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Secreted Giardia intestinalis cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines
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.ORCID iD: 0000-0001-7091-5570
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Univ Bern, Vetsuisse Fac, Inst Parasitol, Bern, Switzerland.
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2018 (English)In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 9, no 1, p. 879-894Article in journal (Refereed) Published
Abstract [en]

Giardiasis is a common diarrheal disease caused by the protozoan parasite Giardia intestinalis. Cysteine proteases (CPs) are acknowledged as virulence factors in Giardia but their specific role in the molecular pathogenesis of disease is not known. Herein, we aimed to characterize the three main secreted CPs (CP14019, CP16160 and CP16779), which were identified by mass spectrometry in the medium during interaction with intestinal epithelial cells (IECs) in vitro. First, the CPs were epitope-tagged and localized to the endoplasmic reticulum and cytoplasmic vesicle-like structures. Second, we showed that recombinant CPs, expressed in Pichia pastoris, are more active in acidic environment (pH 5.5-6) and we determined the kinetic parameters using fluorogenic substrates. Third, excretory-secretory proteins (ESPs) from Giardia trophozoites affect the localization of apical junctional complex (AJC) proteins and recombinant CPs cleave or re-localize the AJC proteins (claudin-1 and -4, occludin, JAM-1, beta-catenin and E-cadherin) of IECs. Finally, we showed that the ESPs and recombinant CPs can degrade several chemokines, including CXCL1, CXCL2, CXCL3, IL-8, CCL2, and CCL20, which are up-regulated in IECs during Giardia-host cell interactions. This is the first study that characterizes the role of specific CPs secreted from Giardia and our results collectively indicate their roles in the disruption of the intestinal epithelial barrier and modulating immune responses during Giardia infections.

Place, publisher, year, edition, pages
2018. Vol. 9, no 1, p. 879-894
Keywords [en]
parasite, diarrhea, tight junction, chemokine, intestinal barrier, secretion, cathepsin B, Host-pathogen interactions
National Category
Infectious Medicine Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-355478DOI: 10.1080/21505594.2018.1451284ISI: 000431949700001PubMedID: 29726306OAI: oai:DiVA.org:uu-355478DiVA, id: diva2:1229228
Funder
Swedish Research Council, 2012-03364Available from: 2018-06-29 Created: 2018-06-29 Last updated: 2019-01-10Bibliographically 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

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Liu, JingyiMa'ayeh, Showgy Y.Peirasmaki, DimitraHellman, LarsSvärd, Staffan G.

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