Phosphoglycolipid Antibiotics Research Articles

Определение флавофосфолипола в лекарственных препаратах для ветеринарного применения методом диффузии в агар

Flavophospholipol is a phosphoglycolipid antibiotic that inhibits the development of gram-positive microorganisms in the gastrointestinal tract, slows down the growth of gram-negative bacteria Salmonella spp. and E.coli, without having a negative effect on lactobacilli and bifidobacteria, helps to reduce the extrachromosomal resistance of microorganisms to antibacterial agents, increasing their effectiveness. Preparations based on flavophospholipol are used for piglets and pigs, cattle and calves, rabbits and fur-bearing animals, poultry for prophylactic purposes in case of bacterial diseases, the pathogens of which are sensitive to flavofoslipol. In recent years, drugs based on flavophospholipol have been used in veterinary practice. In this regard, it is relevant to develop a methodology for the quantitative determination of flavophospholipol in medicinal preparations for veterinary use using the microbiological method (agar diffusion method). The authors established test strains capable of exhibiting growth inhibition under the action of flavophospholipol and its seed dose. By selecting various combinations, a nutrient substrate and its component composition were selected, depending on the needs of the microorganism, the incubation conditions and the concentration of the standard of the tested antibiotic were determined. The optimal experimental conditions for determining the quantitative determination of flavophospholipol in antimicrobial preparations by the agar diffusion method were established.

Adaptive Optimization Boosted the Production of Moenomycin A in the Microbial Chassis Streptomyces albus J1074.

Great efforts have been made to improve Streptomyces chassis for efficient production of targeted natural products. Moenomycin family antibiotics, represented by moenomycin (Moe) and nosokomycin, are phosphoglycolipid antibiotics that display extraordinary inhibition against Gram-positive bacteria. Herein, we assembled a completed 34 kb hybrid biosynthetic gene cluster (BGC) of moenomycin A (moe-BGC) based on a 24 kb nosokomycin analogue biosynthetic gene cluster (noso-BGC). The heterologous expression of the hybrid moe-BGC in Streptomyces albus J1074 achieved the production of moenomycin A in the recombinant strain LX01 with a yield of 12.1 ± 2 mg/L. Further strong promoter refactoring to improve the transcriptional levels of all of the functional genes in strain LX02 enhanced the production of moenomycin A by 58%. However, the yield improvement of moenomycin A resulted in a dramatic 38% decrease in the chassis biomass compared with the control strain. To improve the weak physiological tolerance to moenomycin A of the chassis, another copy of the gene salb-PBP2 (P238N&F200D), encoding peptidoglycan biosynthetic protein PBP2, was introduced into the chassis strain, producing strain LX03. Cell growth was restored, and the fermentation titer of moenomycin A was 130% higher than that of LX01. Additionally, the production of moenomycin A in strain LX03 was further elevated by 45% to 40.0 ± 3 mg/L after media optimization. These results suggested that the adaptive optimization strategy of strong promoter refactoring in the BGC plus physiological tolerance in the chassis was an efficient approach for obtaining the desired natural products with high titers.

TN5-BASED TRANSPOSON MUTAGENESIS OF STREPTOMYCES GHANAENSIS ATCC14672: SEARCHING FOR NOVEL REGULATORS OF MOENOMYCIN PRODUCTION

Streptomyces ghanaensis ATCC14672 is the only genetically characterized natural producer of moenomycin A (MmA), a unique phosphoglycolipid antibiotic capable of direct inhibition of peptidoglycan glycosyltransferases. At the moment there are no drugs on the market that would target this step of peptidoglycan formation. There is much interest in development of MmA-based drugs to combat growing threat of multidrug resistant Gram-positive infections. Consequently, there is also major interest in development of microbiological approaches towards production of this natural compound via submerged cultivation. Currently available ATCC14672 strains accumulate minute quantities of MmA, which fueled the investigations of genetic control of its biosynthesis over the last decade. Much of our understanding of the regulation of MmA production stems from homology-based approaches. Namely, homologs of known pleiotropic regulators of antibiotic production were searched in ATCC14672 genome and functionally characterized. Although this approach was largely successful, it left out of the focus those regulators of MmA production that share no similarity to the regulators identified in model streptomycetes. In this work we report an attempt to discover novel genes involved in regulation of moenomycin biosynthesis via the use of Streptomyces codon-adapted transposon Tn5, which represents an unbiased way to find genuinely new regualtors. After a primary screening of transposon library we were able to identify five Tn mutants that differed in morphology and/or total antibiotic production. We identified Tn5 insertion sites in these mutants through sequencing and re-analyzed their moenomycin production levels, revealing two strains with increased total antibiotic activity. The genes affected in Tn5 mutants were cloned into expression vectors and introduced back into wild type (ATCC14672) in order to study the effects of their increased dosage on the strain. One of the genes, ssfg_04565, exerted negative effects on antibiotic activity when introduced on the plasmid. Possible functions of the identified genes in the context of secondary metabolism are discussed.

Elucidation of the genetic mechanisms contributing to moenomycin resistance in actinobacteria

Aim. Moenomycins are phosphoglycolipid antibiotics produced almost exclusively by representatives of genus Streptomyces. These antibiotics directly inhibit peptidoglycan glycosyltransferases and are extremely active against cocci. Here we studied how antibiotic-producing actinobacteria protect themselves from toxic action of moenomycins. Methods. Microbiological and molecular genetic approaches were combined to reveal intrinsic levels and distribution of moenomycin resistance across actinobacteria genera, and to pinpoint genes contributing to moenomycin resistance in model strain Streptomyces coelicolor M145. Results. Out of 51 actinobacterial species (90 % of which Streptomyces) being tested, only Streptomyces albus J1074 turned out to be highly susceptible to moenomycin A, although resistant variants can be facilely raised. Several classes of mutations increased level of susceptibility of S. coelicolor to moenomycin, although in no case the latter was equal to what we observed in J1074 strain. Conclusions. Moenomycin resistance is widespread across actinobacteria, and it most likely is caused by a combination factors, such as richly decorated cell wall and organization of divisome apparatus. It is possible that moenomycin resistance mechanisms operating in actinobacteria and pathogenic cocci are different.
 Keywords: moenomycin, antibiotic resistance, peptidoglycan.

A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus.

Moenomycins are phosphoglycolipid antibiotics notable for their extreme potency, unique mode of action, and proven record of use in animal nutrition without selection for resistant microflora. There is a keen interest in manipulation of structures of moenomycins in order to better understand their structure-activity relationships and to generate improved analogs. Only two almost identical moenomycin biosynthetic gene clusters are known, limiting our knowledge of the evolution of moenomycin pathways and our ability to genetically diversify them. Here, we report a novel gene cluster (tchm) that directs production of the phosphoglycolipid teichomycin in Actinoplanes teichomyceticus. Its overall genetic architecture is significantly different from that of the moenomycin biosynthesis (moe) gene clusters of Streptomyces ghanaensis and Streptomyces clavuligerus, featuring multiple gene rearrangements and two novel structural genes. Involvement of the tchm cluster in teichomycin biosynthesis was confirmed via heterologous co-expression of amidotransferase tchmH5 and moe genes. Our work sets the background for further engineering of moenomycins and for deeper inquiries into the evolution of this fascinating biosynthetic pathway.

Testing the utility of site-specific recombinases for manipulations of genome of moenomycin producer Streptomyces ghanaensis ATCC14672.

Streptomyces ghanaensis ATCC14672 is the producer of phosphoglycolipid antibiotics moenomycins that for almost 40 years were used worldwide as an animal feed additive. As the use of moenomycins narrows down (due to bans in the EU and some other countries), it opens the opportunity to develop much-needed antibiotics against Gram-positive human pathogens, such as cocci. It is desirable to develop ATCC14672 strains accumulating only certain members of moenomycin family which would facilitate their purification, analysis and/or chemical modification. Here we tested site-specific recombinases (SSRs) as a tool to manipulate the genome of ATCC14672 and to achieve aforementioned goals. We show that of three SSRs tested--Cre, Dre, and Flp--the first two efficiently catalyzed recombination reactions, while Flp showed no activity in ATCC14672 cells. Cre recombinase can be reused at least three times to modify ATCC14672 genome without detrimental effects, such as large-scale inversions or deletions. Properties of the generated strains and SSRs are discussed.

Development of bacterial transglycosylase inhibitors as new antibiotics: Moenomycin A treatment for drug-resistant Helicobacter pylori

The problem of multidrug-resistant Helicobacter pylori requires new antibiotics development. We have evaluated a potential antibiotics, moenomycin A, which is classified as a phosphoglycolipid antibiotics that targets transglycosylase and is previously thought to be limited in Gram-positive bacteria. Herein, we report the activity of moenomycin A against multidrug-resistant H. pylori and the isolates from patients with different gastrointestinal diseases.

MoeH5: a natural glycorandomizer from the moenomycin biosynthetic pathway

The biosynthesis of the phosphoglycolipid antibiotic moenomycin A attracts the attention of researchers hoping to develop new moenomycin-based antibiotics against multidrug resistant Gram-positive infections. There is detailed understanding of most steps of this biosynthetic pathway in Streptomyces ghanaensis (ATCC14672), except for the ultimate stage, where a single pentasaccharide intermediate is converted into a set of unusually modified final products. Here we report that only one gene, moeH5, encoding a homologue of the glutamine amidotransferase (GAT) enzyme superfamily, is responsible for the observed diversity of terminally decorated moenomycins. Genetic and biochemical evidence support the idea that MoeH5 is a novel member of the GAT superfamily, whose homologues are involved in the synthesis of various secondary metabolites as well as K and O antigens of bacterial lipopolysaccharide. Our results provide insights into the mechanism of MoeH5 and its counterparts, and give us a new tool for the diversification of phosphoglycolipid antibiotics.

The molecular biology of moenomycins: towards novel antibiotics based on inhibition of bacterial peptidoglycan glycosyltransferases

Moenomycins are phosphoglycolipid antibiotics and the only known natural product inhibitors of peptidoglycan glycosytransferases (PGTs). Techniques that would allow facile diversification of the moenomycin structure would facilitate the development of novel antibiotics, which are urgently needed in the wake of multidrug resistant bacterial infections. The cloning and initial characterization of the moenomycin biosynthetic genes has already redefined the minimal moenomycin pharmacophore and now opens the door for the biocombinatorial generation of bioactive moenomycin fragments. Here, we highlight the importance of research on the genetic mechanisms that regulate moenomycin biosynthesis and that confer moenomycin resistance to bacteria in the development of novel anti-infectives based on PGT inhibition.

Complete Characterization of the Seventeen Step Moenomycin Biosynthetic Pathway

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here, we report a comprehensive set of genetic and enzymatic experiments that establish functions for the 17 moenomycin biosynthetic genes involved in the synthesis of moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogues. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogues can be explored.

Influence of flavomycin on ruminal fermentation and microbial populations in sheep

Flavomycin is a phosphoglycolipid antibiotic that promotes growth in ruminants. The aim of this study was to characterize the effects of flavomycin on ruminal micro-organisms and their metabolic consequences. In sheep receiving a mixed grass hay/concentrate diet, inclusion of 20 mg flavomycin day(-1) decreased ruminal ammonia and total volatile fatty acid concentrations (P<0.001), but the acetate : propionate ratio was unchanged. Ruminal pH tended to be lower with flavomycin, and ammonia-production rates of ruminal digesta from control animals measured in vitro tended to be inhibited by flavomycin. Pure-culture studies indicated that anaerobic fungi, protozoa and most bacterial species were insensitive to flavomycin. Fusobacterium necrophorum was the most sensitive species tested, along with some high-activity ammonia-producing (HAP) species. Effects on F. necrophorum in vivo were inconsistent due to large inter-animal variation. HAP numbers appeared to be decreased. Changes in the rumen bacterial-community structure were assessed by using denaturing-gradient gel electrophoresis (DGGE) analysis of rumen digesta 16S rRNA. DGGE profiles differed from animal to animal, but remained consistent from day to day. The community structure changed when flavomycin was introduced. The roles of F. necrophorum and HAP species in ammonia formation and of F. necrophorum in the invasion of wall tissue are consistent with the observed effects of flavomycin on ruminal ammonia formation and, in other studies, on decreasing tissue-turnover rates.

Aptamers that recognize the lipid moiety of the antibiotic moenomycin A.

Moenomycin A is an amphiphilic phosphoglycolipid antibiotic that interferes with the transglycosylation step in peptidoglycan biosynthesis. The antibiotic consists of a branched pentasaccharide moiety, connected to the moenocinol lipid via a glycerophosphate linker. We have previously described the selection of aptamers that require the lipid group and the disaccharide epitopes of the oligosaccharide moiety for moenomycin binding. Here we report that the enriched moenomycin-binding library contains sequences that evolved for specific recognition of the unpolar lipid group of the antibiotic. These results suggest that the evolution of hydrophobic binding pockets in RNA molecules may be much more common than previously assumed.

ChemInform Abstract: Synthesis of 2‐(Trimethylsilyl)ethyl 4‐C‐methyl‐β‐D‐ glucopyranuronamide. A Derivative Suitable as Building Block in Synthesis of Phosphoglycolipid Antibiotics.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis of 2-(Trimethylsilyl)ethyl 4-C-methyl-β- d-gluco-pyranuronamide. a derivative suitable as building block in synthesis of phosphoglycolipid antibiotics

The title compound, a protected derivative of a unique component in the moenuronamide group of phosphoglycolipid antibiotics has been synthesised in eleven steps and 30 % total yield from 2(trimethylsilyl) ethyl-β- d-galactopyranoside ( 6). The key step was a stereroselective addition of methylmagnesium chloride to ketone 11 to form the 4-C-methyl-glucopyranoside 12.

5206405 2-alkenyloxy-3 (phosphono) oxypropanoic acids derived from the cleavage of phosphoglycolipid antibiotics: Werner Aretz, Dirk Bottger, Gerhard Seibert, Alois Tumulka, Peter Welzel, Kurt Hobert, Federal Republic of Germany assigned to Hoechst Aktiengesellschaft

5206405 2-alkenyloxy-3 (phosphono) oxypropanoic acids derived from the cleavage of phosphoglycolipid antibiotics: Werner Aretz, Dirk Bottger, Gerhard Seibert, Alois Tumulka, Peter Welzel, Kurt Hobert, Federal Republic of Germany assigned to Hoechst Aktiengesellschaft

The effect of flavomycin on the synthesis and transfer of lipid-linked saccharides in pig brain.

Particulate membrane fractions from pig brain catalyse the synthesis of lipid-linked sugar derivatives of the dolichyl phosphate pathway. Flavomycin, a phosphoglycolipid antibiotic produced by various species of streptomycetes, interferes with the formation of these glycolipids to a different extent. The formation of dolichyl phosphate glucose was shown to be most susceptible to the antibiotic, being blocked by about 50% in the presence of 0.2mm-flavomycin, whereas the synthesis of dolichyl diphosphate N-acetylglucosamine, dolichyl diphosphate chitobiose and dolichyl diphosphate chitobiosyl mannose required higher concentrations to achieve a comparable inhibition. Although the formation of dolichyl phosphate mannose was hardly affected, the accumulation of oligosaccharides with five to seven sugar units was observed, when dolichyl diphosphate oligosaccharides were synthesized with GDP-[(14)C]mannose in the presence of 1mm-flavomycin. This indicates that the inhibition of the synthesis of larger-sized oligosaccharides, known to be mediated by lipid-bound mannose, was not caused by an actual deficiency in dolichyl phosphate mannose. At flavomycin concentrations that inhibited the formation of dolichyl phosphate glucose by 50%, the transfer of lipid-linked saccharides to either the hexapeptide Tyr-Asn-Gly-Thr-Ser-Val or endogenous protein acceptors was hardly influenced. The mode of action of flavomycin is still obscure, but seems not to be of a competitive nature, since the inhibition was unaffected by increasing concentrations of dolichyl phosphate. Some evidence indicates that, besides a direct interaction of the antibiotic with some transferases, a non-specific incorporation into the membrane and alteration of its properties might be responsible for those inhibitory effects on all enzymes which were observed at high concentrations of flavomycin.

Inhibition of lipid-linked mannose and mannoprotein synthesis in yeast by diumycin in vitro.

Diumycin, a phosphoglycolipid antibiotic, inhibits different mannosyl transfer reactions in yeast. Using membrane preparations, the drug effectively inhibited the formation of dolichyl phosphate mannose (DolP-Man); 50% inhibition was observed at approximately 10 microgram/ml. To a lesser extent also mannosyl transfer from DolP-Man to protein decreased in presence of diumycin. Both mannosyl transfer to protein-serine/threonine acceptor sites as well as into positions within the asparagine-linked polymannose part of the yeast mannoprotein are inhibited to about 60% under conditions where DolP-Man formation is blocked. DolP-Man synthesis as well as mannosyl transfer from DolP-Man to protein are also inhibited by diumycin using solubilized enzymes and exogenous acceptor substrates. Glycosyltransfer reactions from GDP-mannose either to protein-serine/threonine-linked mannose (formation of short manno-oligosaccharides) or to dolichyl-diphosphate-linked chitobiose (formation of lipid-linked trisaccharide) are not inhibited by diumycin under conditions where DolP-Man synthesis is blocked by the antibiotic. The inhibitory action of diumycin on DolP-Man formation does not seem to be competitive with respect to dolichyl phosphate, since it cannot be overcome by higher concentrations of dolichyl phosphate.

Pholipomycin, a new member of phosphoglycolipid antibiotics. III. Biological properties.

Pholipomycin, a new member of the phosphoglycolipid antibiotics, was primarily active against gram-positive bacteria including clinically isolated resistant bacteria. It differed from other members of the antibiotic family as it also demonstrated activity against gram-negative bacteria. Pholipomycin protected mice from infection with Staphylococcus aureus and was nontoxic to mice having an LD50 (i.v.) of 600 mg/kg. Besides possessing anti-microbiol activity, pholipomycin, when administered orally, appeared to promote growth in chickens and pigs.

Pholipomycin, a new member of phosphoglycolipid antibiotics. I. Taxonomy of producing organism and fermentation and isolation of pholipomycin.

Pholipomycin is a new member of the phosphoglycolipid family of antibiotics. Taxonomic studies of the producing organism revealed that it has morphologically characteristic aerial mycelia in which two to three spores are borne usually on short and clavate side branches. The species name, Streptomyces lividoclavatus, has been proposed. Pholipomycin is produced mainly in the solid residue of the fermentation culture broth and is isolated by methanol extraction of the mycelial cake followed by purification on ion-exchange resin and silica gel chromatography.