SMART MODE:

NORMAL GENOMIC

• Leone P et al.
• X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form.
• J Biol Chem. 2015; 290: 13191-201
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• beta-Barrel pore-forming toxins (beta-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a beta-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of beta-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other beta-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.

• Unno H, Goda S, Hatakeyama T
• Hemolytic lectin CEL-III heptamerizes via a large structural transition from alpha-helices to a beta-barrel during the transmembrane pore formation process.
• J Biol Chem. 2014; 289: 12805-12
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• CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long beta-barrel as a transmembrane pore. This beta-barrel is composed of 14 beta-strands resulting from a large structural transition of alpha-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these alpha-helices triggered their structural transition into a beta-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane beta-barrel in a position perpendicular to the plane of the lipid bilayer.

• Xu C, Wang BC, Yu Z, Sun M
• Structural insights into Bacillus thuringiensis Cry, Cyt and parasporin toxins.
• Toxins (Basel). 2014; 6: 2732-70
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• Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type alpha-PFTs, Cyt toxin type beta-PFTs and aerolysin type beta-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively.

• Los FC, Randis TM, Aroian RV, Ratner AJ
• Role of pore-forming toxins in bacterial infectious diseases.
• Microbiol Mol Biol Rev. 2013; 77: 173-207
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• Pore-forming toxins (PFTs) are the most common bacterial cytotoxic proteins and are required for virulence in a large number of important pathogens, including Streptococcus pneumoniae, group A and B streptococci, Staphylococcus aureus, Escherichia coli, and Mycobacterium tuberculosis. PFTs generally disrupt host cell membranes, but they can have additional effects independent of pore formation. Substantial effort has been devoted to understanding the molecular mechanisms underlying the functions of certain model PFTs. Likewise, specific host pathways mediating survival and immune responses in the face of toxin-mediated cellular damage have been delineated. However, less is known about the overall functions of PFTs during infection in vivo. This review focuses on common themes in the area of PFT biology, with an emphasis on studies addressing the roles of PFTs in in vivo and ex vivo models of colonization or infection. Common functions of PFTs include disruption of epithelial barrier function and evasion of host immune responses, which contribute to bacterial growth and spreading. The widespread nature of PFTs make this group of toxins an attractive target for the development of new virulence-targeted therapies that may have broad activity against human pathogens.

• Guo Z
• Synthetic Studies of Glycosylphosphatidylinositol (GPI) Anchors and GPI-Anchored Peptides, Glycopeptides, and Proteins.
• Curr Org Synth. 2013; 10: 366-383
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• Glycosylphosphatidylinositol (GPI) anchorage of proteins and glycoproteins onto the cell surface is ubiquitous in eukaryotes, and GPI-anchored proteins and glycoproteins play an important role in many biological processes. To study GPI anchorage and explore the functions of GPIs and GPI-anchored proteins and glycoproteins, it is essential to have access to these molecules in homogeneous and structurally defined forms. This review is focused on the progress that our laboratory has made towards the chemical and chemoenzymatic synthesis of structurally defined GPI anchors and GPI-anchored peptides, glycopeptides, and proteins. Briefly, highly convergent strategies were developed for GPI synthesis and were employed to successfully synthesize a number of GPIs, including those carrying unsaturated lipids and other useful functionalities such as the azido and alkynyl groups. The latter enabled further site-specific modification of GPIs by click chemistry. GPI-linked peptides, glycopeptides, and proteins were prepared by regioselective chemical coupling of properly protected GPIs and peptides/glycopeptides or through site-specific ligation of synthetic GPIs and peptides/glycopeptides/proteins under the influence of sortase A. The investigation of interactions between GPI anchors and pore-forming bacterial toxins by means of synthetic GPI anchors and GPI analogs is also discussed.

• Yan XX et al.
• Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens.
• MBio. 2013; 4: 1913-1913
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• Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a beta-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 A. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a beta-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 A, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

• Fennessey CM, Ivie SE, McClain MS
• Coenzyme depletion by members of the aerolysin family of pore-forming toxins leads to diminished ATP levels and cell death.
• Mol Biosyst. 2012; 8: 2097-105
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• Recent studies demonstrated that a variety of bacterial pore-forming toxins induce cell death through a process of programmed necrosis characterized by the rapid depletion of cellular ATP. However, events leading to the necrosis and depletion of ATP are not thoroughly understood. We demonstrate that ATP-depletion induced by two pore-forming toxins, the Clostridium perfringens epsilon-toxin and the Aeromonas hydrophila aerolysin toxin, is associated with decreased mitochondrial membrane potential and opening of the mitochondrial permeability transition pore. To gain further insight into the toxin-induced metabolic changes contributing to necrosis and depletion of ATP, we analyzed the biochemical profiles of 251 distinct compounds by GC/MS or LC/MS/MS following exposure of a human kidney cell line to the epsilon-toxin. As expected, numerous biochemicals were seen to increase or decrease in response to epsilon-toxin. However, the pattern of these changes was consistent with the toxin-induced disruption of major energy-producing pathways in the cell including disruptions to the beta-oxidation of lipids. In particular, treatment with epsilon-toxin led to decreased levels of key coenzymes required for energy production including carnitine, NAD (and NADH), and coenzyme A. Independent biochemical assays confirmed that epsilon-toxin and aerolysin induced the rapid decrease of these coenzymes or their synthetic precursors. Incubation of cells with NADH or carnitine-enriched medium helped protect cells from toxin-induced ATP depletion and cell death. Collectively, these results demonstrate that members of the aerolysin family of pore-forming toxins lead to decreased levels of essential coenzymes required for energy production. The resulting loss of energy substrates is expected to contribute to dissipation of the mitochondrial membrane potential, opening of the mitochondrial permeability transition pore, and ultimately cell death.

• Seuring C et al.
• The mechanism of toxicity in HET-S/HET-s prion incompatibility.
• PLoS Biol. 2012; 10: 1001451-1001451
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• The HET-s protein from the filamentous fungus Podospora anserina is a prion involved in a cell death reaction termed heterokaryon incompatibility. This reaction is observed at the point of contact between two genetically distinct strains when one harbors a HET-s prion (in the form of amyloid aggregates) and the other expresses a soluble HET-S protein (96% identical to HET-s). How the HET-s prion interaction with HET-S brings about cell death remains unknown; however, it was recently shown that this interaction leads to a relocalization of HET-S from the cytoplasm to the cell periphery and that this change is associated with cell death. Here, we present detailed insights into this mechanism in which a non-toxic HET-s prion converts a soluble HET-S protein into an integral membrane protein that destabilizes membranes. We observed liposomal membrane defects of approximately 10 up to 60 nm in size in transmission electron microscopy images of freeze-fractured proteoliposomes that were formed in mixtures of HET-S and HET-s amyloids. In liposome leakage assays, HET-S has an innate ability to associate with and disrupt lipid membranes and that this activity is greatly enhanced when HET-S is exposed to HET-s amyloids. Solid-state nuclear magnetic resonance (NMR) analyses revealed that HET-s induces the prion-forming domain of HET-S to adopt the beta-solenoid fold (previously observed in HET-s) and this change disrupts the globular HeLo domain. These data indicate that upon interaction with a HET-s prion, the HET-S HeLo domain partially unfolds, thereby exposing a previously buried approximately 34-residue N-terminal transmembrane segment. The liberation of this segment targets HET-S to the membrane where it further oligomerizes, leading to a loss of membrane integrity. HET-S thus appears to display features that are reminiscent of pore-forming toxins.

• De Colibus L et al.
• Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition.
• Structure. 2012; 20: 1498-507
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• Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.

• Muzard J, Martinho M, Mathe J, Bockelmann U, Viasnoff V
• DNA translocation and unzipping through a nanopore: some geometrical effects.
• Biophys J. 2010; 98: 2170-8
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• This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric alpha-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.

• Castilho MC et al.
• High frequency of hemolytic and cytotoxic activity in Aeromonas spp. isolated from clinical, food and environmental in Rio de Janeiro, Brazil.
• Antonie Van Leeuwenhoek. 2009; 96: 53-61
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• Molecular study of aerolysin and cytotonic enterotoxin genes by PCR and colony blot hybridization was performed in 117 strains of Aeromonas spp. isolated from different sources. Homogeneous distribution of these genes in A. hydrophila complex strains was observed. For A. caviae and A. sobria complex strains, aerolysin genes were more frequent than cytotonic enterotoxins genes. Of 64 A. caviae complex strains, only one (1.5%) amplified the 451 bp product for the aer gene, however, the same primers detected a 400 bp product in 50 (78%) strains. This product was sequenced and had two short regions with homology to several hemolysin genes. The genotype aer (+)/aerA(+)/hly (+)/ast (+)/alt (+) was detected in six A. hydrophila strains from food and environmental source. The most common genotype found in A. hydrophila strains was hly (+) (85%) and aerA(+) (78.7%), while in A. caviae complex strains was aerA(+) (32.8%). All A. veronii complex sobria strains were aer (+)/aerA(+). All A. caviae and A. hydrophila were positive when tested with aer probe using the colony blot test. Thirty-seven percent of A. hydrophila and 53% of A. caviae tested were positive for ast probe. Eighty-nine percent of samples were cytotoxic in Vero cells. Our data demonstrated that Aeromonas spp. can harbor and express virulence genes and reinforce the potential of Aeromonas as a human pathogen.

• Madegowda M, Eswaramoorthy S, Burley SK, Swaminathan S
• X-ray crystal structure of the B component of Hemolysin BL from Bacillus cereus.
• Proteins. 2008; 71: 534-40
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• Bacillus cereus Hemolysin BL enterotoxin, a ternary complex of three proteins, is the causative agent of food poisoning and requires all three components for virulence. The X-ray structure of the binding domain of HBL suggests that it may form a pore similar to other soluble channel forming proteins. A putative pathway of pore formation is discussed.

• Vilches S, Wilhelms M, Yu HB, Leung KY, Tomas JM, Merino S
• Aeromonas hydrophila AH-3 AexT is an ADP-ribosylating toxin secreted through the type III secretion system.
• Microb Pathog. 2008; 44: 1-12
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• We cloned and sequenced an ADP-ribosylating toxin (AexT) from a mesophilic Aeromonas hydrophila strain AH-3 with a type III secretion system (T3SS). This toxin only showed homology, in genes and proteins, with the first half of A. salmonicida AexT. The A. hydrophila AexT showed ADP-ribosyltransferase activity, translocation through the T3SS system, and this A. hydrophila T3SS system is inducible under calcium-depleted conditions. The A. hydrophila aexT mutant showed a slight reduction in their virulence assayed by several methods when compared to the wild-type strain, while an A. hydrophila T3SS mutant is highly reduced in virulence on the same assays. The A. hydrophila AexT is the first described and the smallest T3SS effector toxin found in mesophilic Aeromonas with a functional T3SS.

• Sierra JC et al.
• Biological characterization of a new type III secretion system effector from a clinical isolate of Aeromonas hydrophila-part II.
• Microb Pathog. 2007; 43: 147-60
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• We recently identified a novel type III secretion system (T3SS) effector, AexU, from a diarrheal isolate SSU of Aeromonas hydrophila, and demonstrated that mice infected with the DeltaaexU mutant were significantly protected from mortality. Although the NH(2)-terminal domain of this toxin exhibits homology to AexT of A. salmonicida, a fish pathogen, and ExoT/S of Pseudomonas aeruginosa, the COOH-terminal domain of AexU is unique, with no homology to any known proteins in the NCBI database. In this study, we purified the full-length AexU and its NH(2)-terminal (amino acid residues 1-231) and COOH-terminal (amino acid residues 232-512) domains after expression of their corresponding genes in Escherichia coli as histidine-tag fusion proteins using the bacteriophage T7 RNA polymerase/promoter-based pET-30a vector system. The full-length and NH(2)- and COOH-terminal domains of AexU exhibited ADP-ribosyltransferase activity, with the former two exhibiting much higher activity than the latter. These different forms of AexU were also successfully expressed and produced in the HeLa Tet-Off cell system using a pBI-EGFP vector, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and intracellular staining of the toxin using flow cytometric analysis. Production of AexU in HeLa cells resulted in possible actin reorganization and cell rounding, as determined by phalloidin staining and confocal microscopy. Based on electron microscopy, the toxin also caused chromatin condensation, which is indicative of apoptosis. Apoptosis of HeLa cells expressing and producing AexU was confirmed by 7-amino actinomycin D (7-AAD) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] assays, by detection of cytoplasmic histone-associated DNA fragments, and by activation of caspases 3 and 9. These effects were much more pronounced in host cells that expressed and produced the full-length or NH(2)-terminal domain of AexU, compared to those that expressed and produced the COOH-terminal domain or the vector alone. This study represents the first characterization of this novel T3SS effector.

• Sha J et al.
• Further characterization of a type III secretion system (T3SS) and of a new effector protein from a clinical isolate of Aeromonas hydrophila--part I.
• Microb Pathog. 2007; 43: 127-46
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• A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.

• Erova TE et al.
• Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila.
• FEMS Microbiol Lett. 2007; 275: 301-11
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• A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.

• Eifler N et al.
• Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
• EMBO J. 2006; 25: 2652-61
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• ClyA is a pore-forming toxin from virulent Escherichia coli and Salmonella enterica strains. Here, we show that the intrinsic hemolytic activity of ClyA is independent of its redox state, and that the assembly of both reduced and oxidized ClyA to the ring-shaped oligomer is triggered by contact with lipid or detergent. A rate-limiting conformational transition in membrane-bound ClyA monomers precedes their assembly to the functional pore. We obtained a three-dimensional model of the detergent-induced oligomeric complex at 12 A resolution by combining cryo- and negative stain electron microscopy with mass measurements by scanning transmission electron microscopy. The model reveals that 13 ClyA monomers assemble into a cylinder with a hydrophobic cap region, which may be critical for membrane insertion.

• Shin DJ, Lee JJ, Choy HE, Hong Y
• Generation and characterization of Clostridium septicum alpha toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria.
• Biochem Biophys Res Commun. 2004; 324: 753-60
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• Glycosylphosphatidylinositol (GPI) anchors various proteins to the membrane of eukaryotic cells. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that is primarily due to the lack of GPI-anchored proteins on the surface of blood cells. To detect the GPI-deficient cells in PNH patients, we modified alpha toxin, a pore-forming toxin of the Gram-positive bacterium Clostridium septicum. We first showed that aerolysin, a homologous toxin from Aeromonas hydrophila, bound to both of Chinese hamster ovary cells deficient of N-glycan maturation as well as GPI biosynthesis at a significant level. However, alpha toxin bound to the mutant cells of N-glycosylation, but not to GPI-deficient cells. It suggested that alpha toxin could be used as a specific probe to differentiate only GPI-deficient cells. As a diagnostic probe, alpha toxin must be the least cytotoxic while maintaining its affinity for GPI. Thus, we constructed several mutants. Of these, the mutants carrying the Y155G or S189C/S238C substitutions bound to GPI as well as the wild-type toxin. These mutants also efficiently underwent proteolytic activation and aggregated into oligomers on the cell surface, which are events that precede the formation of a pore in the host cell membrane, leading to cell death. Nevertheless, these mutants almost completely failed to kill host cells. It was revealed that the substitutions affect the events that follow oligomerization. The S189C/S238C mutant toxin differentiated GPI-deficient granulocyte and PMN, but not red blood cells, of a PNH patient from GPI-positive cells at least as sensitively as the commercial monoclonal antibodies that recognize the CD59 or CD55 GPI proteins on blood cells. Thus, this modified bacterial toxin can be employed instead of costly monoclonal antibodies to diagnose PNH patients.

• Song T, Toma C, Nakasone N, Iwanaga M
• Aerolysin is activated by metalloprotease in Aeromonas veronii biovar sobria.
• J Med Microbiol. 2004; 53: 477-82
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• Aeromonas veronii is an opportunistic human pathogen that causes diarrhoea and extraintestinal infections, such as wound infection and septicaemia. An A. veronii protease (AVP) from a biovar sobria strain, AeG1, was partially purified and characterized. Mature AVP hydrolysed casein but not elastin, and protease activity was inhibited by metalloprotease inhibitors. Nucleotide sequence analysis showed that AVP belongs to the thermolysin family of proteases. An AVP-deficient mutant was constructed to investigate the role of AVP in aerolysin activation. Western blot analysis using anti-aerolysin antisera revealed that proaerolysin (52 kDa) in the AVP-deficient mutant was not completely activated to mature aerolysin (47 kDa) as seen in the wild-type strain. The AVP-deficient mutant showed lower cytotoxic and haemolytic activities than wild type. AVP and proaerolysin had no haemolytic activity; however, activity appeared after incubating both proteins. Taken together, these results suggested that AVP plays an indirect role in virulence through activating aerolysin, which is an essential step for cytotoxic activity.

• Rausell C et al.
• Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel.
• J Biol Chem. 2004; 279: 55168-75
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• The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.

• Masson L, Schwab G, Mazza A, Brousseau R, Potvin L, Schwartz JL
• A novel Bacillus thuringiensis (PS149B1) containing a Cry34Ab1/Cry35Ab1 binary toxin specific for the western corn rootworm Diabrotica virgifera virgifera LeConte forms ion channels in lipid membranes.
• Biochemistry. 2004; 43: 12349-57
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• The binary Bacillus thuringiensis PS149B1 insecticidal crystal (Cry) protein is comprised of two components, Cry34Ab1, a 14-kDa protein, and Cry35Ab1, a 44-kDa protein, the combination of which forms a novel binary toxin active on western corn rootworm larvae. The permeabilizing behavior of the native binary toxin and its two individual components expressed as recombinant proteins was studied using calcein efflux determination in liposomes and by ion channel activity measurements in planar lipid bilayers (PLBs). Data obtained with solubilized native PS149B1 binary protein revealed it to be a pore-forming toxin that can permeabilize liposomes and form ion channels ( approximately 300-900 pS) in PLBs at pH 5.5 but not pH 9.0. The 14-kDa component of the toxin also formed ion channels ( approximately 15-300 pS) at pH 5.5 but did not insert easily in PLBs. While the 44-kDa moiety did seldomly form resolvable ion channels ( approximately 15-750 pS) in PLBs, it did destabilize the membranes. It showed pH-dependent truncation to a stable 40-kDa protein. The purified 40-kDa truncated product formed channels ( approximately 10-450 pS) in PLBs at pH 5.5. At that same pH, while a 3:1 molar mixture (14:44 kDa) of the individual components of the toxin induced channel activity that resembled that of the 14-kDa component alone, the 3:1 molar mixture of the 14-kDa component and 40-kDa truncated product induced channel activity ( approximately 20-800 pS) similar to that of PS149B1 in planar lipid bilayers. We conclude that the overall membrane permeabilization process of Cry34Ab1/Cry35Ab1 is a result of ion channel formation.

• Fukushima K, Ikehara Y, Kanai M, Kochibe N, Kuroki M, Yamashita K
• A beta-N-acetylglucosaminyl phosphate diester residue is attached to the glycosylphosphatidylinositol anchor of human placental alkaline phosphatase: a target of the channel-forming toxin aerolysin.
• J Biol Chem. 2003; 278: 36296-303
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• Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitous in eukaryotes. The minimum conserved GPI core structure of all GPI-anchored glycans has been determined as EtN-PO4-6Manalpha1-2Manalpha1-6Manalpha1-4GlcN-myo-inositol-PO3H. Human placental alkaline phosphatase (AP) has been reported to be a GPI-anchored membrane protein. AP carries one N-glycan, (NeuAcalpha2-->3)2Gal2GlcNAc2Man3GlcNAc(+/-Fuc)GlcNAc, and a GPI anchor, which contains an ethanolamine phosphate diester group, as a side chain. However, we found that both sialidase-treated soluble AP (sAP) and its GPI-anchored glycan bound to a Psathyrella velutina lectin (PVL)-Sepharose column, which binds beta-GlcNAc residues. PVL binding of asialo-sAP and its GPI-anchored glycan was diminished by digestion with diplococcal beta-N-acetylhexosaminidase or by mild acid treatment. After sequential digestion of asialo-sAP with beta-N-acetylhexosaminidase and acid phosphatase, the elution patterns on chromatofocusing gels were changed in accordance with the negative charges of phosphate residues. Trypsin-digested sAP was analyzed by liquid chromatography/electrospray ionization mass spectrometry, and the structures of two glycopeptides with GPI-anchored glycans were confirmed as peptide-EtN-PO4-6Manalpha1-->2(GlcNAcbeta1-PO4-->6)Manalpha1-6(+/-EtN-PO4-->)Mana lpha1-->4GlcN, which may be produced by endo-alpha-glucosaminidase. In addition to AP, GPI-anchored carcinoembryonic antigen, cholinesterase, and Tamm-Horsfall glycoprotein also bound to a PVL-Sepharose column, suggesting that the beta-N-acetylglucosaminyl phosphate diester residue is widely distributed in human GPI-anchored glycans. Furthermore, we found that the beta-N-acetylglucosaminyl phosphate diester residue is important for GPI anchor recognition of aerolysin, a channel-forming toxin derived from Aeromonas hydrophila.

• Menestrina G et al.
• Ion channels and bacterial infection: the case of beta-barrel pore-forming protein toxins of Staphylococcus aureus.
• FEBS Lett. 2003; 552: 54-60
• Display abstract

• Staphylococcus aureus strains causing human pathologies produce several toxins, including a pore-forming protein family formed by the single-component alpha-hemolysin and the bicomponent leukocidins and gamma-hemolysins. The last comprise two protein elements, S and F, that co-operatively form the active toxin. alpha-Hemolysin is always expressed by S. aureus strains, whereas bicomponent leukotoxins are more specifically involved in a few diseases. X-ray crystallography of the alpha-hemolysin pore has shown it is a mushroom-shaped, hollow heptamer, almost entirely consisting of beta-structure. Monomeric F subunits have a very similar core structure, except for the transmembrane stem domain which has to refold during pore formation. Large deletions in this domain abolished activity, whereas shorter deletions sometimes improved it, possibly by removing some of the interactions stabilizing the folded structure. Even before stem extension is completed, the formation of an oligomeric pre-pore can trigger Ca(2+)-mediated activation of some white cells, initiating an inflammatory response. Within the bicomponent toxins, gamma-hemolysins define three proteins (HlgA, HlgB, HlgC) that can generate two toxins: HlgA+HlgB and HlgC+HlgB. Like alpha-hemolysin they form pores in planar bilayers with similar conductance, but opposite selectivity (cation instead of anion) for the presence of negative charges in the ion pathway. gamma-Hemolysin pores seem to be organized as alpha-hemolysin, but should contain an even number of each component, alternating in a 1:1 stoichiometry.

• Fujii Y, Nomura T, Yokoyama R, Shinoda S, Okamoto K
• Studies of the mechanism of action of the aerolysin-like hemolysin of Aeromonas sobria in stimulating T84 cells to produce cyclic AMP.
• Infect Immun. 2003; 71: 1557-60
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• We previously reported that the aerolysin-like hemolysin of Aeromonas sobria stimulates T84 cells to produce cyclic AMP, which then emerges in the culture medium. In order to clarify the mechanism of action of the hemolysin, we examined the involvement of adenosine nucleotide. The results show that the hemolysin stimulates T84 cells to release ATP, which is then converted to adenosine by ectonucleotidase. The adenosine generated might stimulate the P1 adenosine receptors of T84 cells to produce cyclic AMP.

• Tsitrin Y et al.
• Conversion of a transmembrane to a water-soluble protein complex by a single point mutation.
• Nat Struct Biol. 2002; 9: 729-33
• Display abstract

• Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.

• Jedrzejas MJ
• Pneumococcal virulence factors: structure and function.
• Microbiol Mol Biol Rev. 2001; 65: 187-207
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• The overall goal for this review is to summarize the current body of knowledge about the structure and function of major known antigens of Streptococcus pneumoniae, a major gram-positive bacterial pathogen of humans. This information is then related to the role of these proteins in pneumococcal pathogenesis and in the development of new vaccines and/or other antimicrobial agents. S. pneumoniae is the most common cause of fatal community-acquired pneumonia in the elderly and is also one of the most common causes of middle ear infections and meningitis in children. The present vaccine for the pneumococcus consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, who are normally at low risk of serious disease, it is only about 60% effective in the elderly. In children younger than 2 years the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the pneumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets. Essentially all of these antigens have been or are being investigated on a structural level in addition to being characterized biochemically. Recently, three-dimensional structures for hyaluronate lyase and pneumococcal surface antigen A became available from X-ray crystallography determinations. Also, modeling studies based on biophysical measurements provided more information about the structures of pneumolysin and pneumococcal surface protein A. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines as an alternative to polysaccharide-based vaccines for the treatment of pneumococcal disease.

• Barry R, Moore S, Alonso A, Ausio J, Buckley JT
• The channel-forming protein proaerolysin remains a dimer at low concentrations in solution.
• J Biol Chem. 2001; 276: 551-4
• Display abstract

• Proaerolysin, the proform of the channel-forming protein aerolysin, is secreted as a dimer by Aeromonas sp. The protein also exists as a dimer in the crystal, as well as in solution, at least at concentrations in the region of 500 microg/ml. Recently it has been argued that proaerolysin becomes monomeric at concentrations below 100 microg/ml and that only the monomeric form of the protoxin can bind to cell surface receptors (Fivaz, M., Velluz, M.-C., and van der Goot, F. G. (1999) J. Biol. Chem. 274, 37705-37708). Here we show, using non-denaturing polyacrylamide electrophoresis, chemical cross-linking, and analytical ultracentrifugation, that proaerolysin remains dimeric at the lowest concentrations of the protein that we measured (less than 5 microg/ml) and that the dimeric protoxin is quite capable of receptor binding.

• Burr SE, Diep DB, Buckley JT
• Type II secretion by Aeromonas salmonicida: evidence for two periplasmic pools of proaerolysin.
• J Bacteriol. 2001; 183: 5956-63
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• Aeromonas salmonicida containing the cloned gene for proaerolysin secretes the protein via the type II secretory pathway. Here we show that altering a region near the beginning of aerA led to a dramatic increase in the amount of proaerolysin that was produced and that a large amount of the protein was cell associated. All of the cell-associated protein had crossed the cytoplasmic membrane, because the signal sequence had been removed, and all of it was accessible to processing by trypsin during osmotic shock. Enlargement of the periplasm was observed by electron microscopy in overproducing cells, likely caused by the osmotic effect of the very large concentrations of accumulated proaerolysin. Immunogold electron microscopy localized nearly all of the proaerolysin in the enlarged periplasm; however, only half of the protoxin was released from the cells by osmotic shocking. Cross-linking studies showed that this fraction contained normal dimeric proaerolysin but that proaerolysin in the fraction that was not shockable had not dimerized, although it appeared to be correctly folded. Both periplasmic fractions were secreted by the cells; however, the nonshockable fraction was secreted much more slowly than the shockable fraction. We estimated a rate for maximal secretion of proaerolysin from the bacteria that was much lower than the rates that have been estimated for inner membrane transit, which suggests that transit across the outer membrane is rate limiting and may account for the periplasmic accumulation of the protein. Finally, we show that overproduction of proaerolysin inhibited the release of the protease that is secreted by A. salmonicida.

• Fivaz M, Abrami L, Tsitrin Y, van der Goot FG
• Aerolysin from Aeromonas hydrophila and related toxins.
• Curr Top Microbiol Immunol. 2001; 257: 35-52

• Abrami L, Fivaz M, van der Goot FG
• Surface dynamics of aerolysin on the plasma membrane of living cells.
• Int J Med Microbiol. 2000; 290: 363-7
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• Aerolysin secreted by the human pathogen Aeromonas hydrophila belongs to a group of bacterial toxins that are hemolytic and form channels in biological membranes. The toxin is secreted as an inactive precursor proaerolysin that must be proteolytically processed at its C-terminus to become active. The toxin then polymerizes into a heptameric ring that is amphipathic and can insert into a lipid bilayer and form a pore. We have examined these various steps at the surface of target cells. The toxin binds to specific receptors. Various receptors have been identified, all of which are anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI)-anchored moiety. The GPI anchor confers to the protein that is linked to it two usual properties: (i) the protein has a higher lateral mobility in a phospholipid bilayer than its transmembrane counterpart, (ii) the protein has the capacity to transiently associate with cholesterol-glycosphingolipid-rich microdomains. We have shown that both these properties of GPI-anchored proteins are exploited by proaerolysin bound to its receptor. The high lateral mobility within the phosphoglyceride region of the plasma membrane favors the encounter of the protoxin with its converting enzyme furin. The ability to associate with microdomains on the other hand favors the oligomerization process presumably by concentrating the toxin locally.

• Abrami L, Fivaz M, van der Goot FG
• Adventures of a pore-forming toxin at the target cell surface.
• Trends Microbiol. 2000; 8: 168-72
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• The past three years have shed light on how the pore-forming toxin aerolysin binds to its target cell and then hijacks cellular devices to promote its own polymerization and pore formation. This selective permeabilization of the plasma membrane has unexpected intracellular consequences that might explain the importance of aerolysin in Aeromonas pathogenicity.

• Lesieur C, Frutiger S, Hughes G, Kellner R, Pattus F, van der Goot FG
• Increased stability upon heptamerization of the pore-forming toxin aerolysin.
• J Biol Chem. 1999; 274: 36722-8
• Display abstract

• Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH(2)-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180-307 and 401-427, were identified, by limited proteolysis, NH(2)-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of beta structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.

• Fivaz M, Velluz MC, van der Goot FG
• Dimer dissociation of the pore-forming toxin aerolysin precedes receptor binding.
• J Biol Chem. 1999; 274: 37705-8
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• The pore-forming toxin aerolysin is secreted by Aeromonas hydrophila as an inactive precursor. Based on chemical cross-linking and gel filtration, we show here that proaerolysin exists as a monomer at low concentrations but is dimeric above 0.1 mg/ml. At intermediate concentrations, monomers and dimers appeared to be in rapid equilibrium. All together our data indicate that, at low concentrations, the toxin is a monomer and that this species is competent for receptor binding. In contrast, a mutant toxin that forms a covalent dimer was unable to bind to target cells.

• Merzlyak PG, Yuldasheva LN, Rodrigues CG, Carneiro CM, Krasilnikov OV, Bezrukov SM
• Polymeric nonelectrolytes to probe pore geometry: application to the alpha-toxin transmembrane channel.
• Biophys J. 1999; 77: 3023-33
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• Asymmetrical (one-sided) application of penetrating water-soluble polymers, polyethylene glycols (PEGs), to a well-defined channel formed by Staphylococcus aureus alpha-toxin is shown to probe channel pore geometry in more detail than their symmetrical (two-sided) application. Polymers added to the cis side of the planar lipid membrane (the side of protein addition) affect channel conductance differently than polymers added to the trans side. Because a satisfactory theory quantitatively describing PEG partitioning into a channel pore does not exist, we apply the simple empirical rules proposed previously (, J. Membr. Biol. 161:83-92) to gauge the size of pore openings as well as the size and position of constrictions along the pore axis. We estimate the radii of the two openings of the channel to be practically identical and equal to 1. 2-1.3 nm. Two apparent constrictions with radii of approximately 0. 9 nm and approximately 0.6-0.7 nm are inferred to be present in the channel lumen, the larger one being closer to the cis side. These structural findings agree well with crystallographic data on the channel structure (, Science. 274:1859-1866) and verify the practicality of polymer probing. The general features of PEG partitioning are examined using available theoretical considerations, assuming there is no attraction between PEG and the channel lumen. It is shown that the sharp dependence of the partition coefficient on polymer molecular weight found under both symmetrical and asymmetrical polymer application can be rationalized within a "hard sphere nonideal solution model." This finding is rather surprising because PEG forms highly flexible coils in water with a Kuhn length of only several Angstroms.

• Khan AA, Kim E, Cerniglia CE
• Molecular cloning, nucleotide sequence, and expression in Escherichia coli of a hemolytic toxin (aerolysin) gene from Aeromonas trota.
• Appl Environ Microbiol. 1998; 64: 2473-8
• Display abstract

• Aeromonas trota AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn5Km1 to generate a hemolysin-deficient mutant, designated strain AK253. Southern blotting data indicated that an 8.7-kb NotI fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn5Km1. The 8.7-kb NotI DNA fragment was cloned into the vector pGEM5Zf(-) by selecting for kanamycin resistance, and the resultant clone, pAK71, showed aerolysin activity in Escherichia coli JM109. The nucleotide sequence of the aerA gene, located on the 1.8-kb ApaI-EcoRI fragment, was determined to consist of 1,479 bp and to have an ATG initiation codon and a TAA termination codon. An in vitro coupled transcription-translation analysis of the 1.8-kb region suggested that the aerA gene codes for a 54-kDa protein, in agreement with nucleotide sequence data. The deduced amino acid sequence of the aerA gene product of A. trota exhibited 99% homology with the amino acid sequence of the aerA product of Aeromonas sobria AB3 and 57% homology with the amino acid sequences of the products of the aerA genes of Aeromonas salmonicida 17-2 and A. sobria 33.

• Krovacek K, Huang K, Sternberg S, Svenson SB
• Aeromonas hydrophila septicaemia in a grey seal (Halichoerus grypus) from the Baltic Sea: a case study.
• Comp Immunol Microbiol Infect Dis. 1998; 21: 43-9
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• Aeromonas hydrophila septicaemia in a grey seal (Halichoerus grypus) from the Swedish part of the Baltic Sea is reported. The pathogen was isolated from both lung and spleen specimens. All of the A. hydrophila isolates produced haemolysin and Vero active cytotoxin. The aerolysin gene was found in all tested isolates as evidenced by the polymerase chain reaction (PCR) technique. Also, all isolates tested showed identical patterns of biochemical and antibiotic resistance. As Aeromonas spp. commonly occur in aquatic environments, we suggest that organisms from this genus may also play an important role as opportunistic pathogens in morbillivirus infected seals.

• Fujii Y et al.
• Purification and characterization of enterotoxin produced by Aeromonas sobria.
• Microbiol Immunol. 1998; 42: 703-14
• Display abstract

• We purified the toxin of Aeromonas sobria capable of inducing a positive response in the mouse intestinal loop assay. The purified toxin showed a positive response not only in the loop assay but also in a hemolytic assay. Subsequently, we cloned the toxin gene and demonstrated that the product of this gene possessed both hemolytic and enterotoxic activities. These results showed that the enterotoxin of A. sobria possesses hemolytic activity. Nucleotide sequence determination of the toxin gene and amino acid sequence analysis of the purified toxin revealed that it is synthesized as a precursor composed of 488 amino acid residues, and that the 24 amino-terminal amino acid residues of the precursor is removed in the mature toxin. As antiserum against the purified toxin neutralized the fluid accumulation induced by living cells not only of A. sobria but also of A. hydrophila, this and antigenically related toxin(s) are thought to play an essential role in the induction of diarrhea by these organisms. The toxin-injured Chinese hamster ovary (CHO) cells induced the release of intracellular lactose dehydrogenase (LDH). The release of LDH from CHO cells and the lysis of erythrocytes by the toxin were repressed by the addition of dextran to the reaction solution, indicating that the toxin forms pores in the membranes and that the cells were injured by the osmotic gradient developed due to pore formation. However, the histopathological examination of intestinal cells exposed to the toxin showed that it caused fluid accumulation in the mouse intestinal loop without causing cellular damage.

• Rossjohn J et al.
• Movement of a loop in domain 3 of aerolysin is required for channel formation.
• Biochemistry. 1998; 37: 741-6
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• Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the protein before oligomerization can proceed. In order to determine if movement actually occurs, strategically located amino acids in the loop and in the sheet were replaced with cysteines by site-directed mutagenesis. A double mutant was produced in which the new cysteines, at position 253 on the loop and position 300 in the sheet, were close enough together to allow formation of a disulfide bridge. The double mutant was unable to oligomerize, and it was completely inactive, showing not only that the bridge had formed but also that movement of the loop was essential for formation of the oligomer. The existence of the bridge was confirmed by X-ray crystallography. The reduced form of the protein and the single mutants T253C and A300C were as active as wild type, indicating that the amino acid replacements themselves had no functional consequences. Labeling studies using an environment-sensitive fluorescent sulfhydryl-reactive probe confirmed that the structure of the protein changes in the loop region as a consequence of proteolytic activation of proaerolysin, a step which also must precede oligomerization.

• Abrami L et al.
• The pore-forming toxin proaerolysin is activated by furin.
• J Biol Chem. 1998; 273: 32656-61
• Display abstract

• Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A. Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin. We also investigated the potential role of these enzymes in the in vivo activation of the protoxin. We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin. The furin inhibitor alpha1-antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells. The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin. Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.

• Palmer M, Harris R, Freytag C, Kehoe M, Tranum-Jensen J, Bhakdi S
• Assembly mechanism of the oligomeric streptolysin O pore: the early membrane lesion is lined by a free edge of the lipid membrane and is extended gradually during oligomerization.
• EMBO J. 1998; 17: 1598-605
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• Streptolysin O (SLO) is a bacterial exotoxin that binds to cell membranes containing cholesterol and then oligomerizes to form large pores. Along with rings, arc-shaped oligomers form on membranes. It has been suggested that each arc represents an incompletely assembled oligomer and constitutes a functional pore, faced on the opposite side by a free edge of the lipid membrane. We sought functional evidence in support of this idea by using an oligomerization-deficient, non-lytic mutant of SLO. This protein, which was created by chemical modification of a single mutant cysteine (T250C) with N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid, formed hybrid oligomers with active SLO on membranes. However, incorporation of the modified T250C mutant inhibited subsequent oligomerization, so that the hybrid oligomers were reduced in size. These appeared as typical arc lesions in the electron microscope. They formed pores that permitted passage of NaCl and calcein but restricted permeation of large dextran molecules. The data indicate that the SLO pore is formed gradually during oligomerization, implying that pores lined by protein on one side and an edge of free lipid on the other may be created in the plasma membrane. Intentional manipulation of the pore size may extend the utility of SLO as a tool in cell biological experiments.

• Rossjohn J et al.
• Aerolysin--a paradigm for membrane insertion of beta-sheet protein toxins?
• J Struct Biol. 1998; 121: 92-100
• Display abstract

• The determination of the crystal structure of the bacterial protein proaerolysin provided the first view of a pore-forming toxin constructed mainly from beta-sheet. The structure that was obtained and subsequent crystallographic and biochemical studies have together allowed us to explain how the toxin is transformed from a water-soluble dimer to a heptameric transmembrane pore. Recent discoveries of structural similarities between aerolysin and other toxins suggest that the structure/function studies we have made may prove useful in understanding the actions of a number of pore-forming proteins.

• Sowdhamini R, Mitchell TJ, Andrew PW, Morgan PJ
• Structural and functional analogy between pneumolysin and proaerolysin.
• Protein Eng. 1997; 10: 207-15
• Display abstract

• Pneumolysin and proaerolysin are bacterial toxins that form pores in host cells by oligomerization. We propose that they may have similar structures despite a poor sequence identity. The crystal structure of proaerolysin reveals a protein composed of four domains, arranged in the shape of an elongated comma. Electron microscopy of the pneumolysin monomer shows a similar arrangement of domains. The sequence of pneumolysin recognizes the template of proaerolysin from a library of protein folds. A three-dimensional model of pneumolysin has been constructed by the comparative approach using the structure of proaerolysin. This model, together with results on the activity of site-specific mutants and the positions of antigenic sites, has been used to propose functional roles of individual domains.

• Kuhnert P, Heyberger-Meyer B, Burnens AP, Nicolet J, Frey J
• Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.
• Appl Environ Microbiol. 1997; 63: 2258-65
• Display abstract

• The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.

• Letellier L, Howard SP, Buckley JT
• Studies on the energetics of proaerolysin secretion across the outer membrane of Aeromonas species. Evidence for a requirement for both the protonmotive force and ATP.
• J Biol Chem. 1997; 272: 11109-13
• Display abstract

• Aeromonas spp. secrete the channel-forming protein proaerolysin across their inner and outer membranes in separate steps using the general secretion pathway. Here we show that treating A. hydrophila or A. salmonicida with the protonophore carbonyl cyanide m-chorophenyl hydrazone blocks the second step in transport, secretion across the outer membrane from the periplasm, under conditions where the ATP levels in the cell are no different than the levels in control, secreting cells. A threshold for DeltaPsi was observed in the region of 120 mV, below which secretion by both species was inhibited. Treatment of cells with arsenate, which lowered ATP levels but did not affect DeltaPsi, also reduced secretion from the periplasm, an indication that there is an ATP requirement for this step independent of the requirement for DeltaPsi. Secretion across the outer membrane was also arrested by increasing the osmotic pressure of the medium, even though cellular ATP levels and DeltaPsi were not affected. This may be due to disruption of some necessary association between the inner and outer membranes.

• Swift S et al.
• Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules.
• J Bacteriol. 1997; 179: 5271-81
• Display abstract

• Spent culture supernatants from both Aeromonas hydrophila and Aeromonas salmonicida activate a range of biosensors responsive to N-acylhomoserine lactones (AHLs). The genes for a quorum sensing signal generator and a response regulator were cloned from each Aeromonas species and termed ahyRI and asaRI, respectively. Protein sequence homology analysis places the gene products within the growing family of LuxRI homologs. ahyR and asaR are transcribed divergently from ahyI and asaI, respectively, and in both Aeromonas species, the genes downstream have been identified by DNA sequence and PCR analysis. Downstream of both ahyI and asaI is a gene with close homology to iciA, an inhibitor of chromosome replication in Escherichia coli, a finding which implies that in Aeromonas, cell division may be linked to quorum sensing. The major signal molecule synthesized via both AhyI and AsaI was purified from spent culture supernatants and identified as N-(butanoyl)-L-homoserine lactone (BHL) by thin-layer chromatography, high-pressure liquid chromatography analysis, and mass spectrometry. In addition, a second, minor AHL, N-hexanoyl-L-homoserine lactone, was identified. Transcriptional reporter studies with ahyI::luxCDABE fusions indicate that AhyR and BHL are both required for ahyI transcription. For A. salmonicida, although the addition of exogenous BHL gives only a small stimulation of the production of serine protease with comparison to the control culture, the incorporation of a longer-chain AHL, N-(3-oxodecanoyl)-L-homoserine lactone, reduced the final level (by approximately 50%) and delayed the appearance (from an A650 of 0.9 in the control to an A650 of 1.2 in the test) of protease in the culture supernatant. These data add A. hydrophila and A. salmonicida to the growing family of gram-negative bacteria now known to control gene expression through quorum sensing.

• Parker MW, van der Goot FG, Buckley JT
• Aerolysin--the ins and outs of a model channel-forming toxin.
• Mol Microbiol. 1996; 19: 205-12
• Display abstract

• Aerolysin is one of a large group of bacterial proteins that can kill target cells by forming discrete channels in their plasma membranes. The toxin has many properties in common with the porins of the Gram-negative bacterial outer membrane, including an extensive amount of beta-structure, a high proportion of hydrophilic amino acid side-chains and no hydrophobic stretches in the primary structure. It also oligomerizes to produce an insertion-competent state. Aerolysin is secreted as a dimer by members of the Aeromonas family. It binds to a high-affinity receptor on the target cell that has recently been shown to be a glycosylphosphatidylinositol-anchored glycoprotein. Binding is followed by heptamerization to form a structure that we propose contains a beta-barrel which can insert into the membrane and produce a channel.

• Moniatte M, van der Goot FG, Buckley JT, Pattus F, van Dorsselaer A
• Characterisation of the heptameric pore-forming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry.
• FEBS Lett. 1996; 384: 269-72
• Display abstract

• Aerolysin, a virulence factor secreted by Aeromonas hydrophila, is representative of a group of beta-sheet toxins that must form stable homooligomers in order to be able to insert into biological membranes and generate channels. Electron microscopy and image analysis of two-dimensional membrane crystals had previously revealed a structure with 7-fold symmetry suggesting that aerolysin forms heptameric oligomers [Wilmsen et al. (1992) EMBO J. 11, 2457-2463]. However, this unusual molecularity of the channel remained to be confirmed by an independent method since low-resolution electron crystallography had led to artefactual data for other pore-forming toxins. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to measure the mass of the aerolysin oligomer preparation. A mass of 333 850 Da was measured, fitting very well with a heptameric complex (expected mass: 332 300 Da). These results confirm the earlier evidence that the aerolysin oligomer is a heptamer and also show that MALDI-TOF mass spectrometry could be a valuable tool to study non-covalent association of proteins.

• Blundell TL, Srinivasan N
• Symmetry, stability, and dynamics of multidomain and multicomponent protein systems.
• Proc Natl Acad Sci U S A. 1996; 93: 14243-8
• Display abstract

• Symmetry is commonly observed in many biological systems. Here we discuss representative examples of the role of symmetry in structural molecular biology. Point group symmetries are observed in many protein oligomers whose three-dimensional atomic structures have been elucidated by x-ray crystallography. Approximate symmetry also occurs in multidomain proteins. Symmetry often confers stability on the molecular system and results in economical usage of basic components to build the macromolecular structure. Symmetry is also associated with cooperativity. Mild perturbation from perfect symmetry may be essential in some systems for dynamic functions.

• Shiro M, Ueda M, Kawaguchi T, Arai M
• Cloning of a cluster of chitinase genes from Aeromonas sp. No. 10S-24.
• Biochim Biophys Acta. 1996; 1305: 44-8
• Display abstract

• A gene encoding chitinases from Aeromonas sp. No. 10S-24 was cloned into Escherichia coli DH5 alpha using pUC19, and its nucleotides were sequenced. The chitinase gene was clustered in ORFs (open reading frame) 1 to 4, in a 8-kb fragment of DNA. ORF-1 consisted of 1608 bp encoding 535 amino acid residues, and ORF-2 consisted of 1425 bp encoding 474 amino acid residues. ORF-3 was 1617 bp long and encodes a protein consisting of 538 amino acids. ORF-4 encodes 287 amino acids of the N-terminal region. The amino acid sequences of ORF-1 and ORF-3 share sequence homology with chitinase D from Bacillus circulans, and chitinase A and B from Streptomyces lividans. The amino acid sequence of ORF-2 shared sequence homology with chitinase II from Aeromonas sp. No. 10S-24, and chitinase from Saccharopolyspora erythraea. A region of the sequence starting from Ala-28 of the amino acid sequence of ORF-3 coincided with the N-terminal amino acid sequence of chitinase III from Aeromonas sp. No. 10S-24.

• Tschodrich-Rotter M, Kubitscheck U, Ugochukwu G, Buckley JT, Peters R
• Optical single-channel analysis of the aerolysin pore in erythrocyte membranes.
• Biophys J. 1996; 70: 723-32
• Display abstract

• Scanning microphotolysis (Scamp), a recently developed photobleaching technique, was used to analyze the transport of two small organic anions and one inorganic cation through single pores formed in human erythrocyte membranes by the channel-forming toxin aerolysin secreted by Aeromonas species. The transport rate constants of erythrocyte ghosts carrying a single aerolysin pore were determined to be (1.83 +/- 0.43) x 10(-3) s-1 for Lucifer yellow, (0.33 +/- 0.10) x 10(-3) s-1 for carboxyfluorescein, and (8.20 +/- 2.30) x 10(-3) s-1 for Ca2+. The radius of the aerolysin pore was derived from the rate constants to be 19-23 A, taking steric hindrance and viscous drag into account. The size of the Ca2+ rate constant implies that at physiological extracellular Ca2+ concentrations (> 1 mM) the intracellular Ca2+ concentration would be elevated to the critical level of > 1 microM in much less than a second after formation of a single aerolysin pore in the plasma membrane. Thus changes in the levels of Ca2+ or other critical intracellular components may be more likely to cause cell death than osmotic imbalance.

• Ballard J, Crabtree J, Roe BA, Tweten RK
• The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin.
• Infect Immun. 1995; 63: 340-4
• Display abstract

• The gene for Clostridium septicum alpha-toxin was cloned and expressed in Escherichia coli from C. septicum BX96. The toxin was determined to be 443 amino acids in length, with a 31-residue signal peptide that was removed from the toxin during secretion. No extended hydrophobic regions were observed in the mature toxin sequence. Expression of alpha-toxin in E. coli BL21 resulted in the production of ATpro, which was identical to native toxin from C. septicum with respect to activity and activation. The proteolytic activation site for alpha-toxin was determined to be on the carboxy-terminal side of arginine 398, which lies within the sequence KKRRGKR-398SVD. Previous work showing similarities in activation and mechanism between alpha-toxin and Aeromonas hydrophila aerolysin was extended to the primary structures of both toxins. The DNA-derived primary sequence of alpha-toxin exhibited 27% identity and 72% similarity over a 387-residue region with the primary structure of the A. hydrophila aerolysin toxin, a level of similarity heretofore unobserved between toxins produced by a gram-positive organism and a gram-negative organism.

• Baloda SB, Krovacek K, Eriksson L, Linne T, Mansson I
• Detection of aerolysin gene in Aeromonas strains isolated from drinking water, fish and foods by the polymerase chain reaction.
• Comp Immunol Microbiol Infect Dis. 1995; 18: 17-26
• Display abstract

• A polymerase chain reaction (PCR) technique was used to assay the presence of the aerolysin gene in a total of 89 Aeromonas hydrophila and A. sobria strains isolated from drinking water, fish and foods. These strains were also characterized for the production of virulence factors such as haemolysin, protease and cytotoxin. The primers used in the PCR targeted a 209-bp fragment of the aer gene coding for the beta-haemolysin and detected template DNA only in haemolytic A. hydrophila strains. The cell-free culture supernatants of these aerolysin-positive A. hydrophila strains were also cytotoxic to the HeLa and McCoy cells. The haemolytic A. sobria and non-haemolytic A. hydrophila were consistently negative in the PCR assay. Primer specificity was determined in the PCR by using a control haemolytic Escherichia coli, Streptococcus pyogenes and a restriction endonuclease assay. The PCR clearly identified the aerolysin-producing strains of A. hydrophila and may have application as a rapid species-specific virulence test.

• Hilger M, Braun V
• Superlytic hemolysin mutants of Serratia marcescens.
• J Bacteriol. 1995; 177: 7202-9
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• Hemolysis by Serratia marcescens is caused by two proteins, ShlA and ShlB. ShlA is the hemolysin proper, and ShlB transports ShlA through the outer membrane, whereby ShlA is converted into a hemolysin. Superhemolytic ShlA derivatives that displayed 7- to 20-fold higher activities than wild-type ShlA were isolated. ShlA80 carried the single amino acid replacement of G to D at position 326 (G326D), ShlA87 carried S386N, and ShlA80III carried G326D and N236D. Superhemolysis was attributed to the greater stability of the mutant ShlA derivatives because they aggregated less than the wild-type hemolysin, which lost activity within 3 min at 20 degrees C. In contrast to the highly hemolytic wild-type ShlA at 0 degrees C, the hyperlytic hemolysins were nonhemolytic at 0 degrees C, suggesting that the hyperlytic derivatives differed from wild-type ShlA in adsorption to and insertion into the erythrocyte membrane. However, the size of the pores formed at 20 degrees C by superhemolytic hemolysins could not be distinguished from that of wild-type ShlA. In addition to the N-terminal sequence up to residue 238, previously identified to be important for activation and secretion, sites 326 and 386 contribute to hemolysin activity since they are contained in regions that participate in hemolysin inactivation through aggregation.

• Hardie KR, Schulze A, Parker MW, Buckley JT
• Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation.
• Mol Microbiol. 1995; 17: 1035-44
• Display abstract

• Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp. in separate steps. To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida, as well as Vibrio cholerae. Replacing Met-41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge. A double mutant was also made, in which Gly-202 and Ile-445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded. The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants. Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted. We observed in the cells, but they were not secreted. We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur. The proton ionophore CCCP reduced release of the folded proteins. Unoxidized protein was secreted by cells grown in beta-mercaptoethanol and by a dsbA mutant of V. cholerae, indicating that disulphide bond formation may not be essential for release.

• Parker MW et al.
• Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states.
• Nature. 1994; 367: 292-5
• Display abstract

• Aerolysin is chiefly responsible for the pathogenicity of Aeromonas hydrophila, a bacterium associated with diarrhoeal diseases and deep wound infections. Like many other microbial toxins, the protein changes in a multistep process from a completely water-soluble form to produce a transmembrane channel that destroys sensitive cells by breaking their permeability barriers. Here we describe the structure of proaerolysin determined by X-ray crystallography at 2.8 A resolution. The protoxin (M(r) 52,000) adopts a novel protein fold. Images of an aerolysin oligomer derived from electron microscopy have assisted in constructing a model of the membrane channel and have led to the proposal of a scheme to account for insertion of the protein into lipid bilayers to form ion channels.

• van der Goot FG, Hardie KR, Parker MW, Buckley JT
• The C-terminal peptide produced upon proteolytic activation of the cytolytic toxin aerolysin is not involved in channel formation.
• J Biol Chem. 1994; 269: 30496-501
• Display abstract

• The channel-forming toxin aerolysin is secreted by Aeromonas hydrophila as a protoxin that can be activated by nicking with endoproteinase Lys-C after Lys-427 near the C terminus of the protein. The fate of the 43-amino acid peptide distal to the activation site was investigated. A cysteine was introduced into the C-terminal region by replacing Ile-445, and another replaced Gly-202, which is on the proximal side of the activation site. In a double mutant, the two new cysteines were close enough in the folded molecule to form an intrachain 202-445 disulfide bond. Tryptophan fluorescence measurements on wild type and the 2 single cysteine mutants indicated that activation results in exposure of at least 1 tryptophan residue, leading to the conclusion that the peptide moves with respect to the protein when it is produced. This was supported by the observation that upon activation there was a decrease in energy transfer between a tryptophan in the bulk of the protein and a probe attached to Cys-445. The peptide could be separated from active toxin by several methods, indicating that it leaves the protein when it is produced, and that it plays no further role in the process of channel formation.

• Riddihough G
• Picture story. From protoxin to pore.
• Nat Struct Biol. 1994; 1: 80-80

• Perelle S, Gibert M, Boquet P, Popoff MR
• Characterization of Clostridium perfringens iota-toxin genes and expression in Escherichia coli.
• Infect Immun. 1993; 61: 5147-56
• Display abstract

• The iota toxin which is produced by Clostridium perfringens type E, is a binary toxin consisting of two independent polypeptides: Ia, which is an ADP-ribosyltransferase, and Ib, which is involved in the binding and internalization of the toxin into the cell. Two degenerate oligonucleotide probes deduced from partial amino acid sequence of each component of C. spiroforme toxin, which is closely related to the iota toxin, were used to clone three overlapping DNA fragments containing the iota-toxin genes from C. perfringens type E plasmid DNA. Two genes, in the same orientation, coding for Ia (387 amino acids) and Ib (875 amino acids) and separated by 243 noncoding nucleotides were identified. A predicted signal peptide was found for each component, and the secreted Ib displays two domains, the propeptide (172 amino acids) and the mature protein (664 amino acids). The Ia gene has been expressed in Escherichia coli and C. perfringens, under the control of its own promoter. The recombinant polypeptide obtained was recognized by Ia antibodies and ADP-ribosylated actin. The expression of the Ib gene was obtained in E. coli harboring a recombinant plasmid encompassing the putative promoter upstream of the Ia gene and the Ia and Ib genes. Two residues which have been found to be involved in the NAD+ binding site of diphtheria and pseudomonas toxins are conserved in the predicted Ia sequence (Glu-14 and Trp-19). The predicted amino acid Ib sequence shows 33.9% identity with and 54.4% similarity to the protective antigen of the anthrax toxin complex. In particular, the central region of Ib, which contains a predicted transmembrane segment (Leu-292 to Ser-308), presents 45% identity with the corresponding protective antigen sequence which is involved in the translocation of the toxin across the cell membrane.

• Hirono I, Aoki T
• Cloning and characterization of three hemolysin genes from Aeromonas salmonicida.
• Microb Pathog. 1993; 15: 269-82
• Display abstract

• Two hemolysin genes (ASH3 and ASH4) of Aeromonas salmonicida strain 17-2 and one (ASH1) of A. salmonicida ATCC14174 were cloned into the cosmid vector charomid 9-36 in Escherichia coli DH1. The overall amino acid sequence of the ASH3 was similar to that of the aerolysins of Aeromonas hydrophila and Aeromonas sobria, and hemolysins AHH3, AHH4, and AHH5 of A. hydrophila, and hemolysin ASA1 of A. sobria. The sequence of ASH4 was similar to that of the AHH1 hemolysin of A. hydrophila. The ASH4 hemolysin contains some homologous sequence regions of the Vibrio vulnificus and Vibrio cholerae cytolysin-hemolysin. Both ASH3 and ASH4 DNA probes reacted with all 104 strains of A. salmonicida, whereas the ASH1 DNA probe did not hybridize with any of the 104 strains studied except strain ATCC14174. ASH1 and ASH3 were broad spectrum hemolysins with most activity against rabbit and horse erythrocytes, respectively, whereas ASH4 hemolysin did not lyse bovine and horse erythrocytes. ASH3 and ASH4, but not ASH1, were activated by trypsin.

• Giladi H, Wang WX, Oppenheim AB
• Isolation and characterization of the hupA gene coding for HU of Aeromonas proteolytica.
• Nucleic Acids Res. 1992; 20: 4092-4092

• Sansom MS, Kerr ID, Mellor IR
• Ion channels formed by amphipathic helical peptides. A molecular modelling study.
• Eur Biophys J. 1991; 20: 229-40
• Display abstract

• Channel forming peptides (CFPs) are amphipathic peptides, of length ca. 20 residues, which adopt an alpha-helical conformation in the presence of lipid bilayers and form ion channels with electrophysiological properties comparable to those of ion channel proteins. We have modelled CFP channels as bundles of parallel trans-bilayer helices surrounding a central ion-permeable pore. Ion-channel interactions have been explored via accessible surface area calculations, and via evaluation of changes in van der Waals and electrostatic energies as a K+ ion is translated along the length of the pore. Two CFPs have been modelled: (a) zervamicin-A1-16, a synthetic apolar peptaibol related to alamethicin, and (b) delta-toxin from Staphylococcus aureus. Both of these CFPs have previously been shown to form ion channels in planar lipid bilayers, and have been shown to have predominantly helical conformations. Zervamicin-A1-16 channels were modelled as bundles of 4 to 8 parallel helices. Two related helix bundle geometries were explored. K(+)-channel interactions have been shown to involve exposed backbone carbonyl oxygen atoms. delta-Toxin channels were modelled as bundles of 6 parallel helices. Residues Q3, D11 and D18 generate favourable K(+)-channel interactions. Rotation of W15 about its C beta-C gamma bond has been shown to be capable of occluding the central pore, and is discussed as a possible model for sidechain conformational changes in relation to ion channel gating.

• Wong KR, McLean DM, Buckley JT
• Cloned aerolysin of Aeromonas hydrophila is exported by a wild-type marine Vibrio strain but remains periplasmic in pleiotropic export mutants.
• J Bacteriol. 1990; 172: 372-6
• Display abstract

• With a wide host range vector, the structural gene aerA for the hole-forming extracellular protein aerolysin of Aeromonas hydrophila was cloned into the marine Vibrio sp. strain 60 and into three pleiotropic export mutants (epr mutants). The parent strain and all of the mutants were able to express the protein with the aerA promoter in the plasmid. The parent strain exported proaerolysin into the medium, while all of the mutants accumulated the protoxin in their periplasms. Two of the mutants also accumulated protease; however, as we have found earlier with A. hydrophila, the periplasmic form of proaerolysin in the Vibrio sp. must somehow be protected from proteolysis because it was not converted to active toxin until the cells were shocked. Conversion could be prevented by adding o-phenanthroline to the solutions used in shocking. These results show that the export pathway in the marine Vibrio sp. is very similar to the pathway in A. hydrophila.

• Lai HC, Hu ST, Liu ST, Tsai WC
• [Clinical Aeromonas hydrophila contain the DNA fragments with nucleotide sequence similar to those of hemolysin and cholera-toxin gene].
• Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi. 1990; 23: 126-33
• Display abstract

• The artificially synthesized aquatic Aeromonas hydrophila partial aerolysin gene DNA (Ae, containing 48 oligonucleotides), and partial cholera-toxin gene DNA fragments (C1 and C2, containing 34 and 19 oligonucleotide, respectively) were used as probes to determine their DNA sequence homology to the chromosome DNA of 191 strains of the clinically isolated A. hydrophila, using the colony hybridization test. The three probes had a positive reaction with clinical A. hydrophila strains. Several positive strains of this organism were further screened by cutting reaction with restriction endonuclease BamHI and EcoRI, the alkaline Southern transfer, and DNA hybridization test. Some strains were found to be positive in reacting with Ae and C1, but negative with C2. The findings led to the conclusion that the DNA sequences were homologous among the clinical A. hydrophila toxin gene, cholera-toxin gene and aquatic A. hydrophila aerolysin gene.

• Rose JM, Houston CW, Coppenhaver DH, Dixon JD, Kurosky A
• Purification and chemical characterization of a cholera toxin-cross-reactive cytolytic enterotoxin produced by a human isolate of Aeromonas hydrophila.
• Infect Immun. 1989; 57: 1165-9
• Display abstract

• A bacterial protein toxin possessing hemolytic, enterotoxic, and cytotoxic activities as well as cross-reactivity to cholera toxin was purified from culture filtrates of a human diarrheal isolate of Aeromonas hydrophila (SSU). This cytolytic enterotoxin was purified by ammonium sulfate precipitation, hydrophobic chromatography using phenyl-Sepharose, anion-exchange chromatography on DEAE-Bio-Gel A, and size-exclusion high-performance liquid chromatography. The factor was a single polypeptide with an apparent molecular weight of 52,000 as determined by polyacrylamide gel electrophoresis. Automated amino acid sequence analysis confirmed that the toxin was a single chain and established a 25-residue N-terminal segment which was identical to that of aerolysin purified from culture supernatants of A. hydrophila isolate Ah65 originally obtained from rainbow trout as reported by Howard et al. (S. P. Howard, W. J. Garland, M. J. Green, and J. T. Buckley, J. Bacteriol. 169:2869-2871, 1987). However, the amino acid compositional analysis of the toxin produced by our human isolate (SSU) differed significantly from that of the Ah65 isolate. Taken together, these results strongly indicated that several toxic phenomena associated with A. hydrophila (SSU) culture filtrates, including hemolysis, cytotoxicity, and enterotoxicity as well as cross-reactivity to cholera toxin, all can occur on a single polypeptide. In addition, these results underline the fact that although aerolysin-related toxins isolated from culture filtrates of A. hydrophila are biologically similar, significant chemical and immunological differences may exist between toxins produced by individual isolates.

• Gu XR, Giroux S, Cedergren R
• The nucleotide sequence of the argT locus of Aeromonas hydrophila.
• Nucleic Acids Res. 1988; 16: 10936-10936

• Howard SP, Buckley JT
• Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila.
• Mol Gen Genet. 1986; 204: 289-95
• Display abstract

• The structural gene for the hemolytic toxin aerolysin has been cloned into the plasmid vectors pBR322 and pEMBL8+. The gene was localized on the hybrid plasmids by analysis of plasmids generated by transposon mutagenesis. The sequence of the first 683 bases of an insert in pEMBL8+ was determined and shown to encode the amino terminus of the protein as well as a typical signal sequence of 23 amino acids. Aerolysin is produced by E. coli cells containing the cloned aerolysin gene and it is processed normally by removal of the signal sequence, however it is not released from the cell. The protein appears to be translocated across the inner membrane of E. coli as its signal sequence is removed and the processed protein can be released by osmotic shock.

• Eisenberg D, Schwarz E, Komaromy M, Wall R
• Analysis of membrane and surface protein sequences with the hydrophobic moment plot.
• J Mol Biol. 1984; 179: 125-42
• Display abstract

• An algorithm has been developed which identifies alpha-helices involved in the interactions of membrane proteins with lipid bilayers and which distinguishes them from helices in soluble proteins. The membrane-associated helices are then classified with the aid of the hydrophobic moment plot, on which the hydrophobic moment of each helix is plotted as a function of its hydrophobicity. The magnitude of hydrophobic moment measures the amphiphilicity of the helix (and hence its tendency to seek a surface between hydrophobic and hydrophilic phases), and the hydrophobicity measures its affinity for the membrane interior. Segments of membrane proteins in alpha-helices tend to fall in one of three regions of a hydrophobic moment plot: (1) monomeric transmembrane anchors (class I HLA transmembrane sequences) lie in the region of highest hydrophobicity and smallest hydrophobic moment; (2) helices presumed to be paired (such as the transmembrane M segments of surface immunoglobulins) and helices which are bundled together in membranes (such as bacteriorhodopsin) fall in the adjacent region with higher hydrophobic moment and smaller hydrophobicity; and (3) helices from surface-seeking proteins (such as melittin) fall in the region with still higher hydrophobic moment. alpha-Helices from globular proteins mainly fall in a region of lower mean hydrophobicity and hydrophobic moment. Application of these methods to the sequence of diphtheria toxin suggests four transmembrane helices and a surface-seeking helix in fragment B, the moiety known to have transmembrane function.

• Pearson TW, Saya LE, Howard SP, Buckley JT
• The use of aerolysin toxin as an aid for visualization of low numbers of African trypanosomes in whole blood.
• Acta Trop. 1982; 39: 73-7
• Display abstract

• A method was developed for detection of low numbers of African trypanosomes in whole blood. The method is based on selective lysis of erythrocytes and leukocytes by aerolysin, a toxin produced by the bacterium Aeromonas hydrophila. African trypanosomes do not bind the toxin and their viability and motility are therefore unaffected by this treatment.

• Kyte J, Doolittle RF
• A simple method for displaying the hydropathic character of a protein.
• J Mol Biol. 1982; 157: 105-32

• Lee HM, Riordan JF
• Does carboxypeptidase Y have intrinsic endopeptidase activity?
• Biochem Biophys Res Commun. 1978; 85: 1135-42

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