The role of the amino terminus in the kinetics and assembly of alpha-hemolysin of Staphylococcus aureus.
The nature of the involvement of an intact NH2 terminus in the assembly of alpha-hemolysin of Staphylococcus aureus was reinvestigated. For the first time, a deletion of the first four amino acids at the NH2 terminus of alpha-hemolysin yielded a novel mutant that undergoes all of the conformational changes to form a lytic pore. The experimental evidence shows unequivocally that the mutant toxin forms heat- and sodium dodecyl sulfate-stable heptameric oligomers. The concentration required to achieve 50% lysis of red blood cells is around 58-116 ng/ml, and the time taken to achieve lysis to the same extent as that of intact toxin is considerably longer. Transmission electron microscopic studies also suggest that the pores formed by this deletion mutant are similar to those by the full-length toxin. This is in contrast to the previously reported 2- and 11-amino acid deletions that failed to proceed further from a presumed prefinal nonlytic pore to a lytic pore. Studies on the kinetics of assembly indicate that this mutant can form heat- and sodium dodecyl sulfate-stable oligomers as fast as full-length alpha-hemolysin but that pore opening is slowed down. The data strongly suggest that these amino acids (Ala-Asp-Ser-Asp) are involved in the final stages of assembly of alpha-hemolysin in target membranes.
Vandana
S
S
Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India.
Raje
M
M
Krishnasastry
M V
MV
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry* / Q000633:toxicity; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli; D005098:Exotoxins / Q000096:biosynthesis / Q000737:chemistry; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000633:toxicity; D006461:Hemolysis; D007700:Kinetics; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000633:toxicity; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D017931:DNA Primers; D004269:DNA, Bacterial; D005098:Exotoxins; D006460:Hemolysin Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C021091:staphylococcal alpha-toxin