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Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S
Umeå University, Faculty of Medicine, Molecular Biology.
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. , p. 51
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 851
Keywords [en]
Cell biology, Pseudomonas aeruginosa, ADP-ribosylation, GAP, Ras superfamily, NAD, ExoS, 14-3-3
Keywords [sv]
Cellbiologi
National Category
Cell and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
URN: urn:nbn:se:umu:diva-121ISBN: 91-7305-505-0 (print)OAI: oai:DiVA.org:umu-121DiVA, id: diva2:140498
Public defence
2003-10-31, Betula, 6M, Umeå, 09:00
Opponent
Supervisors
Available from: 2003-01-01 Created: 2003-01-01 Last updated: 2018-03-15Bibliographically approved
List of papers
1. Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras
Open this publication in new window or tab >>Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras
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2000 (English)In: Biochemical Journal, Vol. 347, p. 217-222Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-2453 (URN)
Available from: 2003-01-01 Created: 2003-01-01 Last updated: 2018-06-09Bibliographically approved
2. Exoenzyme T of Pseudomonas aeruginosa elicits cytotoxicity without interfering with Ras signal transduction
Open this publication in new window or tab >>Exoenzyme T of Pseudomonas aeruginosa elicits cytotoxicity without interfering with Ras signal transduction
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2001 (English)In: Cellular Microbiology, Vol. 3, no 4, p. 237-46Article in journal (Refereed) Published
Abstract [en]

One virulence strategy used by the opportunistic pathogen Pseudomonas aeruginosa is to target toxic proteins into eukaryotic cells by a type III secretion mechanism. Two of these proteins, ExoS and ExoT, show 75% homology on amino acid level. However, compared with ExoS, ExoT exhibits highly reduced ADP-ribosylating activity and the role of ExoT in pathogenesis is poorly understood. To study the biological effect of ExoT, we used a strategy by which ExoT was delivered into host cells by the heterologous type III secretion system ofYersinia pseudotuberculosis. ExoT was found to induce a rounded cell morphology and to mediate disruption of actin microfilaments, similar to that induced by an ADP-ribosylation defective ExoS (E381A) and the related cytotoxin YopE of Y. pseudotuberculosis. In contrast to ExoS, ExoT had no major effect on cell viability and did not modify or inactivate Ras by ADP-ribosylation in vivo. However, similar to ExoS and YopE, ExoT exhibited GAP (GTPase activating protein) activity on RhoA GTPase in vitro. Interestingly, ExoT(R149K), deficient for GAP activity, still caused a morphological change of HeLa cells. Based on our findings, we suggest that the ADP-ribosylating activity of ExoT target another, as yet unidentified, host protein that is distinct from Ras.

Place, publisher, year, edition, pages
John Wiley & Sons, 2001
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-3309 (URN)10.1046/j.1462-5822.2001.00108.x (DOI)
Available from: 2003-12-23 Created: 2003-12-23 Last updated: 2018-06-09Bibliographically approved
3. Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo
Open this publication in new window or tab >>Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo
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2002 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 367, no 3, p. 617-28Article in journal (Refereed) Published
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.

Keywords
bacterial toxin, cytotoxicity, cystic fibrosis, GTP-binding protein, Pseudomonas aeruginosa
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-3311 (URN)10.1042/BJ20020714 (DOI)
Available from: 2003-12-23 Created: 2003-12-23 Last updated: 2018-06-09Bibliographically approved
4. 14-3-3 proteins are required for the inhibition fo Ras by exoenzyme S
Open this publication in new window or tab >>14-3-3 proteins are required for the inhibition fo Ras by exoenzyme S
2000 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 349, p. 697-701Article in journal (Refereed) Published
Abstract [en]

14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine ligands, such as Raf-l kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where 'Xaa' represents 'any residue', and 'pSer' is 'phosphoserine'). However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ib alpha and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS. Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259). In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo. Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.

Place, publisher, year, edition, pages
Portland Press, 2000
National Category
Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-2456 (URN)10.1042/bj3490697 (DOI)000088712000005 ()10903129 (PubMedID)
Available from: 2003-01-01 Created: 2003-01-01 Last updated: 2018-12-05Bibliographically approved
5. A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo
Open this publication in new window or tab >>A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo
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2002 (English)In: European Journal of Biochemistry, Vol. 269, no 20, p. 4921-4929Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-2457 (URN)
Available from: 2003-01-01 Created: 2003-01-01 Last updated: 2018-06-09Bibliographically approved

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