Aerolysin from Aeromonas hydrophila and Related Toxins

Fivaz, Marc; Abrami, Laurence; Tsitrin, Yulia; Goot, F. Gisou van der

doi:10.1007/978-3-642-56508-3_3

Cited by 29 publications

(28 citation statements)

References 68 publications

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“…2a). Blast analysis showed the hemolytic clone to carry a new member of the aerolysin gene family, a class of pore-forming exotoxins secreted by aeromonads (11). Compared to known aerolysins, the protein (accession number EU258894) demonstrated an average Clustal nucleotide alignment score of 71.1 (range, 68 to 78) and an average amino acid score of 73.2 (range, 68 to 79) (16).…”

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confidence: 99%

Rapid DNA Library Construction for Functional Genomic and Metagenomic Screening

Schmitz

Daniel

Collin

et al. 2008

Appl Environ Microbiol

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A rapid protocol was developed for constructing plasmid libraries from small quantities of genomic/ metagenomic DNA. The technique utilizes linker amplification with topoisomerase cloning and allows for inducible transcription in Escherichia coli. As proof of principle, several anti-Bacillus lysins were cloned from bacteriophage genomes and an aerolysin was cloned from a metagenomic sample.The field of metagenomics offers unique perspectives on unculturable microorganisms and their biosynthetic products (4,13,17,19). Overall, metagenomic research can be subdivided into two categories. In bioinformatic studies, environmental DNA is sequenced to gain insight into a sample's diversity and phylogeny (18,20). By contrast, in functional metagenomics, environmental genes are expressed within a host and clones are screened for phenotype acquisition (9,12). A potential limiting factor in either case is the initial amount of DNA, for instance, if the purity/quantity of the sample is low or if the analysis involves a subset of an environmental population. An example of the latter is shown in viral metagenomics, in which phage particles are isolated prior to DNA extraction (5, 6). Random amplification has proven important in these situations, with a prominent method being the linker-amplified shotgun library (LASL) approach (2, 3). Here, DNA (ϳ1 g or less) is fragmented, short linkers are attached, and PCR is conducted with primers targeting the linkers.We report here a modified LASL approach combining linker amplification with topoisomerase cloning (15). The technique is particularly well suited for functional screening, as it rapidly generates expression libraries with gene-sized inserts. These libraries are referred to as expressible LASLs (E-LASLs), and their utility was demonstrated with genomic DNA from four Bacillus bacteriophages isolated from bat guano and metagenomic DNA from the gut contents of an earthworm (Eisenia hortensis). For all samples, 100 ng of DNA was fragmented with Tsp509I (consensus sequence AATT; 0.01 or 0.1 U enzyme; 50-l reaction mixture volume in NEB buffer 2; 1 min of digestion at 65°C). Following phenol-chloroform extraction and ethanol precipitation, the DNA was ligated to 40 ng of linker sequence, with a complementary 5Ј overhang (AATTC GGCTCGAG, where the overhang is underlined). The ligation mixture was used as the template for Taq-based PCR (1 l template per 50-l reaction mixture volume; linker-targeted primer [CCATGACTCGAGCCGAATT]; PCR conditions of 95°C for 1 min; 95°C for 30 s, 55°C for 30 s, and 72°C for 5 min [40 times], and 72°C for 10 min).The resultant E-LASL length distributions ranged from 500 bp to Ͼ4 kb (Fig. 1). For each E-LASL, 1 volume of raw PCR product was added to 3 volumes of distilled water, 1 volume of 1ϫ salt solution, and 1 volume of topoisomerase-conjugated pBAD plasmid (pBAD TOPO TA expression kit; Invitrogen). Following a 1-min room temperature incubation, the mixtures were transformed into competent Escherichia coli TOP10 (Invitrogen) and plated onto LB-ampicill...

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confidence: 99%

Rapid DNA Library Construction for Functional Genomic and Metagenomic Screening

Schmitz

Daniel

Collin

et al. 2008

Appl Environ Microbiol

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“…To determine whether EMP2 localized to vesicles containing GPI-APs, double immunostaining was performed in wild-type or EMP2-overexpressing cells (Figure 6). We used the bacterial toxin aerolysin, which selectively recognizes the core glycan-peptide of the GPI anchor, to measure all GPI-APs (Parker et al, 1996;Diep et al, 1998;Nelson et al, 1999;Fivaz et al, 2001). In wild-type NIH3T3 cells, EMP2 and GPI-APs were mainly confined to cytoplasmic compartments, and partial colocalization was observed in a perinuclear region typical of endoplasmic reticulum (ER)/Golgi staining (Figure 6, C and D).…”

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confidence: 99%

The Tetraspan Protein EMP2 Modulates the Surface Expression of Caveolins and Glycosylphosphatidyl Inositol-linked Proteins

Wadehra

Goodglick

Braun

2004

MBoC

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Caveolae are a subset of lipid rafts enriched in glycosphingolipids and cholesterol-rich domains, but selectively lacking glycosylphosphatidyl inositol-anchored proteins (GPI-APs). Caveolin proteins are the organizing component of caveolae, but the corresponding proteins for other classes of lipid rafts are poorly defined. Epithelial membrane protein-2 (EMP2), a member of the four-transmembrane superfamily, facilitates plasma membrane delivery of certain integrins. In this study, we found by laser confocal microscopy that EMP2 was associated with GPI-APs (detected by the GPI-AP binding bacterial toxin proaerolysin). Biochemical membrane fractionation and methyl-␤-cyclodextrin treatment demonstrated that this association occurred within lipid rafts. EMP2 did not associate with caveolin-bearing membrane structures, and recombinant overexpression of EMP2 in NIH3T3 cells decreased caveolin-1 and caveolin-2 protein levels while increasing the surface expression of GPI-APs. Conversely, a ribozyme construct that specifically cleaves the EMP2 transcript reduced surface GPI-APs and increased caveolin protein expression. These findings suggest that EMP2 facilitates the formation and surface trafficking of lipid rafts bearing GPI-APs, and reduces caveolin expression, resulting in impaired formation of caveolae. INTRODUCTIONAn emerging structural concept for plasma membranes in mammalian cells is the liquid-ordered microdomain. These domains are distinct from the "fluid mosaic" model in that the "liquid-ordered" membranes are more ordered and less fluid than the bulk plasma membrane (Brown and London, 1998b;Brown and London, 2000;Galbiati et al., 2001a). Each domain can be defined by its protein and lipid compositions, and it is becoming increasingly clear that these distinct compartments mediate a number of cellular responses from adhesion and signaling to cellular activation (Brown and London, 1998a;Galbiati et al., 2001a).Lipid rafts, defined as the detergent-resistant fraction of the plasma membrane, are 50-to 350-nm domains rich in cholesterol, glycosphingolipids, sphingolipids, saturated phospholipids, and glycosylphosphatidyl inositol-anchored proteins (GPI-APs) (Gruenberg and Maxfield, 1995;Mukherjee et al., 1997;Melkonian et al., 1999;Abrami et al., 2001;Stuermer et al., 2001). These dynamic structures also include protein components, reflecting a large (Ͼ100) but distinctive set of protein species (Claas et al., 2001). Ubiquitously expressed, lipid rafts have been associated with the sorting of proteins bound for the cell surface and the organizing of signaling molecules for efficient transduction (Robinson, 1997;Schmidt et al., 2001).One important subset of lipid rafts are caveolae (Schnitzer et al., 1995). Caveolae are 50-to 100-nm membrane invaginations comprised of cholesterol, glycosphingolipids, and at least one member of the caveolin family of proteins. To date, three members of the caveolin family have been identified. Caveolin-1 is the principal component of caveolae in the majority of cell types and is r...

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“…Previous work has shown that VCC is secreted as a protoxin that can bind to target cells. In this regard, it is similar to several other pore-forming toxins including aerolysin and Clostridium septicum -toxin [34][35][36][37]. Activation of cell-bound pro-VCC can occur through cleavage at multiple sites and through the action of both endogenous and exogenous proteases.…”

Section: Discussionmentioning

confidence: 99%

Pro-inflammatory feedback activation cycle evoked by attack of Vibrio cholerae cytolysin on human neutrophil granulocytes

Valeva

Walev

Weis

et al. 2007

Med Microbiol Immunol

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Vibrio cholerae cytolysin (VCC) is a pore-forming toxin that is secreted in precursor form (pro-VCC) and requires proteolytic cleavage in order to attain membrane-permeabilizing properties. Pro-VCC can be activated both in solution and membrane-bound state. Processing of membrane-bound pro-VCC can in turn be achieved through the action of both cell-associated and soluble proteases. The current investigation describes the interaction of VCC with human neutrophil granulocytes. It is shown that pro-VCC binds to these cells and is cleaved by cell-bound serine proteases. Membrane permeabilization leads to granulocyte activation, as witnessed by the generation of reactive oxygen metabolites and liberation of granule constituents. A mutant toxin with unaltered binding properties but devoid of pore-forming activity did not elicit these effects. The secreted proteases cleave and activate further bound- and non-bound pro-VCC. A positive feedback loop is thus created that results in enhanced cytotoxicity towards both the targeted granulocytes and towards bystander cells that are not primarily killed by the protoxin. Thus, activation of neutrophil granulocytes by VCC fuels a positive feedback cycle that will cripple immune defence, augment inflammation, and enhance the cytotoxic action of the toxin on neighbouring tissue cells.

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Aerolysin from Aeromonas hydrophila and Related Toxins

Cited by 29 publications

References 68 publications

Rapid DNA Library Construction for Functional Genomic and Metagenomic Screening

Rapid DNA Library Construction for Functional Genomic and Metagenomic Screening

The Tetraspan Protein EMP2 Modulates the Surface Expression of Caveolins and Glycosylphosphatidyl Inositol-linked Proteins

Pro-inflammatory feedback activation cycle evoked by attack of Vibrio cholerae cytolysin on human neutrophil granulocytes

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