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Characterization of secreted Giardia intestinalis cysteine proteases
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, 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 [en]
Giardia intestinalis, ESPs, CPs, apical junction, chemokines, phage display, immunoglobulins, defensins, mucin.
National Category
Microbiology
Research subject
Biology with specialization in Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-372997ISBN: 978-91-513-0551-6 (print)OAI: oai:DiVA.org:uu-372997DiVA, id: diva2:1277339
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
List of papers
1. 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
2. Secreted Giardia intestinalis cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines
Open this publication in new window or tab >>Secreted Giardia intestinalis cysteine proteases disrupt intestinal epithelial cell junctional complexes and degrade chemokines
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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.

Keywords
parasite, diarrhea, tight junction, chemokine, intestinal barrier, secretion, cathepsin B, Host-pathogen interactions
National Category
Infectious Medicine Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-355478 (URN)10.1080/21505594.2018.1451284 (DOI)000431949700001 ()29726306 (PubMedID)
Funder
Swedish Research Council, 2012-03364
Available from: 2018-06-29 Created: 2018-06-29 Last updated: 2019-01-10Bibliographically approved
3. Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins
Open this publication in new window or tab >>Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins
2019 (English)In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 227, p. 29-38Article in journal (Refereed) Published
Abstract [en]

Giardia intestinalis is a protozoan parasite and the causative agent of giardiasis, a common diarrheal disease. Cysteine protease (CP) activities have been suggested to be involved in Giardia's pathogenesis and we have recently identified and characterized three secreted Giardia CPs; CP14019, CP16160 and CP16779. Here we have studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The phage display analyses showed that CP16160 has both chymase and tryptase activity and a broad substrate specificity. This was verified using recombinant protein substrates containing different variants of the cleavage sites. Phage display analyses of CP14019 and CP16779 failed but the substrate specificity of CP14019 and CP16779 was tested using the recombinant substrates generated for CP16160. CP16160 and CP14019 showed similar substrate specificity, while CP16779 has a slightly different substrate specificity. The consensus sequence for cleavage by CP16160, obtained from phage display analyses, was used in an in silico screen of the human intestinal proteome for detection of potential targets. Immunoglobulins, including IgA and IgG and defensins (α-HD6 and β-HD1) were predicted to be targets and they were shown to be cleaved by the recombinant CPs in vitro. Our results suggest that the secreted Giardia CPs are key players in the interaction with host cells during Giardia infections since they can cleave several components of the human mucosal defense machinery.

Keywords
Cysteine protease, Defensins, Diarrhea, Immunoglobulins, Parasite, Phage display
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-372982 (URN)10.1016/j.molbiopara.2018.10.004 (DOI)000457660900006 ()30458129 (PubMedID)
Funder
Swedish Research Council, 2017-02918
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-03-05Bibliographically approved
4. MUC2 mucin is cleaved by Giardia intestinalis cysteine protease14019
Open this publication in new window or tab >>MUC2 mucin is cleaved by Giardia intestinalis cysteine protease14019
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-372985 (URN)
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-01-10

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