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Exoenzyme S of Pseudomonas aeruginosa: cellular targets and interaction with 14-3-3
Umeå University, Faculty of Medicine, Medical Biosciences.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pseudomonas aeruginosa is an opportunistic pathogen that is a serious problem for immuno-compromised patients. Toxins such as exoenzyme (Exo) S, ExoT, ExoY and ExoU are secreted and translocated from the bacteria into the eukaryotic cell via the bacterial encoded type III secretion system. Our research focuses on ExoS, a bifunctional toxin comprising a Rho-GTPase-activating protein domain (RhoGAP) and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain termed MLD. In this study, cell lines expressing activated forms of various components of the Ras signaling pathway have been used to understand the functional and mechanical activation of ExoS-ADP-ribosyltransferase activity and to reveal its cellular targets in the cell. Our observations suggested that Ras GTPase is the dominant target by which ExoS mediates cell death and activated Ras is able to protect cells against cell death, regardless of whether it has been ADP-ribosylated by ExoS.

It has been reported that the 14-3-3 cofactor protein is required for ADP-ribosyltransferase activity of ExoS and a phosphorylation-independent interaction occurs between 14-3-3 and the C-terminal part of ExoS. We have undertaken a deeper analysis including structural and biological investigation of this interaction. Our results suggested that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity. Structural analysis showed that ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. Our structure was supported by biochemical and cytotoxicity analyses, which revealed that the substitution of important residues of ExoS significantly weakens the ability of ExoS to modify endogenous targets such as RAS/RAP1 and to induce cell death. Further, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. Leucine residues-422, 423, 426, and 428 of ExoS are important for the interaction with the ″roof″ of the amphiphatic groove of 14-3-3.

In conclusion, we show the mechanism of cell signal transduction pathways affected upon ExoS infection and also demonstrate that the hydrophobic residues of ExoS in 14-3-3 interaction motif have a significant role for ExoS enzymatic activity.

Place, publisher, year, edition, pages
Umeå: Medicinsk biovetenskap , 2007. , 67 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1125
Keyword [en]
ExoS, ADP-ribosylation, 14-3-3 protein, Pseudomonas aeruginosa, Small GTPases, RAS, RAP
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1411ISBN: 978-91-7264-414-4 (print)OAI: oai:DiVA.org:umu-1411DiVA: diva2:140943
Public defence
2007-11-16, Betula, 6M, Norrland Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2007-10-30 Created: 2007-10-30 Last updated: 2009-09-25Bibliographically approved
List of papers
1. Exoenzyme S of Pseudomonas aeruginosa is not able to induce apoptosis when cells express activated proteins, such as Ras or protein kinase B/Akt
Open this publication in new window or tab >>Exoenzyme S of Pseudomonas aeruginosa is not able to induce apoptosis when cells express activated proteins, such as Ras or protein kinase B/Akt
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2006 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 8, no 5, 815-822 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-2695 (URN)
Available from: 2007-10-30 Created: 2007-10-30 Last updated: 2017-12-14Bibliographically approved
2. Delineation of exoenzyme S residues that mediate the interaction with 14-3-3 and its biological activity.
Open this publication in new window or tab >>Delineation of exoenzyme S residues that mediate the interaction with 14-3-3 and its biological activity.
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2006 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 3, 638-646 p.Article in journal (Refereed) Published
Keyword
14-3-3 Proteins/*physiology, ADP Ribose Transferases/metabolism/pharmacology/*physiology, Amino Acid Motifs, Bacterial Toxins/metabolism/pharmacology, Cell Death/drug effects, Cells; Cultured, Enzyme Activation, Hela Cells, Humans, Leucine/chemistry/metabolism, Phosphorylation, Protein Binding, Pseudomonas aeruginosa/enzymology, Signal Transduction/physiology
Identifiers
urn:nbn:se:umu:diva-15367 (URN)10.1111/j.1742-4658.2005.05100.x (DOI)16420486 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2017-12-14Bibliographically approved
3. Phosphorylation-independent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis.
Open this publication in new window or tab >>Phosphorylation-independent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis.
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2007 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 26, no 3, 902-913 p.Article in journal (Refereed) Published
Abstract [en]

14-3-3 proteins are phosphoserine/phosphothreonine-recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14-3-3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation-independent interaction between 14-3-3 and exoenzyme S (ExoS), an ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. The 1.5 A crystal structure is supported by cytotoxicity analysis, which reveals that substitution of the corresponding hydrophobic residues significantly weakens the ability of ExoS to modify the endogenous targets RAS/RAP1 and to induce cell death. Furthermore, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. In conclusion, we show that ExoS binds 14-3-3 in a novel reversed orientation that is primarily dependent on hydrophobic residues. This interaction is phosphorylation independent and is required for the function of ExoS.

Keyword
14-3-3 Proteins/*genetics/*metabolism, ADP Ribose Transferases/*metabolism, Animals, Bacterial Toxins/*metabolism, Blotting; Western, Crystallography, DNA Primers, Female, Hela Cells, Humans, Mice, Mice; Inbred BALB C, Models; Molecular, Molecular Conformation, Mutation/genetics, Pneumonia/*microbiology, Protein Binding, Pseudomonas aeruginosa/enzymology/*pathogenicity, rap1 GTP-Binding Proteins/metabolism
Identifiers
urn:nbn:se:umu:diva-18052 (URN)10.1038/sj.emboj.7601530 (DOI)17235285 (PubMedID)
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2017-12-14Bibliographically approved
4. Electrostatic interaction is of secondary importance for the interaction between ExoS and 14-3-3
Open this publication in new window or tab >>Electrostatic interaction is of secondary importance for the interaction between ExoS and 14-3-3
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2698 (URN)
Available from: 2007-10-30 Created: 2007-10-30 Last updated: 2010-01-13Bibliographically approved

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