uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Cleavage specificity of recombinant Giardia intestinalis cysteine proteases: Degradation of immunoglobulins and defensins
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.ORCID iD: 0000-0003-1459-3815
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.ORCID iD: 0000-0002-7392-1746
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.

Place, publisher, year, edition, pages
2019. Vol. 227, p. 29-38
Keywords [en]
Cysteine protease, Defensins, Diarrhea, Immunoglobulins, Parasite, Phage display
National Category
Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-372982DOI: 10.1016/j.molbiopara.2018.10.004ISI: 000457660900006PubMedID: 30458129OAI: oai:DiVA.org:uu-372982DiVA, id: diva2:1277262
Funder
Swedish Research Council, 2017-02918Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-03-05Bibliographically 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

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMed

Authority records BETA

Liu, JingyiFu, ZhirongHellman, LarsSvärd, Staffan G.

Search in DiVA

By author/editor
Liu, JingyiFu, ZhirongHellman, LarsSvärd, Staffan G.
By organisation
Microbiology
In the same journal
Molecular and biochemical parasitology (Print)
Microbiology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 46 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf