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Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Medicine, Molecular Biology.
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2002 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 367, no 3, 617-28 p.Article in journal (Refereed) Published
Description
Abstract [en]

Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.

Place, publisher, year, edition, pages
2002. Vol. 367, no 3, 617-28 p.
Keyword [en]
bacterial toxin, cytotoxicity, cystic fibrosis, GTP-binding protein, Pseudomonas aeruginosa
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-3311DOI: 10.1042/BJ20020714OAI: oai:DiVA.org:umu-3311DiVA: diva2:141907
Available from: 2003-12-23 Created: 2003-12-23 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Type III Secretion Mediated Translocation of Effector Exoenzymes by Pseudomonas aeruginosa
Open this publication in new window or tab >>Type III Secretion Mediated Translocation of Effector Exoenzymes by Pseudomonas aeruginosa
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Injektion av gifter via typ III sekretionssystemet hos bakterien Pseudomonas aeruginosa
Publisher
52 p.
Keyword
Cell and molecular biology, Pseudomonas aeruginosa, type III secretion system, ExoS, ExoT, PcrV, PcrG, PopB, PopD, PopN, type IV pili, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-174 (URN)91-7305-545-X (ISBN)
Public defence
2003-11-28, Major Groove, 6L, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2003-12-23 Created: 2003-12-23 Last updated: 2010-02-24Bibliographically approved
2. Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S
Open this publication in new window or tab >>Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP-ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity in vitro. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of Yersinia pseudotuberculosis. We found that Ras was ADP-ribosylated in vivo and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily in vivo, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by P. aeruginosa. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 in vivo, and that ExoT shows GAP activity towards RhoA in vitro.

The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids 424DALDL428 on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS in vivo.

Place, publisher, year, edition, pages
Umeå universitet, 2003. 51 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 851
Keyword
Cell biology, Pseudomonas aeruginosa, ADP-ribosylation, GAP, Ras superfamily, NAD, ExoS, 14-3-3, Cellbiologi
National Category
Cell and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:umu:diva-121 (URN)91-7305-505-0 (ISBN)
Public defence
2003-10-31, Betula, 6M, Umeå, 09:00
Opponent
Supervisors
Available from: 2003-01-01 Created: 2003-01-01 Last updated: 2012-06-27Bibliographically approved

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