Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in
            <i>Escherichia coli</i>

Wai, Sun Nyunt; Westermark, Marie; Oscarsson, Jan; Jaß, Jana; Maier, Elke; Benz, Roland; Uhlin, Bernt Eric

doi:10.1128/jb.185.18.5491-5499.2003

Cited by 50 publications

(68 citation statements)

References 33 publications

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“…Furthermore, ClyA is not subject to any signal sequence cleavage and there is no evidence of any other posttranslational modification (3,8,23,30,45). The finding that ClyA accumulates in the periplasmic space when overproduced in E. coli has indicated that the ClyA secretion pathway includes toxin translocation to the periplasm (3,23,42,43,47). It has also been demonstrated that ClyA can subsequently be released from E. coli cells by blebbing of outer-membrane vesicles (42).…”

mentioning

confidence: 76%

Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli

et al. 2010

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Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N-and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix ␣A1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix ␣G, the "␤-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

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confidence: 76%

Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli

et al. 2010

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“…Cytoplasmic, periplasmic, and membrane fractions were obtained following procedures described previously (38).…”

Section: Methodsmentioning

confidence: 99%

Active Cytotoxic Necrotizing Factor 1 Associated with Outer Membrane Vesicles from Uropathogenic Escherichia coli

et al. 2006

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Cytotoxic necrotizing factor type 1 (CNF1) is one of the virulence factors produced by uropathogenic Escherichia coli (UPEC). How this toxin is translocated from the bacterial cytoplasm to the surrounding environment is not well understood. Our data suggest that CNF1 may be regarded as a secreted protein, since it could be detected in culture supernatants. Furthermore, we found that CNF1 was tightly associated to outer membrane vesicles, suggesting that such vesicles play a role in the secretion of this protein. Interestingly, vesicle samples containing CNF1 could exert the effects known for this protein on HeLa cell cultures, showing that CNF1 is transported by vesicles in its active form. Taken together, our results strongly suggest that outer membrane vesicles could be a means for the bacteria to deliver CNF1 to the environment and to the infected tissue. In addition, our results indicate that the histone-like nucleoid structuring protein H-NS has a role in the downregulation of CNF1 production and that it affects the outer membrane vesicle release in UPEC strain J96.Uropathogenic Escherichia coli (UPEC) strains are responsible for at least 80% of human urinary tract infections (UTIs), which include urethritis, cystitis, and pyelonephritis (21). UTI is among the most common bacterial infections in humans and has been shown to be an independent risk factor for both bladder cancer and renal cell carcinoma (30). To achieve pathogenicity, the UPEC strains produce many virulence factors such as adhesins (P and type 1 pili), hemolysin, aerobactin siderophore system, a capsule, and cytotoxic necrotizing factor type 1 (CNF1). CNF1 belongs to a group of bacterial necrotic substances which include the dermonecrotic toxin of Bordetella spp. (14), the virulence plasmid-encoded CNF2 of E. coli (14,29), and the recently described CNFy from Yersinia pseudotuberculosis (24). Unlike CNFy, whose mechanism of action is specific to RhoA (13), CNF1 affects three proteins of the Rho family of the small GTPases: RhoA, Rac1, and Cdc42. CNF1 deamidates Gln63 of RhoA (or the corresponding Gln61 of Rac1 and Cdc42), leading to the loss of both intrinsic and GAP-stimulated GTPase activities but without affecting the GTP-binding activity (14). This modification renders these GTPases constitutively active, which commonly results in formation of cellular actin stress fibers, filopodia and lamellipodia, with an overall effect of blocking the cytokinesis, leading to giant multinucleated cells (7,23,25). CNF1 is a 115-kDa A-B toxin, in which a catalytic A domain is located in the C-terminal region of about 300 amino acids and where the cell binding B-domain is in the N-terminal region (amino acids 53 to 190) (6,22). No typical signal peptide is found in the CNF1 sequence, and the secretion pathway of this toxin remains unclear. In this work we show that CNF1 is secreted and that it is associated with membrane vesicles. Furthermore, we present evidence that CNF1 is down regulated by H-NS (histone-like nucleoid structuring protein). MATERIALS AN...

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“…Other groups report MV production to be an alternate secretion pathway capable of directing bacterial products, including cytotoxins (10,11), enzymes (5), and DNA (10,12), to prokaryotic and eukaryotic cells. For example, Helicobacter pylori produce vacuolating cytotoxin A-containing MVs (13,14), and E. coli and Salmonella typhi produce cytolysin A-containing MVs (15,16), which are cytotoxic to mammalian cells. P. aeruginosa MVs are found in biofilm matrices (17), contain the quorum-sensing molecule 2-heptyl-3-hydroxy-4-quinolone (18), and fuse with unrelated Gram-negative and Gram-positive bacteria (19,20), suggesting MVs function as signals in biofilm formation and interspecies intraspecies bacterial population biology.…”

Section: Embrane Vesicles (Mvs)mentioning

confidence: 99%

Membrane Vesicles Are Immunogenic Facsimiles of Salmonella typhimurium That Potently Activate Dendritic Cells, Prime B and T Cell Responses, and Stimulate Protective Immunity In Vivo

Alaniz¹,

Deatherage

Lara

et al. 2007

The Journal of Immunology

270

152

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Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-α, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4+ T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4+ T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.

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Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli

Cited by 50 publications

References 33 publications

Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli

Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli

Active Cytotoxic Necrotizing Factor 1 Associated with Outer Membrane Vesicles from Uropathogenic Escherichia coli

Membrane Vesicles Are Immunogenic Facsimiles of Salmonella typhimurium That Potently Activate Dendritic Cells, Prime B and T Cell Responses, and Stimulate Protective Immunity In Vivo

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