Norris E. Allen

Daptomycin disrupts membrane potential in growing Staphylococcus aureus.

Daptomycin (LY146032) caused a calcium-dependent dissipation of the membrane potential (delta psi) in Staphylococcus aureus without noticeably affecting the chemical gradient (delta pH) across the membrane. The effect of daptomycin on membrane energization may account for many of the inhibitory effects on macromolecular biosyntheses and membrane function reported for this antibiotic. Our evidence indicates that the bactericidal activity of daptomycin is dependent on an available delta psi.

Inhibition of peptidoglycan biosynthesis in gram-positive bacteria by LY146032.

LY146032, a cyclic lipopeptide antibiotic, is an inhibitor of cell wall peptidoglycan biosynthesis in gram-positive bacteria. Although LY146032 at relatively high concentrations inhibited the in vitro polymerization of UDP-linked sugar precursors, inhibition of cell wall formation in intact Staphylococcus aureus and Bacillus megaterium cells did not lead to the accumulation of UDP-N-acetyl-muramyl (MurNAc)-peptide(s). Experiments that measured formation of UDP-MurNAc-peptides revealed that LY146032 inhibited the formation of these nucleotide-linked intermediates. This antibiotic had a disruptive effect on membrane permeability as evidenced by the loss of intracellular potassium immediately after exposure to the drug. The lack of any major disruption of the phosphoenolpyruvate:sugar phosphotransferase system indicated that the membrane is not likely a lethal target for this antibiotic. The findings are consistent with a mechanism by which LY146032 inhibits the formation of precursor molecules utilized in peptidoglycan biosynthesis. The observed membrane effects likely result from transit of the inhibitor to its lethal target site.

Inhibition of membrane potential-dependent amino acid transport by daptomycin.

Daptomycin inhibits the formation of UDP-N-acetylmuramyl-pentapeptide in Bacillus megaterium by inhibiting active transport of amino acids incorporated into the pentapeptide. The ability of daptomycin to inhibit active transport and peptidoglycan formation may be due to its ability to disrupt the transmembrane electrochemical gradient.

Characterization of vancomycin resistance in Enterococcus faecium and Enterococcus faecalis.

Vancomycin resistance in Enterococcus faecium 180, a clinical isolate from England, was studied. Resistance to vancomycin was transferable by conjugation to other enterococci. Expression of resistance was inducible and coincided with the appearance of a new membrane protein. Images

Genetic and enzymatic basis of hygromycin B resistance in Escherichia coli.

A plasmid conferring resistance to the aminocyclitol antibiotic hygromycin B was isolated from Escherichia coli. The gene conferring resistance to this drug was cloned in pBR322, and the gene was localized to a fragment of ca. 1,510 base pairs. Resistance to hygromycin B is determined by an aminocyclitol phosphotransferase that modifies hygromycin B and structurally related antibiotics. The specific modification of hygromycin B is a phosphorylation of the hydroxyl on the 4 position of the cyclitol ring (hyosamine). The presence of the phosphotransferase in E. coli correlates with reduced accumulation of [14C]hygromycin B. Images

Macrolide Resistance in Staphylococcus aureus: Inducers of Macrolide Resistance

Several macrolide-, lincosamide-, and streptogramin B-type (MLS) antibiotics were tested as inducers of erythromycin A (EM)-resistant [14C]leucine incorporation. Only 14-membered-ring macrolides having a glycosidically linked 6-deoxy sugar at the C-3 position of the lactone ring and the structurally dissimilar lincosamide, celesticetin, showed inducer activity. Modifications of EM at the C-4″ position of cladinose can apparently destroy the inducer property but do not affect the inhibitory properties of the antibiotic. The findings clearly show that inducer and inhibitor activities can be dissociated and are consistent with the concept that distinct binding/receptor sites are utilized for inhibition of ribosome function and induction of resistance.

Molecular modeling of γ-lactam analogues of β-lactam antibacterial agents: synthesis and biological evaluation of selected penem and carbapenem analoques

Abstract Computational chemistry made possible the prediction of the three-dimensional structures of γ-lactam analogues of penems and carbapenems before the analogues were made. Molecular superpositioning showed that these novel structures with a 7β-acylamino side-chain present the pharmacophoric groups in close spatial similarity to the groups in biologically active cephalosporin and penicillin antibiotics. This suggests that 8-oxo-7-acylamino-1-azabicyclo[3.3.0]-oct-2-ene-2-carboxylates and the 4-thia-analogues can be accommodated in the same active sites of essential bacterial penicillin-binding proteins where cephalosporins and penicillins are recognized. The syntheses of these compounds are reported. The γ-lactams exhibit low, but detectable levels of antibacterial activity and suggest promise that substantial activity can be achieved with other γ-lactams.

Hexapeptide Derivatives of Glycopeptide Antibiotics: Tools for Mechanism of Action Studies

ABSTRACT Hexapeptide (des-N-methylleucyl) derivatives of LY264826 were prepared in order to examine further the role of N-substituted hydrophobic side chains in defining the mechanisms of action of semisynthetic glycopeptide antibiotics. The hexapeptide of LY264826 binds to the cell wall intermediate analog l-Lys-d-Ala-d-Ala with a 100-fold lower affinity than LY264826 and inhibits Micrococcus luteus almost 200-fold more poorly than LY264826 does. Alkylation of the 4-epi-vancosamine moiety of the disaccharide significantly enhanced the antibacterial activity of the hexapeptide. Alkylation did not affect the binding affinity for d-alanyl-d-alanine residues; however, it did enhance dimerization 7,000-fold and enhanced binding to bacterial membrane vesicles noticeably compared with the levels of dimerization and binding for the unsubstituted hexapeptide. The findings from this study complement those presented in an earlier report (N. E. Allen, D. L. LeTourneau, and J. N. Hobbs, Jr., J. Antibiot. 50:677-684, 1997) and are consistent with the conclusion that the enhanced antibacterial activities of semisynthetic glycopeptide antibiotics derive from the ability of the hydrophobic side chain to markedly affect both dimerization and binding to bacterial membranes.

Mechanism of Penicillin-Erythromycin Synergy on Antibiotic-Resistant Staphylococcus aureus

Clinically isolated strains of Staphylococcus aureus that are inducibly resistant to both erythromycin and penicillin were susceptible to a combination of the two antibiotics. The synergistic effect of the combination results from an inhibition of penicillinase induction by erythromycin, sparing penicillin and allowing this drug to inhibit growth. When resistance to erythromycin is constitutive rather than inducible, the combination is no longer synergistic.

Macrolide Resistance in Staphylococcus aureus: Induction of Macrolide-Resistant Protein Synthesis

Induction of resistance to macrolide-, lincosamide-, and streptogramin B-type antibiotics in Staphylococcus aureus was studied by monitoring the appearance of erythromycin A (EM)-resistant [14C]leucine incorporation. Examination of the induction process revealed saturation kinetics and a time course much like that reported for penicillinase in gram-positive bacteria. Induction kinetics in exponentially growing cells were sigmoidal and appeared to reach a maximum and constant rate when growth reached stationary phase. Since the induction of EM-resistant colony-forming ability was complete within 60 min, ribosome modification cannot be limited to a fraction of the population and must occur in essentially every cell. However, EM-resistant growth was expressed in cells where less than half the [14C]leucine-incorporating activity was resistant to EM. This suggests that resistance requires that only a threshold level of ribosome modification be exceeded and that, once exceeded, resistance is dominant to sensitivity.