Additional Antibiotic Inhibitors of Peptidoglycan Synthesis

The increasing resistance of human pathogens to conventional antibiotics presents a growing threat to the chemotherapeutic management of infectious diseases. The lanthionine antibiotics, still unused as therapeutic agents, have recently attracted significant scientific interest as models for targeting and management of bacterial infections. We investigated the action of one member of this class, subtilin, which permeabilizes lipid membranes in a lipid II-dependent manner and binds bactoprenyl pyrophosphate, akin to nisin. The role the C and N termini play in target recognition was investigated in vivo and in vitro by using the natural N-terminally succinylated subtilin as well as enzymatically truncated subtilin variants. Fluorescence dequenching experiments show that subtilin induces leakage in membranes in a lipid II-dependent manner and that N-succinylated subtilin is roughly 75-fold less active. Solid-state nuclear magnetic resonance was used to show that subtilin forms complexes with membrane isoprenyl pyrophosphates. Activity assays in vivo show that the N terminus of subtilin plays a critical role in its activity. Succinylation of the N terminus resulted in a 20-fold decrease in its activity, whereas deletion of N-terminal Trp abolished activity altogether.

Section: Discussionmentioning

confidence: 99%

Molecular Mechanism of Target Recognition by Subtilin, a Class I Lanthionine Antibiotic

Parisot

Carey

Breukink

et al. 2008

“…Inhibition by antibiotics that interfere with the polymerization reactions typically leads to an accumulation of the UDP-MurNAc-pentapeptide precursor in the cytoplasm of intact bacteria (12,14). Surprisingly, however, inhibition by LY146032 in intact S. aureus as well as B. megaterium cells did not lead to detectable accumulation of any radiolabeled precursor.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of peptidoglycan biosynthesis in gram-positive bacteria by LY146032

Allen

Hobbs

Alborn

1987

139

104

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 megatetium 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.

“…4) [30]). Bacitracin, which prevents the recycling of undecaprenyl-pyrophosphate (24), caused 85 to 90% inhibition, as did diumycin, which interferes with the utilization of the GlcNAcMurNAc-(pentapeptide)-P-P-undecaprenol intermediate (7,9). The simultaneous addition of clavulanic acid, a known inhibitor of TEM /3-lactamase (20), brought the penicillin-induced inhibition of peptidoglycan synthesis in the /3-lactamase producer 1L261 from its initially low level to the same value as in the sensitive strain 1L260, although the same concentration of clavulanic acid alone had no inhibitory effect.…”

Section: Resultsmentioning

confidence: 99%

In Vitro Synthesis of Peptidoglycan by β-Lactam-Sensitive and -Resistant Strains of Neisseria gonorrhoeae : Effects of β-Lactam and Other Antibiotics

Brown

Perkins

1979

The synthesis in vitro of peptidoglycan by Neisseria gonorrhoeae was studied in organisms made permeable to nucleotide precursors by treatment with ether.Optimum synthesis occurred at 300C in tris(hydroxymethyl)aminomethane-maleate buffer (0.05 M; pH 6) in the presence of 20 mM Mg2e. The incorporation from uridine 5'-diphosphate-N-acetyl-['4C]glucosamine into peptidoglycan, measured after precipitation of the cells with trichloroacetic acid, was sensitive to the fl-lactam antibiotics, bacitracin, diumycin, and tunicamycin and relatively resistant to spectinomycin and tetracycline. Differences in sensitivity between preparations from a f-lactamase producer and a laboratory segregant derived from it were not great. Synthesized peptidoglycan was also fractionated into sodium dodecyl sulfate-soluble and -insoluble portions. ,B-Lactam antibiotics at concentrations equivalent to the minimal inhibitory concentrations for growth of the organisms did not inhibit peptidoglycan synthesis, but rather caused a small enhancement. At higher concentrations, above about 0.5 ug/ml, incorporation into sodium dodecyl sulfate-insoluble material was progressively inhibited, whereas the amount of sodium dodecyl sulfate-soluble product increased greatly, more than compensating for the loss of the precipitable fraction. Similar observations were made with three strains, and also with the ft-lactam clavulanic acid, normally considered as a f,-lactamase inhibitor rather than as itself an effective antibiotic.Until recently, gonococci had always been observed to be highly sensitive to penicillin, and even though some strains existed that were relatively more resistant this did not prevent penicillin from remaining the preferred treatment. In 1976, strains that had acquired high levels of resistance attributable to their production of a TEM-type f.-lactamase (1,18,19,26) were isolated in various parts of the world, and infections with these could no longer be treated by single massive doses of benzylpenicillin. Later evidence showed that the gene for the 13-lactamase was carried on plasmids of 4.4 x 10' daltons in some strains and 3.2 x 106 daltons in others, but that its base sequence was at least 70% similar in each case (3,21). It was, nevertheless, possible to treat infections with these strains by using fl-lactams resistant to TEM-f-lactamase, for which the fundamental attack on gonococcal wall synthesis could be expected to be the same as for penicillin. f3-Lactams are well known to interfere with the synthesis of bacterial cell walls by preventing the cross-linking of peptidoglycan (4), and there is good evidence that this mechanism is also involved in their action on gonococci (22). As in other organisms, their bactericidal effect is mediated by autolysis (22,32) which is promoted by penicillin action. To gain further insight into the precise way in which,-lactams can kill gonococci, whether or not they bear ,Blactamase genes on their plasmids, we have investigated directly the effect of these antibiotics on the in vitro biosynthesi...