A new bacterial mannosidase for the selective modification of ramoplanin and its derivatives

Ramoplanin is a lipoglycodepsipeptide produced by Actinoplanes sp. ATCC 33076 and active on bacterial cell wall biosynthesis by binding to Lipid II. A screening of an actinomycetes collection was performed to select enzymatic activities able to introduce specific modifications in the ramoplanin molecule. An extracellular mannosidase from Streptomyces GE 91081 was found to selectively remove one mannose unit from ramoplanin and tetrahydroramoplanin to give the corresponding mannosyl aglycones. These molecules show an improved microbiological activity versus some resistant staphylococci and streptococci, and are useful intermediates in the synthesis of novel ramoplanin-like antibiotics. The biotransformation of ramoplanin has been optimised to improve molar conversion and the transformation reaction rate.     

A new bacterial mannosidase for the selective modification of ramoplanin and its derivatives

Ramoplanin is a lipoglycodepsipeptide produced by Actinoplanes sp. ATCC 33076 and active on bacterial cell wall biosynthesis by binding to Lipid II. A screening of an actinomycetes collection was performed to select enzymatic activities able to introduce specific modifications in the ramoplanin molecule. An extracellular mannosidase from Streptomyces GE 91081 was found to selectively remove one mannose unit from ramoplanin and tetrahydroramoplanin to give the corresponding mannosyl aglycones. These molecules show an improved microbiological activity versus some resistant staphylococci and streptococci, and are useful intermediates in the synthesis of novel ramoplanin-like antibiotics. The biotransformation of ramoplanin has been optimised to improve molar conversion and the transformation reaction rate.     

Chemistry and biology of the ramoplanin family of peptide antibiotics

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.     

The mechanism of action of ramoplanin and enduracidin

The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by ramoplanin, but there is no definitive information about whether one step is inhibited preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin and the related compound enduracidin have an intrinsic preference for one step over the other. Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These results provide compelling evidence that ramoplanin's and enduracidin's primary cellular target is the transglycosylation step of peptidoglycan biosynthesis.     

Docking on Lipid II—A Widespread Mechanism for Potent Bactericidal Activities of Antibiotic Peptides

Natural product antibiotics usually target the major biosynthetic pathways of bacterial cells and the search for new targets outside these pathways has proven very difficult. Cell wall biosynthesis maybe the most prominent antibiotic target, and ß-lactams are among the clinically most relevant antibiotics. Among cell wall biosynthesis inhibitors, glycopeptide antibiotics are a second group of important drugs, which bind to the peptidoglycan building block lipid II and prevent the incorporation of the monomeric unit into polymeric cell wall. However, lipid II acts as a docking molecule for many more naturally occurring antibiotics from diverse chemical classes and likely is the most targeted molecule in antibacterial mechanisms. We summarize current knowledge on lipid II binding antibiotics and explain, on the levels of mechanisms and resistance development, why lipid II is such a prominent target, and thus provide insights for the design of new antibiotic drugs.     

In situ assay for identifying inhibitors of bacterial transglycosylase

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.     

Differential antibacterial activity of moenomycin analogues on gram-positive bacteria

The moenomycin trisaccharide degradation product and synthetic disaccharide analogues based on the disaccharide core were bactericidal to gram-positive bacteria, inhibited lipid II polymerization, and inhibited cell wall synthesis in Enterococcus faecalis. Truncating moenomycin to the trisaccharide, and building upon the core disaccharide have both led to molecules possessing properties not shared with their respective parent structures.     

Characterization of the bifunctional glycosyltransferase/acyltransferase penicillin-binding protein 4 of Listeria monocytogenes

Multimodular penicillin-binding proteins (PBPs) are essential enzymes responsible for bacterial cell wall peptidoglycan (PG) assembly. Their glycosyltransferase activity catalyzes glycan chain elongation from lipid II substrate (undecaprenyl-pyrophosphoryl-N-acetylglucosamine-N-acetylmuramic acid-pentapeptide), and their transpeptidase activity catalyzes cross-linking between peptides carried by two adjacent glycan chains. Listeria monocytogenes is a food-borne pathogen which exerts its virulence through secreted and cell wall PG-associated virulence factors. This bacterium has five PBPs, including two bifunctional glycosyltransferase/transpeptidase class A PBPs, namely, PBP1 and PBP4. We have expressed and purified the latter and have shown that it binds penicillin and catalyzes in vitro glycan chain polymerization with an efficiency of 1,400 M(-1) s(-1) from Escherichia coli lipid II substrate. PBP4 also catalyzes the aminolysis (d-Ala as acceptor) and hydrolysis of the thiolester donor substrate benzoyl-Gly-thioglycolate, indicating that PBP4 possesses both transpeptidase and carboxypeptidase activities. Disruption of the gene lmo2229 encoding PBP4 in L. monocytogenes EGD did not have any significant effect on growth rate, peptidoglycan composition, cell morphology, or sensitivity to beta-lactam antibiotics but did increase the resistance of the mutant to moenomycin.     

Substrate analogues to study cell-wall biosynthesis and its inhibition

It has been known for more than 30 years that Lipid II is an intermediate in peptidoglycan synthesis. Recently, it has become apparent that it is also an important target of numerous antibiotics, including the glycopeptides, the lantibiotics and ramoplanin. It is also utilized by sortases in the construction of Gram-positive cell walls. Recent progress has been made in the synthesis of peptidoglycan intermediates that can be used to study enzymes which make peptidoglycan. These intermediates also enable studies to probe the mechanism of action of a variety of substrate-binding antibiotics.     

Studies on the biosynthesis of the lipodepsipeptide antibiotic Ramoplanin A2

Ramoplanin, a non-ribosomally synthesized peptide antibiotic, is highly effective against several drug-resistant Gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Recently, the biosynthetic cluster from the ramoplanin producer Actinoplanes ATCC 33076 was sequenced, revealing an unusual architecture of fatty acid and non-ribosomal peptide synthetase biosynthetic genes (NRPSs). The first steps towards understanding how these biosynthetic enzymes cooperatively interact to produce the depsipeptide product are expression and isolation of each enzyme to probe its specificity and function. Here we describe the successful production of soluble enzymes from within the ramoplanin locus and the confirmation of their specific role in biosynthesis. These methods may be broadly applicable to the production of biosynthetic enzymes from other natural product biosynthetic gene clusters, especially those that have been refractory to production in heterologous hosts despite standard expression optimization methods.     

Induction of vancomycin resistance in Enterococcus faecium by inhibition of transglycosylation

Vancomycin resistance has recently been recognized among clinical isolates of enterococci. Resistance is inducible, and associated with production of a novel 39 kDa membrane protein. The mechanism by which exposure to vancomycin, which does not penetrate the cell membrane, induces resistance is unknown. In the vancomycin resistant strain Enterococcus faecium 228, resistance was also inducible by moenomycin, suggesting that inhibition of the transglycosylation step in peptidoglycan synthesis may be required for induction of resistance. Cytoplasmic pools of peptidoglycan precursors were increased after exposure to vancomycin or moenomycin, representing a potential means for regulation of induction.     

Functional characterization of penicillin-binding protein 1b from Streptococcus pneumoniae

The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by beta-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.     

Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

Enterococcus faecalis virulence requires cell wall-associated proteins, including the sortase-assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the E. faecalis cell cycle, we found that cell surface Ebp do not co-localize with newly synthesized PG. Instead, surface-exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the E. faecalis diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis.     

In vivo and in vitro action of new antibiotics interfering with the utilization of N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide

Recent literature on the antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP suggested an interaction with murein synthesis. Incubation of sensitive strains from Bacillus cereus and Staphylococcus aureus in a "wall medium" containing labeled l-alanine showed that all four antibiotics inhibited the incorporation of alanine into murein and gave rise to accumulation of radioactive uridine diphosphate-N-acetyl-muramyl (UDP-MurNAc)-pentapeptide. Peptidoglycan was synthesized when the particulate enzyme of B. stearothermophilus was incubated with the murein precursors UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-MurNAc-pentapeptide. The newly formed polymer was less accessible for lysozyme and more strongly bound to the acceptor than the same product from the Escherichia coli particulate enzyme. After incubation in the presence of penicillin, a greater part of the peptidoglycan was lysozyme sensitive and more loosely bound to the acceptor. The antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP inhibited peptidoglycan synthesis by the B. stearothermophilus particulate enzyme. The rate of synthesis of GlcNAc-MurNAc(-pentapeptide)-P-P-phospholipid was independent from the addition of these antibiotics, but its utilization was strongly inhibited. With the present results, it is not possible to distinguish the mechanisms of action of enduracidin, moenomycin, prasinomycin, and 11.837 RP from the mechanisms of action of vancomycin and ristocetin.     

Moenomycin: An inhibitor of C55-isoprenoid-alcohol kinase from Staphylococcus aureus

The effect of a variety of structurally diverse antibiotics on C₅₅-isoprenoid-alcohol kinase (EC 2.7.1.66) from Staphylococcus aureus has been examined. Only moenomycin was found to be inhibitory (K_i = 0.2 mM).Moenocinol, The C₂₅ lipid component of moenomycin, did not serve as a substrate of kinase reaction and was less inhibitory than the intact antibiotic.It is concluded that the observed inhibition may be due to the structural similarity between C₅₅-isoprenylmonophosphate and the substituted moenocinol moiety of moenomycin.