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Exoenzyme T of Pseudomonas aeruginosa elicits cytotoxicity without interfering with Ras signal transduction
Department of Microbiology, FOI NBC-Defence, Umeå.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
Department of Microbiology, FOI NBC-Defence, Umeå.
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2001 (Engelska)Ingår i: Cellular Microbiology, Vol. 3, nr 4, s. 237-46Artikel i tidskrift (Refereegranskat) Published
Fritextbeskrivning
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.

Ort, förlag, år, upplaga, sidor
John Wiley & Sons, 2001. Vol. 3, nr 4, s. 237-46
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-3309DOI: 10.1046/j.1462-5822.2001.00108.xOAI: oai:DiVA.org:umu-3309DiVA, id: diva2:141905
Tillgänglig från: 2003-12-23 Skapad: 2003-12-23 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
Ingår i avhandling
1. Type III Secretion Mediated Translocation of Effector Exoenzymes by Pseudomonas aeruginosa
Öppna denna publikation i ny flik eller fönster >>Type III Secretion Mediated Translocation of Effector Exoenzymes by Pseudomonas aeruginosa
2003 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Alternativ titel[sv]
Injektion av gifter via typ III sekretionssystemet hos bakterien Pseudomonas aeruginosa
Förlag
s. 52
Nyckelord
Cell and molecular biology, Pseudomonas aeruginosa, type III secretion system, ExoS, ExoT, PcrV, PcrG, PopB, PopD, PopN, type IV pili, Cell- och molekylärbiologi
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
molekylärbiologi
Identifikatorer
urn:nbn:se:umu:diva-174 (URN)91-7305-545-X (ISBN)
Disputation
2003-11-28, Major Groove, 6L, Norrlands Universitetssjukhus, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2003-12-23 Skapad: 2003-12-23 Senast uppdaterad: 2019-01-24Bibliografiskt granskad
2. Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S
Öppna denna publikation i ny flik eller fönster >>Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S
2003 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Umeå universitet, 2003. s. 51
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 851
Nyckelord
Cell biology, Pseudomonas aeruginosa, ADP-ribosylation, GAP, Ras superfamily, NAD, ExoS, 14-3-3, Cellbiologi
Nationell ämneskategori
Cell- och molekylärbiologi
Forskningsämne
molekylär cellbiologi
Identifikatorer
urn:nbn:se:umu:diva-121 (URN)91-7305-505-0 (ISBN)
Disputation
2003-10-31, Betula, 6M, Umeå, 09:00
Opponent
Handledare
Tillgänglig från: 2003-01-01 Skapad: 2003-01-01 Senast uppdaterad: 2019-01-21Bibliografiskt granskad

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Sundin, CharlottaHallberg, BengtForsberg, Åke

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Cell- och molekylärbiologi

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