The Role of Penicillin-Binding Proteins in the Action of Cephalosporins against Escherichia coli and Salmonella typhimurium

The binding of two radioactively-labelled cephalosporins, LY97962 and LY121998, to the penicillin-binding proteins of Escherichia coli and Salmonella typhimurium has been studied in detail. The concentrations of the cephalosporins that resulted in 50% saturation of the penicillin-binding proteins were measured in growing cells as well as in preparations of disrupted cell envelopes. This technique has allowed the crypticity of an organism towards a particular β-lactam to be measured directly, i.e. the extent to which a penicillin-binding protein is accessible in intact cells. The cephalosporin LY97962 appeared to have no difficulty in penetrating the outer layers of the cell whereas access of cephalosporin LY121998 to the penicillin-binding proteins appeared to be somewhat restricted. At concentrations below the minimum growth inhibitory concentration the two cephalosporins inhibited cell division and caused the cells to grow as long filaments. When growing cells were incubated with the radioactive β-lactam antibiotics at these concentrations, the β-lactams bound almost exclusively to penicillin-binding protein 3. The degree of binding of the cephalosporins to protein 1 A in cells growing in the presence of the minimum growth inhibitory concentrations of antibiotics varied with the duration of the incubation. After exposure for 5 min, very little binding to protein 1 A was apparent, whereas substantial binding was observed after 60–180 min. It was only at the end of this period of growth in the presence of the cephalosporins that lysis of the cells became apparent. This provided evidence that the binding of β-lactams to protein 1 A of gramnegative organisms is an important step in the bactericidal action of certain cephalosporins. Protein 1 B was excluded as a target for the lethal action of these cephalosporins because binding to this protein was observed only at concentrations greater than those needed to inhibit growth of the cells. Microscopical examination of the growth of S. typhimurium in the presence of cefotaxime at sub-lytic concentrations showed that cells grew as filaments for at least six generations without lysis. Observations of the division of the filament into individual cells after removal of the antibiotic showed that the entire length of the filament was viable. It is concluded that inhibition of cell division as a result of the binding of β-lactams to protein 3 of gram-negative organisms is not in itself a lethal event for the cells, but requires concomitant binding of antibiotic to protein 1 A before lysis is initiated.