Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics

Abstract

A biochemical method was developed to quantitatively compare the effectiveness of beta-lactams in triggering murein degradation (autolysin activity) in Escherichia coli. Bacteria prelabeled in their cell walls with radioactive diaminopimelic acid in growth medium were exposed for 10 min to the antibiotics at the appropriate minimal growth inhibitory concentrations and at multiples of these values, and the rate of cell wall degradation was followed during subsequent incubation of the cells in a buffer solution. Beta-lactams with high affinity for the penicillin-binding protein (PBP)-1 were the most effective triggers of autolytic wail degradation; beta-lactams selective for PBP-2 were the poorest; and antibiotics preferentially binding to PBP-3 showed intermediate activities. The relative effectiveness of beta-lactams in autolysin triggering was found to parallel the effectiveness of the same drugs in causing rapid loss of viability, culture lysis, and spheroplast formation. Autolysin triggering was suppressed by inhibitors of protein and ribonucleic acid biosynthesis but not by inhibitors of deoxyribonucleic acid synthesis. The beta-lactam-induced cell wall degradation did not seem to involve a direct stimulation of enzyme activity or synthesis of new enzyme molecules, and murein sacculi isolated from cells that had been preexposed to a triggering dose of beta-lactam treatment exhibited the same sensitivity to crude, homologous autolysins as sacculi prepared from untreated control bacteria. On the basis of these observations, mechanisms are considered for the triggering of E. coli autolysins and for the role of autolytic activity in bacterial spheroplast formation, lysis, and death.

Extracellular proteases increase tolerance of Bacillus subtilis to nafcillin

“…These observations led Tomasz and Waks to suggest that the autolysin activity may be the cause of cell death as well as lysis in pneumococci (24). Similar claims have been made for the effects of penicillin on E. coli (11,12). To characterize the relationship of autolytic enzymes to penicillin-induced killing of B. subtilis, viability loss in the presence of nafcillin for strain QB136 (a protease hyperproducer) and strain SR22 (protease-deficient) was compared ( Fig.…”

The effect of antibiotics on the cell morphology of Legionella pneumophila

“…The effect of methicillin on L. pneumophila can also be compared with that of cephalothin on gram-negative bacteria, in that spheroplast-like formation occurs with high frequency at drug levels near the MIC, but less frequently as drug concentrations are increased (Nishino and Nakazawa, 1976). Methicillin could exert an influence upon a unique penicillin-binding protein in a manner analogous to that of 1-lactam antibiotics binding to the penicillin-binding protein-1 of Escherichia coli (Kitano and Tomasz, 1979), which effect is known to be associated with Fig. 9.…”

Morphological and ultrastructural changes in bacterial cells as an indicator of antibacterial mechanism of action

“…In cephaloridine-treated E. coli, spheroplast formation is observable between 1xMIC and 16xMIC, with 1xMIC cephaloridine converting over 20% of cells to spheroplasts in just 60 minutes [77]. For β-lactams which preferentially target E. coli PBP2 and/or PBP3, other morphological changes ('ovoid cells', 'localized swelling' and 'filaments') occur at low antibiotic concentrations, with spheroplasts only becoming evident when antibiotic concentration is increased to a point where PBPs 1a and 1b become bound [76,78].…”

“…In Escherichia coli, β-lactam-induced spheroplast formation is due to inhibition of penicillin binding proteins 1a and 1b (PBPs 1a and 1b) [75][76][77]. PBPs 1a and 1b are required for peptidoglycan synthesis, and inhibition of these enzymes, in the absence of peptidoglycan hydrolase inhibition, is thought to explain spheroplast formation [75].…”