giardia ESPs had signal peptides (29% for both isolates) and extracellular vesicles were detected in the ESPs fractions, suggesting alternative secretory pathways. Microscopic analyses showed ESPs binding to IECs and partial internalization. Parasite ESPs reduced ERK1/2 and P38 phosphorylation and NF-κB nuclear translocation. Giardia ESPs altered gene expression in IECs, with a transcriptional profile indicating recruitment of immune cells via chemokines, disturbances in glucose homeostasis, cholesterol and lipid metabolism, cell cycle and induction of apoptosis.

CONCLUSIONS:

This is the first study identifying Giardia ESPs and evaluating their effects on IECs. It highlights the importance of host and parasite ESPs during interactions and reveals the intricate cellular responses that can explain disease mechanisms and attenuated inflammatory responses during giardiasis.

National Category
Analytical Chemistry Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-338331 (URN)10.1371/journal.pntd.0006120 (DOI)000419108500030 ()29228011 (PubMedID)
Available from: 2018-01-08 Created: 2018-01-08 Last updated: 2019-04-19Bibliographically approved
3. High Cysteine Proteins are up-regulated during Giardia-host cell interaction.
Open this publication in new window or tab >>High Cysteine Proteins are up-regulated during Giardia-host cell interaction.
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(English)Manuscript (preprint) (Other academic)
Keywords
Giardia, HCMPs, interactions, host-parasite interactions, infection, parasite, RNA-seq
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-381936 (URN)
Available from: 2019-04-16 Created: 2019-04-16 Last updated: 2019-04-19
4. Proteome analyses of Giardia–host cell interactions in vitro.
Open this publication in new window or tab >>Proteome analyses of Giardia–host cell interactions in vitro.
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(English)Manuscript (preprint) (Other academic)
Keywords
Giardia, host-parasite interactions, parasite, infections, proteomics
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-381937 (URN)
Available from: 2019-04-16 Created: 2019-04-16 Last updated: 2019-04-19

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