RESUMO
A microplate, scintillation proximity assay to measure the coupled transglycosylase-transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[(3)H]N-acetylglucosamine in the presence of 40 µM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-D-ala-D-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC50 of 8 nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a 'neutral' combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.
Assuntos
Escherichia coli/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Penicilinas/administração & dosagem , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano/biossíntese , Peptidil Transferases/metabolismo , Vancomicina/administração & dosagem , Bioensaio/instrumentação , Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Interações Medicamentosas , Ativação Enzimática , Desenho de Equipamento , Escherichia coli/efeitos dos fármacos , Miniaturização , Peptídeos/administração & dosagem , Mapeamento de Interação de Proteínas/instrumentaçãoRESUMO
The combination of antibiotics is one of the strategies to combat drug-resistant bacteria, though only a handful of such combinations are in use, such as the ß-lactam combinations. In the present study, the efficacy of a specific sub-inhibitory concentration of cefsulodin with other ß-lactams was evaluated against a range of Gram-negative clinical isolates. This approach increased the sensitivity of the isolates, regardless of the ß-lactamase production. The preferred target and mechanism of action of cefsulodin were identified in laboratory strains of Escherichia coli, by examining the effects of deleting the penicillin-binding protein (PBP) 1a and 1b encoding genes individually. Deletion of PBP1b was involved in sensitizing the bacteria to ß-lactam agents, irrespective of its O-antigen status. Moreover, the use of a sub-inhibitory concentration of cefsulodin in combination with a ß-lactam exerted an effect similar to that one obtained for PBP1b gene deletion. We conclude that the identified ß-lactam/cefsulodin combination works by inhibiting PBP1b (at least partially) despite the involvement of ß-lactamases, and therefore could be extended to a broad range of Gram-negative pathogens.