Relationship between glycopeptide production and resistance in the actinomycete Nonomuraea sp. ATCC 39727.

Glycopeptides and ß-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens. Nonomuraea sp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonical vanHAX genes. Consequently, we investigated the role of the ß-lactam-sensitive D,D-peptidase/D,D-carboxypeptidase encoded by vanYn, the only van-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic in Nonomuraea sp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we varied vanYn gene dosage and expressed vanHatAatXat from the teicoplanin producer Actinoplanes teichomyceticus in Nonomuraea sp. ATCC 39727. Knocking out vanYn, complementing a vanYn mutant, or duplicating vanYn had no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production. Nonomuraea sp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYn activity. The heterologous expression of vanHatAatXat increased A40926 resistance in Nonomuraea sp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. The vanYn-based self-resistance in Nonomuraea sp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants of Enterococcus faecium selected in vitro for high-level resistance to glycopeptides and penicillins.

Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32-07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32-07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

Novel mechanism of glycopeptide resistance in the A40926 producer Nonomuraea sp. ATCC 39727.

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by replacing the C-terminal d-alanyl-d-alanine of lipid II with d-alanyl-d-lactate, thus reducing glycopeptide affinity for cell wall targets. Reorganization of the cell wall in these organisms is directed by the vanHAX gene cluster. Similar self-resistance mechanisms have been reported for glycopeptide-producing actinomycetes. We investigated glycopeptide resistance in Nonomuraea sp. ATCC 39727, the producer of the glycopeptide A40926, which is the precursor of the semisynthetic antibiotic dalbavancin, which is currently in phase III clinical trials. The MIC of Nonomuraea sp. ATCC 39727 toward A40926 during vegetative growth was 4 microg/ml, but this increased to ca. 20 microg/ml during A40926 production. vanHAX gene clusters were not detected in Nonomuraea sp. ATCC 39727 by Southern hybridization or by PCR with degenerate primers. However, the dbv gene cluster for A40926 production contains a gene, vanY (ORF7), potentially encoding an enzyme capable of removing the terminal d-Ala residue of pentapeptide peptidoglycan precursors. Analysis of UDP-linked peptidoglycan precursors in Nonomuraea sp. ATCC 39727 revealed the predominant presence of the tetrapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala and only traces of the pentapeptide UDP-MurNAc-l-Ala-d-Glu-meso-Dap-d-Ala-d-Ala. This suggested a novel mechanism of glycopeptide resistance in Nonomuraea sp. ATCC 39727 that was based on the d,d-carboxypeptidase activity of vanY. Consistent with this, a vanY-null mutant of Nonomuraea sp. ATCC 39727 demonstrated a reduced level of glycopeptide resistance, without affecting A40926 productivity. Heterologous expression of vanY in a sensitive Streptomyces species, Streptomyces venezuelae, resulted in higher levels of glycopeptide resistance.

Determining the structure and mode of action of microbisporicin, a potent lantibiotic active against multiresistant pathogens.

Antibiotics blocking bacterial cell wall assembly (beta-lactams and glycopeptides) are facing a challenge from the progressive spread of resistant pathogens. Lantibiotics are promising candidates to alleviate this problem. Microbisporicin, the most potent antibacterial among known comparable lantibiotics, was discovered during a screening applied to uncommon actinomycetes. It is produced by Microbispora sp. as two similarly active and structurally related polypeptides (A1, 2246-Da and A2, 2230-Da) of 24 amino acids linked by 5 intramolecular thioether bridges. Microbisporicin contains two posttranslational modifications that have never been reported previously in lantibiotics: 5-chloro-trypthopan and mono- (in A2) or bis-hydroxylated (in A1) proline. Consistent with screening criteria, microbisporicin selectively blocks peptidoglycan biosynthesis, causing cytoplasmic UDP-linked precursor accumulation. Considering its spectrum of activity and its efficacy in vivo, microbisporicin represents a promising antibiotic to treat emerging infections.

E40, a novel microbial protease efficiently detoxifying gluten proteins, for the dietary management of gluten intolerance.

Gluten proteins are the causative agent of Celiac Disease (CD), a life-long food intolerance characterized by an autoimmune enteropathy. Inadvertent gluten exposure is frequent even in celiac patients complying with a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is indeed a promising approach to reduce the risk of dietary gluten boost. Here we describe Endopeptidase 40, a novel glutenase discovered as secreted protein from the soil actinomycete Actinoallomurus A8, and its recombinant active form produced by Streptomyces lividans TK24. E40 is resistant to pepsin and trypsin, and active in the acidic pH range 3 to 6. E40 efficiently degrades the most immunogenic 33-mer as well as the whole gliadin proteins, as demonstrated by SDS-PAGE, HPLC, LC-MS/MS, and ELISA. T lymphocytes from duodenal biopsies of celiac patients showed a strongly reduced or absent release of IFN-γ when exposed to gluten digested with E40. Data in gastrointestinal simulated conditions suggest that no toxic peptides are freed during gluten digestion by E40 into the stomach to enter the small intestine, thus counteracting the intestinal inflammatory cascade to occur in CD patients. E40 is proposed as a novel candidate in Oral Enzymatic Therapy for the dietary management of gluten toxicity.

A derivative of the glycopeptide A40926 produced by inactivation of the beta-hydroxylase gene in Nonomuraea sp. ATCC39727.

The actinomycete Nonomuraea sp. ATCC39727 produces the glycopeptide A40926. In the corresponding dbv cluster, ORF28 encodes a putative hydroxylase. A gene replacement mutant of ORF28 in Nonomuraea produces a small amount of an A40926-related metabolite, 16 amu smaller than the parent compound, which was identified as the desoxyderivative of A40926 lacking the beta-hydroxyl group on the tyrosine moiety. This result demonstrates that ORF28 is actually involved in the formation of the beta-hydroxytyrosine residue present in A40926. The formation of an altered glycopeptide and the inability to rescue A40926 production upon feeding free beta-hydroxytyrosine are consistent with the possibility that, in contrast to balhimycin formation, hydroxylation occurs after tyrosine activation by the nonribosomal peptide synthetase.

Extraction and Analysis of Peptidoglycan Cell Wall Precursors.

Extraction and analysis by LC-MS of peptidoglycan precursors represent a valuable method to study antibiotic mode of action and resistance in bacteria. Here, we describe how to apply this method for: (1) testing the action of different classes of antibiotics inhibiting cell wall biosynthesis in Bacillus megaterium; (2) studying the mechanism of self-resistance in mycelial actinomycetes producing glycopeptide antibiotics.

A new assay for the discovery of Bcl-XL inhibitors.

The Bcl-2 family of antiapoptotic proteins is commonly over expressed in many types of human cancer and remains one of the few validated targets. Antiapoptotic family proteins such as Bcl-2 and Bcl-XL function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby prevent release of the apoptotic cascade of events. "BH3-only" members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerization could be used to modulate cell death reinstating apoptosis in cancer cells. An affinity displacement assay based on Bcl-XL/BH3 interaction has been developed. This assay makes use of soluble His-tagged Bcl-XL and fluorescein tagged BH3. Binding is measured as fluorescence associated with magnetic beads. The assay was miniaturized to 96-well microtiter plates and can be employed in high throughput screening (HTS), in addition it is robust enough to be applied to microbial fermentation extracts.

A Novel Microbisporicin Producer Identified by Early Dereplication during Lantibiotic Screening.

With the increasing need of effective antibiotics against multi-drug resistant pathogens, lantibiotics are an attractive option of a new class of molecules. They are ribosomally synthetized and posttranslationally modified peptides possessing potent antimicrobial activity against aerobic and anaerobic Gram-positive pathogens, including those increasingly resistant to ß-lactams and glycopeptides. Some of them (actagardine, mersacidin, planosporicin, and microbisporicin) inhibit cell wall biosynthesis in pathogens and their effect is not antagonized by vancomycin. Hereby, we apply an efficient strategy for lantibiotic screening to 240 members of a newly described genus of filamentous actinomycetes, named Actinoallomurus, that is considered a yet-poorly-exploited promising source for novel bioactive metabolites. By combining antimicrobial differential assay against Staphylococcus aureus and its L-form (also in the presence of a ß-lactamase cocktail or Ac-Lys-D-alanyl-D-alanine tripeptide), with LC-UV-MS dereplication coupled with bioautography, a novel producer of the potent microbisporicin complex was rapidly identified. Under the commercial name of NAI-107, it is currently in late preclinical phase for the treatment of multi-drug resistant Gram-positive pathogens. To our knowledge, this is the first report on a lantibiotic produced by an Actinoallomurus sp. and on a microbisporicin producer not belonging to the Microbispora genus.

Targets and assays for discovering novel antibacterial agents.

The increasing frequency of nosocomial infections due to multi-resistant pathogens exerts a significant toll and calls for novel and better antibiotics. Different approaches can be used in the search for novel antibiotics acting on drug-resistant bacterial pathogens. We present some considerations on valid bacterial targets to be used for searching new antibiotics, and how the information from bacterial genome sequences can assist in choosing the appropriate targets. Other factors to be considered in target selection are the chemical diversity available for screening and its uniqueness. We will conclude discussing our strategy for searching novel antibacterials. This is based on a large collection of microbial extracts as a source of chemical diversity and on the use of specific targets essential for the viability of bacterial pathogens. Two assay strategies have been implemented: a pathway-based assay, where a series of essential bacterial targets is screened in a single assay; and a binding assay, where many targets can be screened individually in the same format.

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes.

Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10(7)-10(9) protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments.

A novel lantibiotic acting on bacterial cell wall synthesis produced by the uncommon actinomycete Planomonospora sp.

Important classes of antibiotics acting on bacterial cell wall biosynthesis, such as beta-lactams and glycopeptides, are used extensively in therapy and are now faced with a challenge because of the progressive spread of resistant pathogens. A discovery program was devised to target novel peptidoglycan biosynthesis inhibitors capable of overcoming these resistance mechanisms. The microbial products were first screened according to their differential activity against Staphylococcus aureus and its L-form. Then, activities insensitive to the addition of a beta-lactamase cocktail or d-alanyl-d-alanine affinity resin were selected. Thirty-five lantibiotics were identified from a library of broth extracts produced by 40,000 uncommon actinomycetes. Five of them showed structural characteristics that did not match with any known microbial metabolite. In this study, we report on the production, structure determination, and biological activity of one of these novel lantibiotics, namely, planosporicin, which is produced by the uncommon actinomycete Planomonospora sp. Planosporicin is a 2194 Da polypeptide originating from 24 proteinogenic amino acids. It contains lanthionine and methyllanthionine amino acids generating five intramolecular thioether bridges. Planosporicin selectively blocks peptidoglycan biosynthesis and causes accumulation of UDP-linked peptidoglycan precursors in growing bacterial cells. On the basis of its mode of action and globular structure, planosporicin can be assigned to the mersacidin (20 amino acids, 1825 Da) and the actagardine (19 amino acids, 1890 Da) subgroup of type B lantibiotics. Considering its spectrum of activity against Gram-positive pathogens of medical importance, including multi-resistant clinical isolates, and its efficacy in vivo, planosporicin represents a potentially new antibiotic to treat emerging pathogens.

Resistance to glycopeptide antibiotics in the teicoplanin producer is mediated by van gene homologue expression directing the synthesis of a modified cell wall peptidoglycan.

Glycopeptide resistance has been studied in detail in enterococci and staphylococci. In these microorganisms, high-level resistance is achieved by replacing the C-terminal D-alanyl-D-alanine of the nascent peptidoglycan with D-alanyl-D-lactate or D-alanyl-D-serine, thus reducing the affinities of glycopeptides for cell wall targets. Reorganization of the cell wall is directed by the expression of the van gene clusters. The identification of van gene homologs in the genomes of several glycopeptide-producing actinomycetes suggests the involvement of a similar self-resistance mechanism to avoid suicide. This report describes a comprehensive study of self-resistance in Actinoplanes teichomyceticus ATCC 31121, the producer of the clinically relevant glycopeptide teicoplanin. A. teichomyceticus ATCC 31121 showed a MIC of teicoplanin of 25 microg/ml and a MIC of vancomycin of 90 microg/ml during vegetative growth. The vanH, vanA, and vanX genes of A. teichomyceticus were found to be organized in an operon whose transcription was constitutive. Analysis of the UDP-linked peptidoglycan precursors revealed the presence of UDP-glycomuramyl pentadepsipeptide terminating in D-alanyl-D-lactate. No trace of precursors ending in d-alanyl-d-alanine was detected. Thus, the van gene complex was transcribed and expressed in the genetic background of A. teichomyceticus and conferred resistance to vancomycin and teicoplanin through the modification of cell wall biosynthesis. During teicoplanin production (maximum productivity, 70 to 80 microg/ml), the MIC of teicoplanin remained in the range of 25 to 35 microg/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 microg/ml.

Candida albicans strains deficient in CHS7, a key regulator of chitin synthase III, exhibit morphogenetic alterations and attenuated virulence.

Chitin is a structural polysaccharide present in most fungal cell walls, whose synthesis depends on a family of enzymic activities named chitin synthases (CSs). The specific role of each of them, as well as of their regulatory proteins, in cell morphogenesis and virulence is not well understood. Here, it is shown that most chitin synthesis in Candida albicans, one of the fungi most commonly isolated from opportunistic mycoses and infections, depends on CHS7. Thus, C. albicans chs7Delta null mutants showed reduced levels of chitin and CS activity, and were resistant to Calcofluor. Despite the sequence similarity and functional relationship with ScChs7p, CaChs7p was unable to restore CSIII activity in a Saccharomyces cerevisiae chs7Delta null mutant, because it was unable to direct ScChs3p export from the endoplasmic reticulum. C. albicans chs7Delta null mutants did not show any defect in growth rate, but yeast cells displayed minor morphogenetic defects affecting septum formation, and showed an increased tendency to form filaments. CaChs7p was not required for germ-tube emission, and null mutant strains underwent the dimorphic transition correctly. However, colony morphology appeared distinctively affected. chs7Delta hyphae were very curved and had irregular lateral walls, resulting in very compact colonies that seemed unable to spread out radially on the surface, unlike the wild-type. This growth pattern may be associated with the reduced virulence and high clearance rate observed when the chs7Delta strain was used in a murine model of infection. Therefore, CaChs7p is required for normal hyphal morphogenesis, suggesting that in C. albicans CSIII plays an important role in maintaining cell wall integrity, being essential when invading surrounding tissues.

A new inhibitor of the transcription-termination factor Rho.

In this study we describe BI-K0058, a new inhibitor of the transcription-termination factor Rho belonging to a different chemical class from bicyclomycin, the only known antibiotic acting on Rho. BI-K0058 inhibits the poly(C)-dependent ATPase activity of Rho with an IC(50) of 25 microM as well as in vitro transcription-termination of two natural substrates, the Salmonella enterica hisG cistron and the f1 phage intergenic region. BI-K0058 does not affect photolabeling of Rho by ATP. The results of gel mobility shift experiments with a natural RNA substrate demonstrate that BI-K0058 inhibits the formation of the ATP-independent high affinity Rho-RNA complex.