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Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells
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.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
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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.

Place, publisher, year, edition, pages
2017. Vol. 11, no 12, article id e0006120
National Category
Analytical Chemistry Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-338331DOI: 10.1371/journal.pntd.0006120ISI: 000419108500030PubMedID: 29228011OAI: oai:DiVA.org:uu-338331DiVA, id: diva2:1171910
Available from: 2018-01-08 Created: 2018-01-08 Last updated: 2019-04-19Bibliographically approved
In thesis
1. Characterization of secreted Giardia intestinalis cysteine proteases
Open this publication in new window or tab >>Characterization of secreted Giardia intestinalis cysteine proteases
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Giardia intestinalis, the causative agent of the diarrheal disease giardiasis, is a protozoan parasite that colonizes the upper small intestine of mammals, including humans. It can be divided into eight genotypes or assemblages (A through H) and only assemblage A and B are infective to humans. Giardiasis is a multi-factorial disease but few giardial virulence factors have been identified and characterized.

In this thesis, we used proteomics to identify the major excretory-secretory products (ESPs) released by Giardia trophozoites of the WB and GS isolates during interaction with intestinal epithelial cells (IECs) in vitro (Paper I). To deepen our understanding of the role of ESPs in giardiasis, we focused on three specific secreted Giardia cysteine proteases (CPs; CP14019, CP16160 and CP16779). All the three CPs are capable of opening the apical junction complexes between IECs to degrade chemokines produced in response to Giardia (Paper II). This can partly explain the induction of symptoms and immunosuppression seen during giardiasis. We further studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The preferred sequences were used to search potential human in vivo targets and a number of candidates were identified, including human immunoglobulins as well as defensins, that were subsequently shown to be efficiently cleaved by the CPs (Paper III). To investigate the involvement of CPs in mucus degradation, we tested the CPs on recombinant MUC2 constructs and full-length MUC2. MUC2 is the major component of the mucus layer in the small intestine. It was shown that CP14019 cleave MUC2 in the N-terminal, suggesting a mechanism that the parasite can use to disrupt/release the mucus gel network and get access to the intestinal epithelium of the host (Paper IV).

In summary, this thesis has studied secreted Giardia CPs and their roles in Giardia infections, providing significant insights into the molecular pathogenesis of giardiasis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1763
Keywords
Giardia intestinalis, ESPs, CPs, apical junction, chemokines, phage display, immunoglobulins, defensins, mucin.
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
urn:nbn:se:uu:diva-372997 (URN)978-91-513-0551-6 (ISBN)
Public defence
2019-02-28, A1:111a, Uppsala Biomedicinska Centrum BMC, Husarg. 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-02-07 Created: 2019-01-10 Last updated: 2019-02-18
2. Studies of Giardia-host interactions: role of cysteine-rich surface proteins.
Open this publication in new window or tab >>Studies of Giardia-host interactions: role of cysteine-rich surface proteins.
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
Giardia, intestinal parasite, parasite infection, host-parasite interaction, virulence factors, HCMPs, cysteine-rich proteins, secretome, proteome
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-382071 (URN)978-91-513-0670-4 (ISBN)
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

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Ma'ayeh, Showgy Y.Liu, JingyiPeirasmaki, DimitraHörnaeus, KatarinaBergström Lind, Sara K.Grabherr, ManfredBergquist, JonasSvärd, Staffan

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