4-nitrophenylphosphorylcholine and Hemolysis

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.

Topics: Amino Acid Substitution; Aspartic Acid; Bacterial Toxins; Calcium-Binding Proteins; Clostridium perfringens; DNA, Bacterial; Egg Yolk; Glutamic Acid; Glycine; Hemolysis; Lysine; Models, Molecular; Mutagenesis, Site-Directed; Phospholipids; Phosphorylcholine; Protein Conformation; Serine; Substrate Specificity; Type C Phospholipases

To examine one possible mechanism of damage to leucocytes and tissue cells in legionellosis, seven species of Legionella were examined for cytolytic activity and for elaboration of phospholipase C, an enzyme that can damage mammalian cell membranes. Cytolysis was assessed using erythrocytes in agar. Phospholipase C was assayed by release of p-nitrophenol from p-nitrophenylphosphorylcholine and of tritiated phosphorylcholine from L-alpha-dipalmitoyl-[choline-methyl-3H]phosphatidylcholine. L. pneumophila, L. bozemanii, L. micdadei, L. dumoffii, L. gormanii, L. longbeachae and L. jordanis all lysed dog red blood cells, which have a high ratio of membrane phosphatidylcholine to sphingomyelin. The same strains hydrolysed varying amounts of p-nitrophenylphosphorylcholine; L. bozemanii exhibited the greatest activity. L. pneumophila, L. bozemanii, L. dumoffii, L. longbeachae and L. jordanis, but not L. micdadei, released tritiated phosphorylcholine from labelled substrate. These results indicate that several species of Legionella possess cytolytic capability; exotoxins with activity may play a role.

Topics: Animals; Cytotoxins; Dogs; Egg Yolk; Erythrocytes; Guinea Pigs; Hemolysis; Humans; Legionella; Phosphatidylcholines; Phosphorylcholine; Rabbits; Sheep; Type C Phospholipases