The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization of<i>Vibrio vulnificus</i>Hemolysin

Kashimoto, Takeshi; Ueno, Shunji; Koga, Takeshi; Fukudome, Shinji; Ehara, Hayato; Komai, Mayumi; Sugiyama, Hiroyuki; Susa, Nobuyuki

doi:10.1128/jb.01049-09

Cited by 17 publications

(24 citation statements)

References 39 publications

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“…The third transition involves a 35°rigid-body rotation of the β-trefoil lectin domain around the loop connecting it to the cytolytic domain. The short helical turn that precedes the β-trefoil linker is anchored within the cytolysin domain by a phenylalanine residue (F455) that is necessary for oligomerization in the related Vibrio vulnificus hemolysin (33). The fourth transition is a movement of the loop that cradles the prestem in the water-soluble structure (residues 191-203) through hydrophobic side-chain interactions (notably L192, Y194, L307, and A309) and backbone hydrogen bonds involving G291 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins

Olson²

2011

Proc. Natl. Acad. Sci. U.S.A.

118

163

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Pore-forming toxins (PFTs) are potent cytolytic agents secreted by pathogenic bacteria that protect microbes against the cellmediated immune system (by targeting phagocytic cells), disrupt epithelial barriers, and liberate materials necessary to sustain growth and colonization. Produced by gram-positive and gramnegative bacteria alike, PFTs are released as water-soluble monomeric or dimeric species, bind specifically to target membranes, and assemble transmembrane channels leading to cell damage and/or lysis. Structural and biophysical analyses of individual steps in the assembly pathway are essential to fully understanding the dynamic process of channel formation. To work toward this goal, we solved by X-ray diffraction the 2.9-Å structure of the 450-kDa heptameric Vibrio cholerae cytolysin (VCC) toxin purified and crystallized in the presence of detergent. This structure, together with our previously determined 2.3-Å structure of the VCC water-soluble monomer, reveals in detail the architectural changes that occur within the channel region and accessory lectin domains during pore formation including substantial rearrangements of hydrogen-bonding networks in the pore-forming amphipathic loops. Interestingly, a ring of tryptophan residues forms the narrowest constriction in the transmembrane channel reminiscent of the phenylalanine clamp identified in anthrax protective antigen [Krantz BA, et al. (2005) Science 309:777-781]. Our work provides an example of a β-barrel PFT (β-PFT) for which soluble and assembled structures are available at high-resolution, providing a template for investigating intermediate steps in assembly.hemolysin | membrane protein | X-ray crystallography | virulence factor

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Section: Resultsmentioning

confidence: 99%

Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins

Olson²

2011

Proc. Natl. Acad. Sci. U.S.A.

118

163

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“…It is likely that the VVH-lectin domain can recognize a carbohydrate chain. In fact, Kashimoto et al have suggested that the VVH-lectin domain may be required to bind to cellular membranes [6]. In this study, MβCD decreased the binding efficiency of VVH to CHO cells (Fig.…”

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confidence: 46%

“…Vibrio vulnificus hemolysin (VVH) is a possible virulence factor which is produced by V. vulnificus [3,12]. This toxin has been reported to cause cytolysis of various eukaryotic cells, as well as erythrocytes [6,12,20]. With regard to the hemolytic or cytotoxic mechanism (s) of VVH, it has been reported that VVH monomer binds to cellular membranes to form oligomers, and that these oligomers form ion-permeable pores which exert hemolysis or cytotoxicity via colloid osmotic shock [8,19].…”

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confidence: 99%

Inhibition of Binding of <i>Vibrio vulnificu</i>s Hemolysin (VVH) by MβCD

et al. 2013

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ABSTRACT. Vibrio vulnificus secrets a pore-forming toxin called Vibrio vulnificus hemolysin (VVH). In this study, we showed that methylbeta-cyclodextrin (MβCD), an oligosaccharide, decreased binding of VVH to Chinese hamster ovary (CHO) cells, resulting in inhibition of its cytotoxicity. When the VVH was incubated with MβCD, cytotoxicity of the toxin was inhibited from 100.3 ± 7.2% to 19.6 ± 5.3%. Binding analysis showed that the amount of VVH on the cells was decreased from 101.4 ± 9.2% to 18.1 ± 8.0% only when MβCD was present in the culture media. Our results indicate that the inhibition of cytotoxicity of VVH by MβCD was due to a decrease in the amount of toxin binding to CHO cells. Vibrio vulnificus, which is a Gram-negative bacterium, typically infects humans suffering from liver cirrhosis, alcoholism, hemochromatosis, immunocompromised conditions or diabetes, resulting in septicemia [5,9,10,17]. Primary septicemia in V. vulnificus infection is caused by the ingestion of contaminated seafood or through wound infection resulting from exposure to contaminated seawater or marine products [10,17]. Vibrio vulnificus hemolysin (VVH) is a possible virulence factor which is produced by V. vulnificus [3,12]. This toxin has been reported to cause cytolysis of various eukaryotic cells, as well as erythrocytes [6,12,20]. With regard to the hemolytic or cytotoxic mechanism (s) of VVH, it has been reported that VVH monomer binds to cellular membranes to form oligomers, and that these oligomers form ion-permeable pores which exert hemolysis or cytotoxicity via colloid osmotic shock [8,19].Methyl-beta-cyclodextrin (MβCD) is an oligosaccharide with a ring-shaped structure, which sequesters cholesterol from cellular membrane, and inhibits formation of lipid rafts by decreasing the contents of membrane cholesterol. Therefore, MβCD is used to analyze the function of lipid rafts which are cholesterol-rich membrane domains. In the process of investigation into the localization of VVH on the cellular membrane using MβCD, when the cells were not washed after MβCD treatment, we found that the cytotoxicity of VVH was inhibited. On the other hand, when the cells were washed to remove the MβCD in the culture media, the cytotoxicity of VVH was not affected [18]. Here, the present study investigated the mechanism by which MβCD inhibits the cytotoxicity of VVH.First, we reconfirmed that the cytotoxicity of VVH was inhibited in CHO and Caco-2 cells by MβCD, when the MβCD was not washed out. These cell lines were cultured as previously described [18], and the purified VVH, prepared by the method of Oh et al. [14], was used in this study. Cytotoxicity was evaluated by measuring a lactate dehydrogenase (LDH) release as previously described with slightly modification [18]. At 1 hr after the culture media were replaced with DMEM containing 8 mM MβCD, the cells were washed or not washed with DMEM. In a pilot study, we confirmed that there was no difference in cytotoxicity of VVH between washing the cells with DMEM containing 8 mM MβCD and not ...

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“…This species commonly produces a hemolytic/cytolytic toxin termed V. vulnificus hemolysin (VVH) (Shinoda et al, 1985;Miyoshi et al, 1993), which has been reported to 10 cause cytolysis of various eukaryotic cells, as well as erythrocytes (Yamanaka et al, 1990;Miyoshi et al, 1993;Kashimoto at al., 2010). For hemolysis, the toxin binds specifically to cholesterol and is subsequently assembled on the membrane to form an oligomer (Yamanaka et al, 1987;Kim and Kim, 2002).…”

Section: Introductionmentioning

confidence: 99%

Inactivation of Vibrio vulnificus hemolysin through mutation of the N- or C-terminus of the lectin-like domain

Miyoshi

Abe

Senoh

et al. 2011

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Vibrio vulnificus is an etiological agent causing serious systemic infections in the immunocompromised humans or cultured eels. This species commonly produces a hemolytic toxin consisting of the cytolysin domain and the lectin-like domain. For hemolysis, the 5 lectin-like domain specifically binds to cholesterol in the erythrocyte membrane, and to form a hollow oligomer, the toxin is subsequently assembled on the membrane. The cytolysin domain is essential for the process to form the oligomer. Three-dimensional structure model revealed that two domains connected linearly and the C-terminus was located near to the joint of the domains. Insertion of amino acid residues between two domains was found to cause 10 inactivation of the toxin. In the C-terminus, deletion, substitution or addition of an amino acid residue also elicited reduction of the activity. However, the cholesterol-binding ability was not affected by the mutations. These results suggest that mutation of the C-or N-terminus of the lectin-like domain may result in blockage of the toxin assembly.

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The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization ofVibrio vulnificusHemolysin

Cited by 17 publications

References 39 publications

Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins

Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins

Inhibition of Binding of <i>Vibrio vulnificu</i>s Hemolysin (VVH) by MβCD

Inactivation of Vibrio vulnificus hemolysin through mutation of the N- or C-terminus of the lectin-like domain

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