scholarly journals Examination of the Coordinate Effects of Pseudomonas aeruginosa ExoS on Rac1

2005 ◽  
Vol 73 (9) ◽  
pp. 5458-5467 ◽  
Author(s):  
Claudia L. Rocha ◽  
Elizabeth A. Rucks ◽  
Deanne M. Vincent ◽  
Joan C. Olson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Fraction ◽  
Low Molecular Weight ◽  
Binding Region ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Adp Ribosyltransferase ◽  
Ht 29

ABSTRACT Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by the Pseudomonas aeruginosa type III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.

scholarly journals ADP-Ribosylation of Rab5 by ExoS ofPseudomonas aeruginosa Affects Endocytosis

2001 ◽  
Vol 69 (9) ◽  
pp. 5329-5334 ◽  
Author(s):  
Alejandro M. Barbieri ◽  
Qun Sha ◽  
Pascale Bette-Bobillo ◽  
Philip D. Stahl ◽  
Michel Vidal
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Horseradish Peroxidase ◽  
Marked Decrease ◽  
Low Molecular Weight ◽  
Strong Inhibition ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Adp Ribosyltransferase ◽  
Stimulation Of

ABSTRACT Pseudomonas aeruginosa exoenzyme S (ExoS) is an ADP-ribosyltransferase that modifies low-molecular-weight GTPases. Here we studied the effect of Rab5 ADP-ribosylation by ExoS on its cellular function, i.e., regulation of early endocytic events. Coculture of CHO cells with P. aeruginosa induced a marked decrease in horseradish peroxidase (HRP) uptake compared to noninfected cells, while coculture with a P. aeruginosa mutant strain that fails to produce ExoS did not lead to any change in HRP uptake. Microinjection of recombinant ExoS into Xenopus oocytes induced strong inhibition of basal HRP uptake by oocytes. Moreover, coinjection of recombinant ExoS with Rab5 abolished the typical stimulation of HRP uptake obtained after GTPase microinjection. Cytosols prepared from injected oocytes were used in an endosome-endosome fusion assay. Cytosol from ExoS-microinjected oocytes was ineffective in promoting endosome-endosome fusion. However, in these conditions, the addition of Rab5 to the assay led to fusion recovery. Finally, we found that the interaction of Rab5 with EEA1 was markedly diminished after Rab5 ADP-ribosylation by ExoS.

scholarly journals Independent and Coordinate Effects of ADP-Ribosyltransferase and GTPase-Activating Activities of Exoenzyme S on HT-29 Epithelial Cell Function

2001 ◽  
Vol 69 (9) ◽  
pp. 5318-5328 ◽  
Author(s):  
Jennifer E. Fraylick ◽  
Jeannine R. La Rocque ◽  
Timothy S. Vincent ◽  
Joan C. Olson
Keyword(s):  
Dna Synthesis ◽  
Cell Function ◽  
Double Mutation ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Cell Rounding ◽  
Inhibition Of Dna Synthesis ◽  
Adp Ribosyltransferase ◽  
Ht 29

ABSTRACT Type III-mediated translocation of exoenzyme S (ExoS) into HT-29 epithelial cells by Pseudomonas aeruginosa causes complex alterations in cell function, including inhibition of DNA synthesis, altered cytoskeletal structure, loss of readherence, microvillus effacement, and interruption of signal transduction. ExoS is a bifunctional protein having both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) functional domains. Comparisons of alterations in HT-29 cell function caused by P. aeruginosastrains that translocate ExoS having GAP or ADPRT mutations allowed the independent and coordinate functions of the two activities to be assessed. An E381A ADPRT mutation revealed that ExoS ADPRT activity was required for effects of ExoS on DNA synthesis and long-term cell rounding. Conversely, the R146A GAP mutation appeared to have little impact on the cellular effects of ExoS. While transient cell rounding was detected following exposure to the E381A mutant, this rounding was eliminated by an E379A-E381A ADPRT double mutation, implying that residual ADPRT activity, rather than GAP activity, was effecting transient cell rounding by the E381A mutant. To explore this possibility, E381A and R146A-E381A mutants were examined for their ability to ADP-ribosylate Ras in vitro or in vivo. While no ADP-ribosylation of Ras was detected by either mutant in vitro, both mutants were able to modify Ras when translocated by the bacteria, with the R146A-E381A mutant causing more efficient modification than the E381A mutant, in association with increased inhibition of DNA synthesis. Comparisons of Ras ADP-ribosylation by wild-type and E381A mutant ExoS by two-dimensional electrophoresis found the former to ADP-ribosylate Ras at two sites, while the latter modified Ras only once. These studies draw attention to the key role of ExoS ADPRT activity in causing the effects of bacterially translocated ExoS on DNA synthesis and cell rounding. In addition, the studies provide insight into the enhancement of ExoS ADPRT activity within the eukaryotic cell microenvironment and into possible modulatory roles that the GAP and ADPRT domains might have on the function of each other.

Interaction Between Mutations in the suppressor of Hairy wing and modifier of mdg4 Genes of Drosophila melunogaster Affecting the Phenotype of gypsy-Induced Mutations

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 425-436 ◽  
Author(s):  
Pavel Georgiev ◽  
Marina Kozycina
Keyword(s):  
Mutagenic Effect ◽  
Induced Mutations ◽  
Binding Region ◽  
Direct Inhibition ◽  
Amino Terminal ◽  
Acidic Domain ◽  
Complete Inactivation ◽  
Insulation Effect ◽  
Gypsy Retrotransposon ◽  
Carboxy Terminal

Abstract The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers located distally from the promoter with respect to the position of the su(Hw)-binding region. Mutations in a second gene, modifier of mdg4, also affect the gypsy-induced phenotype. Two major effects of the mod(mdg4)lul mutation can be distinguished: the interference with insulation by the su(Hw)-binding region and direct inhibition of gene expression that is not dependent on the su(Hw)-binding region position. The mod(mdg4)lul mutation partially suppresses ct6, scD1 and Hw1 mutations, possibly by interfering with the insulation effect of the su(Hw)-binding region. An example of the second effect of mod(mdg4)lul is a complete inactivation of yellow expression in combination with the y  2 allele. Phenotypic analyses of flies with combinations of mod(mdg4)lul and different su(Hw) mutations, or with constructions carrying deletions of the acidic domains of the su(Hw) protein, suggest that the carboxy-terminal acidic domain is important for direct inhibition of yellow transcription in bristles, while the amino-terminal acidic domain is more essential for insulation.

scholarly journals Structure–activity relationships for inhibitors of Pseudomonas aeruginosa exoenzyme S ADP-ribosyltransferase activity

2018 ◽  
Vol 143 ◽  
pp. 568-576 ◽  
Author(s):  
Michael Saleeb ◽  
Charlotta Sundin ◽  
Öznur Aglar ◽  
Ana Filipa Pinto ◽  
Mahsa Ebrahimi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Exoenzyme S ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Adp Ribosyltransferase

scholarly journals ADP-ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo

1999 ◽  
Vol 32 (5) ◽  
pp. 1054-1064 ◽  
Author(s):  
Timothy S. Vincent ◽  
Jennifer E. Fraylick ◽  
Eileen M. McGuffie ◽  
Joan C. Olson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ras Proteins ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Oncogenic Ras

scholarly journals Comparison of the exoS Gene and Protein Expression in Soil and Clinical Isolates of Pseudomonas aeruginosa

2001 ◽  
Vol 69 (4) ◽  
pp. 2198-2210 ◽  
Author(s):  
Michael W. Ferguson ◽  
Jill A. Maxwell ◽  
Timothy S. Vincent ◽  
Jack da Silva ◽  
Joan C. Olson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Structural Gene ◽  
Gene Sequence ◽  
Specific Activity ◽  
Clinical Isolates ◽  
Secretory Process ◽  
Sequence Comparisons ◽  
Exoenzyme S ◽  
Gene And Protein Expression ◽  
Adp Ribosyltransferase

ABSTRACT Exoenzyme S (ExoS) is translocated into eukaryotic cells by the type III secretory process and has been hypothesized to function in conjunction with other virulence factors in the pathogenesis ofPseudomonas aeruginosa. To gain further understanding of how ExoS might contribute to P. aeruginosa survival and virulence, ExoS expression and the structural gene sequence were determined in P. aeruginosa soil isolates and compared with ExoS of clinical isolates. Significantly higher levels of ExoS ADP-ribosyltransferase (ADPRT) activity were detected in culture supernatants of soil isolates compared to those of clinical isolates. The higher levels of ADPRT activity of soil isolates reflected both the increased production of ExoS and the production of ExoS having a higher specific activity. ExoS structural gene sequence comparisons found the gene to be highly conserved among soil and clinical isolates, with the greatest number of nonsynonymous substitutions occurring within the region of ExoS encoding GAP function. The lack of amino acid changes in the ADPRT region in association with a higher specific activity implies that other factors produced by P. aeruginosa or residues outside the ADPRT region are affecting ExoS ADPRT activity. The data are consistent with ExoS being integral to P. aeruginosa survival in the soil and suggest that, in the transition of P. aeruginosa from the soil to certain clinical settings, the loss of ExoS expression is favored.

scholarly journals Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo

2002 ◽  
Vol 367 (3) ◽  
pp. 617-628 ◽  
Author(s):  
Maria L. HENRIKSSON ◽  
Charlotta SUNDIN ◽  
Anna L. JANSSON ◽  
Åke FORSBERG ◽  
Ruth H. PALMER ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Small Gtpase ◽  
Eukaryotic Cells ◽  
Gtpase Activating Protein ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Intracellular Targeting ◽  
Gtp Binding ◽  
Adp Ribosyltransferase

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.

Subcellular localization of iodinated thyroid tubulin

1989 ◽  
Vol 9 (3) ◽  
pp. 375-382
Author(s):  
Alan J. Hargreaves ◽  
Luis Lamas ◽  
Pilar Santisteban ◽  
Jesus Avila
Keyword(s):  
Monoclonal Antibodies ◽  
Golgi Apparatus ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Amino Terminal ◽  
Thyroid Glands ◽  
Carboxy Terminal ◽  
Hydrolysis Of ◽  
Brain Tubulin ◽  
Membrane Tubulin

Subcellular fractions enriched in mitochondria, plasma membranes, microsomes and Golgi apparatus were obtained from thyroid glands of rats injected with I125. Autoradiography of SDS-polyacrylamide gels revealed the presence of a number of radiolabelled proteins in each membrane fraction. One polypeptide, with the same electrophoretic mobility as brain tubulin, was found in all fractions except the plasma membranes and was immunoprecipitated with commercial anti-tubulin monoclonal antibodies. Hydrolysis of Asp-Pro linkages of I125 labelled tubulin with formic acid indicated that there were iodination sites in both the carboxy terminal one third and the amino terminal two thirds of the molecule. These results, together with the absence of iodinated tubulin from the cytosolic fraction, are consistent with the idea that a population of thyroid membrane tubulin is iodinated at multiple sites either just before or after insertion into intracellular membranes where it may act as an anchorage point for microtubule-membrane interactions.

scholarly journals Membrane Localization Contributes to the In Vivo ADP-Ribosylation of Ras by Pseudomonas aeruginosa ExoS

2002 ◽  
Vol 70 (4) ◽  
pp. 2230-2232 ◽  
Author(s):  
Matthew J. Riese ◽  
Joseph T. Barbieri
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Localization ◽  
Protein Distribution ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Type Iii

ABSTRACT Type III-delivered exoenzyme S (ExoS) preferentially ADP-ribosylated membrane-associated His6HRas, relative to its cytosolic derivative His6HRasΔCAAX. This indicates that the subcellular protein distribution contributes to in vivo ADP-ribosylation by ExoS.

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