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1

Electronic Resource

Genetic analysis of exoenzyme S expression by Pseudomonas aeruginosa (1996)

Goranson, Joanne ; Frank, Dara W

Oxford, UK : Blackwell Publishing Ltd

FEMS microbiology letters 135 (1996), S. 0

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ISSN: 1574-6968

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology

Notes: Abstract The dissemination of Pseudomonas aeruginosa to the bloodstream increases the likelihood of developing fatal sepsis. In experimental models, the ability to disseminate is linked to expression of the exoenzyme S pathway. Genetic and biochemical analysis of the pathway has led to the identification of the two structural genes encoding exoenzyme S, exoS and exoT. A key regulator of several loci of the pathway has been identified as a DNA-binding protein with transcriptional activation properties. Preliminary evidence suggests that exoenzyme S and the Yop virulence determinants of yersiniae share homology among proteins involved in their synthesis and secretion. With the addition of exoS and exoT to the molecular arsenal, questions concerning in vivo toxicity and target specificities of exoenzyme S can be directly addressed.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1574-6968.1996.tb07981.x

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2

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Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells (2000)

Pederson, Kristin J. ; Pal, Sangita ; Vallis, Amy J. ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 37 (2000), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N-terminus (residues 1–234) is a GTPase activating protein (GAP) for RhoGTPases, while the C-terminus (residues 232–453) encodes an ADP-ribosyltransferase. Utilizing a series of N-terminal deletion peptides of ExoS and an epitope-tagged full-length ExoS, two independent domains have been identified within the N-terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N-terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90–234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full-length form, which fractionated with membranes, and as an N-terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP-ribosyltransferase activities.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01990.x

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3

Electronic Resource

The exoenzyme S regulon of Pseudomonas aeruginosa (1997)

Frank, Dara W.

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 26 (1997), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Pseudomonas aeruginosa can cause severe life-threatening infections in which the bacterium disseminates rapidly from epithelial colonization sites to the bloodstream. In experimental models, the ability of P. aeruginosa to disseminate is linked to epithelial injury, in vitro cytotoxicity and expression of the exoenzyme S regulon. Using the expression of ExoS as a model, a series of genes that are important for regulation, secretion and, perhaps, intoxication of eukaryotic cells have been identified. Proteins encoded by the exoenzyme S regulon and the Yersinia Yop virulon show a high level of amino acid homology, suggesting that P. aeruginosa may use a contact-mediated translocation mechanism to transfer anti-host factors directly into eukaryotic cells. Potential anti-host factors that may disrupt eukaryotic signal transduction through ADP-ribosylation include ExoS and ExoT. Expression of ExoU, another candidate anti-host factor, has been correlated with acute cytotoxicity and lung epithelial injury. Members of the exoenzyme S regulon represent only a portion of the virulence factor arsenal possessed by P. aeruginosa. It will be important to understand how the exoenzyme S regulon contributes to pathogenesis and whether these factors could serve as potential therapeutic targets.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1997.6251991.x

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4

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ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury (1997)

Finck-Barbançon, Viviane ; Goranson, Joanne ; Zhu, Lei ; [et al.]

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 25 (1997), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non-covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non-cytotoxic derivative PA103 exsA::Ω and several cytotoxic and non-cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70-kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70-kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non-cytotoxic strains or PA103 exsA::Ω. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70-kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non-cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co-ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1997.4891851.x

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5

Electronic Resource

ExoU is a potent intracellular phospholipase (2004)

Sato, Hiromi ; Frank, Dara W.

Oxford, UK : Blackwell Science Ltd

Molecular microbiology 53 (2004), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: The combination of a large genome encoding metabolic versatility and conserved secreted virulence determinants makes Pseudomonas aeruginosa a model pathogen that can be used to study host–parasite interactions in many eukaryotic hosts. One of the virulence regulons that likely plays a role in the ability of P. aeruginosa to avoid innate immune clearance in mammals is a type III secretion system (TTSS). Upon cellular contact, the P. aeruginosa TTSS is capable of delivering a combination of at least four different effector proteins, exoenzyme S (ExoS), ExoT, ExoU, and ExoY. Two of the four translocated proteins, ExoS and ExoU, are cytotoxic to cells during infection and transfection. The mechanism of cytotoxicity of ExoS is unclear. ExoU, however, has recently been characterized as a member of the phospholipase A family of enzymes, possessing at least phospholipase A2 activity. Similar to ExoS, ExoT and ExoY, ExoU requires either a eukaryotic-specific modification or cofactor for its activity in vitro. The biologic effects of minimal expression of ExoU in yeast can be visualized by membrane damage to different organelles and fragmentation of the vacuole. In mammalian cells, the direct injection of ExoU causes irreversible damage to cellular membranes and rapid necrotic death. ExoU likely represents a unique enzyme and is the first identified phopholipase virulence factor that is translocated into the cytosol by TTSS.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04194.x

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6

Electronic Resource

Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway (1996)

Yahr, Timothy L. ; Goranson, Joanne ; Frank, Dara W.

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 22 (1996), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Exoenzyme S is an extracellular ADP-ribosyltransferase of Pseudomonas aeruginosa. Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for exoenzyme S production. Five Tn5 Tc insertion mutants were isolated which exhibited an exoenzyme S-deficient phenotype (388::Tn5 Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn5 Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb EcoRI fragment that is not contiguous with the exoenzyme S trans-regulatory operon. 388::Tn5 Tc 469 and 550 mapped to a region downstream of the trans-regulatory operon which has been previously shown to contain a promoter region that is co-ordinately regulated with exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn5 Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB–L proteins of the yersiniaeYop type III export apparatus. RNase-protection analysis of wild-type and mutant strains indicated that exsD and pscB–L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino- or carboxy-terminal residues were expressed in P. aeruginosa. Deletion analyses indicated that the amino-terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of exoenzyme S and potentially other co-ordinately regulated proteins.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1996.01554.x

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7

Electronic Resource

The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins (1999)

Pederson, Kristin J. ; Vallis, Amy J. ; Aktories, Klaus ; [et al.]

Oxford BSL : Blackwell Science Ltd

Molecular microbiology 32 (1999), S. 0

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ISSN: 1365-2958

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology , Medicine

Notes: Pseudomonas aeruginosa delivers exoenzyme S (ExoS) into the intracellular compartment of eukaryotic cells via a type III secretion pathway. Intracellular delivery of ExoS is cytotoxic for eukaryotic cells and has been shown to ADP-ribosylate Ras in vivo and uncouple a Ras-mediated signal transduction pathway. Functional mapping has localized the FAS-dependent ADP-ribosyltransferase domain to the carboxyl-terminus of ExoS. A transient transfection system was used to examine cellular responses to the amino-terminal 234 amino acids of ExoS (ΔC234). Intracellular expression of ΔC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose-dependent manner. Expression of ΔC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of ΔC234 was not cytotoxic to CHO cells. Carboxyl-terminal deletion proteins of ΔC234 were less efficient in the elicitation of CHO cell rounding than ΔC234. Cytoskeleton rearrangement elicited by ΔC234 was blocked and reversed by the addition of cytotoxic necrotizing factor 1 (CNF-1). CNF-1 catalyses the deamidation of Gln-63 of members of the Rho subfamily of small-molecular-weight GTP-binding proteins, resulting in protein activation. This implies a role for small-molecular-weight GTP-binding proteins in the disruption of actin by ΔC234. Together, these data identify ExoS as a cytotoxin that possesses two functional domains. Intracellular expression of the amino-terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl-terminal domain of ExoS possesses FAS-dependent ADP-ribosyltransferase activity and is cytotoxic to eukaryotic cells.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01359.x

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8

Electronic Resource

Active and passive immunization with the Pseudomonas V antigen protects against type III intoxication and lung injury (1999)

Sawa, Teiji ; Yahr, Timothy L. ; Ohara, Maria ; [et al.]

[s.l.] : Nature America Inc.

Nature medicine 5 (1999), S. 392-398

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ISSN: 1546-170X

Source: Nature Archives 1869 - 2009

Topics: Biology , Medicine

Notes: [Auszug] Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Damage to the lung epithelium is associated with the expression of toxins that are directly injected into eukaryotic cells through a type III-mediated ...

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1038/7391

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Identification of a ubiquitin-binding interface using Rosetta and DEER (2018)

Tessmer, Maxx H. ; Anderson, David M. ; Pickrum, Adam M. ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2018; 115(3): 525-530. Published 2018 Jan 02. doi: 10.1073/pnas.1716861115.

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Publication Date: 2018-01-02

Description: ExoU is a type III-secreted cytotoxin expressing A2 phospholipase activity when injected into eukaryotic target cells by the bacterium Pseudomonas aeruginosa. The enzymatic activity of ExoU is undetectable in vitro unless ubiquitin, a required cofactor, is added to the reaction. The role of ubiquitin in facilitating ExoU enzymatic activity is poorly understood but of significance for designing inhibitors to prevent tissue injury during infections with strains of P. aeruginosa producing this toxin. Most ubiquitin-binding proteins, including ExoU, demonstrate a low (micromolar) affinity for monoubiquitin (monoUb). Additionally, ExoU is a large and dynamic protein, limiting the applicability of traditional structural techniques such as NMR and X-ray crystallography to define this protein–protein interaction. Recent advancements in computational methods, however, have allowed high-resolution protein modeling using sparse data. In this study, we combine double electron–electron resonance (DEER) spectroscopy and Rosetta modeling to identify potential binding interfaces of ExoU and monoUb. The lowest-energy scoring model was tested using biochemical, biophysical, and biological techniques. To verify the binding interface, Rosetta was used to design a panel of mutations to modulate binding, including one variant with enhanced binding affinity. Our analyses show the utility of computational modeling when combined with sensitive biological assays and biophysical approaches that are exquisitely suited for large dynamic proteins.

Print ISSN: 0027-8424

Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General