Site-directed mutagenesis of the hole-forming toxin aerolysin: studies on the roles of histidines in receptor binding and oligomerization of the monomer

Green, Melissa; Jt, Buckley

doi:10.1021/bi00460a031

Cited by 52 publications

(47 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For one of these domains, the receptor-binding domain, previous work carried out on aerolysin (10,(13)(14)(15) as well as experiments conducted in this study provided evidence to its probable location in D1. Based on this knowledge, scanning was initiated in this region and alanine was chosen for substitution due to its minimal side chain.…”

Section: Mutagenesis Strategysupporting

confidence: 62%

Identification of Functional Domains of Clostridium septicum Alpha Toxin

2006

View full text Add to dashboard Cite

Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin belonging to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule based on functional and sequence similarity with aerolysin, for which the crystal structure has been solved. In the present study we have substituted the entire primary structure of AT with alanine or cysteine in order to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain, but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane β-hairpin of the prepore oligomer.Clostridium septicum is a major cause of non-traumatic gas gangrene in humans, a fulminant form of myonecrosis that can progress to a fatal infection in less than 24 hours with reported mortality rates ranging from 67-100% (1-4). Of its many virulence factors, alpha toxin (AT) is the only lethal factor secreted by this organism (5) and it has recently been shown to be absolutely required for virulence of C. septicum (6). AT is classified as a pore-forming toxin and is a member of the aerolysin-like family of pore-forming toxins. AT and aerolysin share a great deal of structural and sequence similarity (72%) (5), which has allowed for the development of a molecular model of AT using the previously solved crystal structure of aerolysin ( Fig. 1) (7,8). Aerolysin is a two lobed protein in which the small lobe is comprised of domain 1 (D1) and the large lobe is comprised of domains 2-4 (D2-D4, Fig. 1). The primary structure of AT exhibits similarity with the large lobe of aerolysin but it lacks the small lobe structure of about 83 amino acids. Both toxins appear to follow a similar ordered path to form pores in cell membranes (5). Following secretion, they bind to receptors on the cell membrane where they are cleaved into their active form by cell surface proteases, usually furin. Once activated, the toxins oligomerize on the cell surface into a prepore complex followed by insertion of a transmembrane β-barrel into the membrane (8).

show abstract

Section: Mutagenesis Strategysupporting

confidence: 62%

Identification of Functional Domains of Clostridium septicum Alpha Toxin

2006

View full text Add to dashboard Cite

show abstract

“…For aerolysin (35,36) from Aeromonas hydrophyla and for the ␣-toxin (37) of Staphylococcus aureus, it has been reported that histidine modification with DEPC or side-directed mutagenesis blocks aggregation and pore formation. The crystal structure of the ␣-toxin revealed that a histidine is involved in oligomerization by promoting electrostatic interactions between two monomers (38).…”

Section: Resultsmentioning

confidence: 99%

Solution Structure of the Pore-forming Protein of Entamoeba histolytica

Hecht

Nuland

Schleinkofer

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Amoebapore A is a 77-residue protein from the protozoan parasite and human pathogen Entamoeba histolytica. Amoebapores lyse both bacteria and eukaryotic cells by pore formation and play a pivotal role in the destruction of host tissues during amoebiasis, one of the most life-threatening parasitic diseases. Amoebapore A belongs to the superfamily of saposin-like proteins that are characterized by a conserved disulfide bond pattern and a fold consisting of five helices. Membrane-permeabilizing effector molecules of mammalian lymphocytes such as porcine NK-lysin and the human granulysin share these structural attributes. Several mechanisms have been proposed to explain how saposin-like proteins form membrane pores. All mechanisms indicate that the surface charge distribution of these proteins is the basis of their membrane binding capacity and pore formation. Here, we have solved the structure of amoebapore A by NMR spectroscopy. We demonstrate that the specific activation step of amoebapore A depends on a pH-dependent dimerization event and is modulated by a surface-exposed histidine residue. Thus, histidine-mediated dimerization is the molecular switch for pore formation and reveals a novel activation mechanism of pore-forming toxins.The protozoan parasite, Entamoeba histolytica, inhabits the colon of infected humans. It is the causative agent of human amoebiasis that often leads to tissue damage, colitis, and extraintestinal abscesses (1). Amoebiasis is the second leading cause of death from parasitic diseases worldwide (2). About 50 million people suffer from invasive amoebiasis, of whom up to 100,000 die annually (3).In the amoebic trophozoite, several factors have been identified that are involved in pathogenesis. In addition to a galactose-/N-acetylgalactosamine-specific lectin on the amoebic surface that mediates adhesion to colonic mucus and host cells (4) and secreted cysteine proteinases that disintegrate tissues by cleaving extracellular matrix proteins (5), a family of membrane-active polypeptides have been discovered (6). These polypeptides exist as three isoforms and are named amoebapore A, B, and C, respectively. They are capable of lysing a broad spectrum of target cells, including human host cells and bacteria. It has been recently shown that trophozoites of E. histolytica lacking amoebapore A, due to transcriptional silencing of the encoding gene, became avirulent (7), demonstrating that this protein is a key pathogenicity factor of the parasite. All three amoebapore isoforms have been isolated and biochemically characterized, and their primary structure has been elucidated by molecular cloning of the genes of their precursors (8 -10). The mature proteins consist of 77 amino acid residues each and are localized within cytoplasmic granules. The overall sequence identity between the three amoebapores is between 35 and 57% (10). Despite the substantial sequence divergence, they possess a characteristic disulfide bond pattern and a single conserved C-terminal histidine residue. The secondary structur...

show abstract

“…H o w e v e r , in c o n t r a s t to a -t o x i n a n d a e r o l y s i n (whose oligomers are stable in SDS [12,17,44], E q T -I I failed to s h o w stable a g g r e g a t e s w h e n p e r m eabilized vesicles w e r e d e t e r g e n t - large oligomers increased by prolonging the reaction with DMS (Fig. 5), but did not change increasing the toxin-SUV incubation time (not shown).…”

Section: Resultsmentioning

confidence: 99%

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

et al. 1993

View full text Add to dashboard Cite

The interaction of Actinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.

show abstract

Site-directed mutagenesis of the hole-forming toxin aerolysin: studies on the roles of histidines in receptor binding and oligomerization of the monomer

Cited by 52 publications

References 15 publications

Identification of Functional Domains of Clostridium septicum Alpha Toxin

Identification of Functional Domains of Clostridium septicum Alpha Toxin

Solution Structure of the Pore-forming Protein of Entamoeba histolytica

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Contact Info

Product

Resources

About