Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor

Goldberg, S L; Murphy, John R.

doi:10.1128/jb.160.1.239-244.1984

Cited by 42 publications

(33 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These include the cholera enterctoxin (CT) (Finkelstein, 1969;1972;Pierce ef ai. 1971), a neuraminidase (Vimr ef ai, 1988), proteases (Schneider and Parker, 1978), DNases (Newland ef ai, 1985;Focareta and Manning, 1987) and haemolysins (Manning et ai, 1984;Goldberg and Murphy, 1984;Richardson efa/., 1986).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the hlyB gene and its role in the production of the EI Tor haemolysin of Vibrio choierae O1

Alm

Manning

1990

Molecular Microbiology

View full text Add to dashboard Cite

El Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood. In addition to the structural gene for the haemolysin (hlyA), we have cloned a second adjacent gene, hlyB. By site-directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid-exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane-associated protein.

show abstract

Section: Introductionmentioning

confidence: 99%

“…which appeared to affect production of the haemoiysin. The absence cf this gene in the clone of Goldberg and Murphy (1984) probably accounts for the long incubation times required to detect activity in E. coli K12.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the hlyB gene and its role in the production of the EI Tor haemolysin of Vibrio choierae O1

Alm

Manning

1990

Molecular Microbiology

View full text Add to dashboard Cite

show abstract

“…These observations suggested: (a) partitioning of the HlyA to lipid vesicles is driven by the tendency of the amphiphilic toxin to reduce energetically unfavorable contacts with water and is not affected significantly by the composition of the vesicles; and (b) partial unfolding of the HlyA at the lipidwater interface precedes and promotes cholesterol-induced oligomerization to an insertion-competent configuration.Vibrio cholerae hemolysin (HlyA), a water-soluble cytolytic protein expressed by many V. cholerae El Tor O1 and non-O1 strains [1,2], belongs to a large, heterologous family of pore-forming toxins (PFT) [3,4] that are ubiquitous in prokaryotic and eukaryotic organisms. The toxin has been cloned and sequenced [5,6]. The HlyA permeabilizes a wide spectrum of eukaryotic cells including human and rabbit erythrocytes [2] and synthetic lipid vesicles [7,8] by forming transmembrane pentameric [9] diffusion channels with a diameter of approximately 1.5 nm.…”

mentioning

confidence: 99%

Vibrio cholerae hemolysin

Chattopadhyay

Bhattacharyya

Banerjee

2002

European Journal of Biochemistry

View full text Add to dashboard Cite

Vibrio cholerae hemolysin (HlyA), a water-soluble protein with a native monomeric relative molecular mass of 65 000, forms transmembrane pentameric channels in target biomembranes. The HlyA binds to lipid vesicles nonspecifically and without saturation; however, self-assembly is triggered specifically by cholesterol. Here we show that the HlyA partitioned quantitatively to amphiphilic media irrespective of their compositions, indicating that the toxin had an amphiphilic surface. Asialofetuin, a b1-galactosyl-terminated glycoprotein, which binds specifically to the HlyA in a lectin-glycoprotein type of interaction and inhibits carbohydrate-independent interaction of the toxin with lipid, reduced effective amphiphilicity of the toxin significantly. Resistance of the HlyA to proteases together with the tryptophan fluorescence emission spectrum suggested a compact structure for the toxin. Fluorescence energy transfer from the HlyA to dansyl-phosphatidylethanolamine required the presence of cholesterol in the lipid bilayer and was synchronous with oligomerization. Phospholipid bilayer without cholesterol caused a partial unfolding of the HlyA monomer as indicated by the transfer of tryptophan residues from the nonpolar core of the protein to a more polar region. These observations suggested: (a) partitioning of the HlyA to lipid vesicles is driven by the tendency of the amphiphilic toxin to reduce energetically unfavorable contacts with water and is not affected significantly by the composition of the vesicles; and (b) partial unfolding of the HlyA at the lipidwater interface precedes and promotes cholesterol-induced oligomerization to an insertion-competent configuration.Keywords: pore-forming toxin; amphiphilicity; lipidinduced conformational change; oligomerization; protein fluorescence.Vibrio cholerae hemolysin (HlyA), a water-soluble cytolytic protein expressed by many V. cholerae El Tor O1 and non-O1 strains [1,2], belongs to a large, heterologous family of pore-forming toxins (PFT) [3,4] that are ubiquitous in prokaryotic and eukaryotic organisms. The toxin has been cloned and sequenced [5,6]. The HlyA permeabilizes a wide spectrum of eukaryotic cells including human and rabbit erythrocytes [2] and synthetic lipid vesicles [7,8] by forming transmembrane pentameric [9] diffusion channels with a diameter of approximately 1.5 nm. In addition to binding specifically to cholesterol [9], the toxin shows a lectin-like property in interacting with b1-galactosyl-terminated glycoconjugates such as asialofetuin and asialothyroglobulin [10]. The purified toxin evokes secretion of fluid in a rabbit ligated ileal loop, suggesting its involvement in pathogenesis of cholera [11].A consensus on the pathway of induction of membrane damage by PFTs postulates a sequence of at least three discrete biochemical events: binding of the toxin monomer to a cell surface receptor, self-assembly to an amphiphilic prepore oligomer and insertion in the lipid bilayer generating a functional pore that mediates passive flux of molecules across ...

show abstract

“…The structural gene hlyA of hemolysin (HlyA) from V cholerue 01 biotype El Tor has been cloned and sequenced independently by two different laboratories [12,131. Its translation product is a precursor protein with a molecular mass of 81 977 Da.…”

mentioning

confidence: 99%

HlyA Hemolysin of Vibrio Cholerae O1 Biotype El Tor

Menzl

Maier

Chakraborty

et al. 1996

European Journal of Biochemistry

View full text Add to dashboard Cite

Hemolysin (HlyA) was concentrated from supernatants of different Vibrio cholerue 01 biotype El Tor strains by ammonium sulfate precipitation. The concentration of the toxin in the supernatants and in the precipitates was quantified using its hemolytic activity. The toxin formed a high molecular-mass band (about 220 kDa) on SDS/PAGE while the toxin monomer had a molecular mass of 60 kDa when it was heated. The addition of the El Tor hemolysin oligomers, but not that of the monomers, to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels, which had long lifetimes at small voltages. The hemolysin channel had a single-channel conductance of 350 pS in 1 M KC1. These results defined hemolysin (HlyA) from V choleme as a channel-forming component with properties similar to other cytolytic toxins. The long lifetime of the channel suggested that the channel-forming oligomer did not show a rapid association/dissociation reaction. At voltages larger than 50 mV, the hemolysin channel was voltage dependent in an asymmetric fashion dependent on the side of its addition. The single-channel conductance of the hemolysin (HlyA) from V cholerue 0 1 biotype El Tor channel was a linear function of the bulk aqueous conductance, which suggested that the toxin forms aqueous channels with an estimated minimum diameter of about 0.7 nm. The hemolysin channel of V cholerue was found to be moderately anion-selective. The pore-forming properties of hemolysin (HlyA) from V cholerue 0 1 biotype El Tor were compared with those of aerolysin of Aeromonus sobriu and atoxin from Staphylococcus uureus. All these cytolytic toxins must probably oligomerize for activity in biological and artificial membranes and form anion-selective channels.Keywords: hemolysin; ion channel; toxin oligomer; lipid bilayer membrane; Vibrio choleru biotype El Tor.The Gram-negative Vibrio cholerue is an intestinal pathogen that is non-invasive and adheres to the apical side of the small intestine [I, 21. The epidemic qualities of this pathogen have recently been dramatically underscore by the reappearance of cholera in the Western Hemisphere after more than 100 years and the emergence of a new strain, the V cholerae 0 -1 39 Bengal strain, that appeared in India and spread rapidly to countries surrounding the Indian Ocean [3]. Virulence factors elaborated by Y cholerae include cholera toxin, colonization factor, toxinco-regulated pilus, zona occuledens toxin and the regulatory elements ToxR, ToxS and ToxT [2].To establish an infection, V cholerue colonizes the intestinal surface and releases cholera toxin composed of two subunits [4, 51. The binding component ( 5 B subunits) binds to the surface and facilitates the entry of the A subunit into the epithelial cells. The A subunit effects an increase of the intracellular level of cyclic AMP. The high level of intracellular AMP stimulates the secretion of chloride and bicarbonate out of the cell and leads to diarrhoea because of the severe loss of isotonic body fluid [I, 21. Chol...

show abstract

Molecular cloning of the hemolysin determinant from Vibrio cholerae El Tor

Cited by 42 publications

References 27 publications

Characterization of the hlyB gene and its role in the production of the EI Tor haemolysin of Vibrio choierae O1

Characterization of the hlyB gene and its role in the production of the EI Tor haemolysin of Vibrio choierae O1

Vibrio cholerae hemolysin

HlyA Hemolysin of Vibrio Cholerae O1 Biotype El Tor

Contact Info

Product

Resources

About