Sophie Lepage

The Penicillin sensory transducer, blar, involved in the inducibility of beta-lactamase synthesis in bacillus licheniformis is embedded in the plasma membrane via a four-alpha-helix bundle

Prediction studies, conformational analyses and membrane‐topology mapping lead to the conclusion that the penicillin sensory transducer, BlaR, involved in the inducibility of β‐lactamase synthesis in Bacillus licheniformis, is embedded in the plasma membrane bilayer via four transmembrane segments TM1–TM4 that form a four‐α‐helix bundle. The extracellular 262‐amino‐acid‐residue polypeptide, S340–R601, that is fused at the carboxy end of TM4, possesses the amino acid sequence signature of a penicilloyl serine transferase. It probably functions as penicillin sensor. As an independent entity, this polypeptide behaves as a high‐affinity penicillin‐binding protein. As a component of the full‐size BlaR, it adopts a different conformation presumably because of interactions with the extracellular 63‐amino‐acid‐residue P53–S115 loop that connects TM2 and TM3. Reception of the penicillin‐induced signal requires a precise conformation of the sensor but it does not involve penicilloylation of the serine residue S402 of motif STYK. Signal transmission through the plasma membrane by the four‐α‐helix bundle may proceed in a way comparable to that of the aspartate receptor, Tar. Signal emission in the cytosol by the intracellular 189‐amino‐acid‐residue Y134–K322 loop that connects TM3 and TM4, may proceed via the activation of a putative metallopeptidase.

When Drug Inactivation Renders the Target Irrelevant to Antibiotic Resistance: A Case Story with Beta-Lactams

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a β‐lactamase‐overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor β‐lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents.

Saturation of Penicillin-Binding Protein 1 by Beta-Lactam Antibiotics in Growing Cells of Bacillus Licheniformis

With the help of a new highly sensitive method allowing the quantification of free penicillin‐binding proteins (PBPs) and of an integrated mathematical model, the progressive saturation of PBP1 by various β‐lactam antibiotics in growing cells of Bacillus licheniformis was studied. Although the results confirmed PBP1 as a major lethal target for these compounds, they also underlined several weaknesses in our present understanding of this phenomenon. In growing cells, but not in resting cells, the penicillin target(s) appeared to be somewhat protected from the action of the inactivators. In vitro experiments indicated that amino acids, peptides and depsipeptides mimicking the peptide moiety of the nascent peptidoglycan significantly interfered with the acylation of PBP1 by the antibiotics. In addition, the level of PBP1 saturation at antibiotic concentrations corresponding to the minimum inhibitory concentrations was not constant, suggesting that additional, presently undiscovered, factors might be necessary to account for the experimental observations.