Piero Sensi is probably known primarily for his role in the discovery of rifamycin and for developing it to be a drug of fundamental importance in the treatment of tuberculosis. He has also contributed to promote screening programs of microbial products and research approaches for antibacterial agents that have been further developed up to the present day. This paper reports a sequence of discovery approaches, failures and successes that spans for about 50 years and is still in progress.

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.

ABSTRACT 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 μg/ml and a MIC of vancomycin of 90 μg/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 μg/ml), the MIC of teicoplanin remained in the range of 25 to 35 μg/ml. Teicoplanin-producing cells were found to be tolerant to high concentrations of exogenously added glycopeptides, which were not bactericidal even at 5,000 μg/ml.

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.

An efficient method for protoplast generation for the uncommon actinomycete Planobispora rosea, the producer of the thiazolylpeptide antibiotic GE2270, was developed using a combination of hen egg white lysozyme and Streptomyces globisporus mutanolysin. This method converted more than 70% of vegetative mycelium to protoplasts, which were then regenerated with 50% efficiency in an optimized medium. When P. rosea protoplasts were efficiently fused, recombination between different antibiotic (streptomycin and gentamicin) resistance markers originated sensitive strains (str(s)gen(s)) at frequencies as high as 18% and double resistant fusants (str(r)gen(r)) at frequencies as high as 29%. Double resistant fusants showed GE2270 productivity intermediate between the productivity of the parental strains. Protoplast generation and fusion in P. rosea makes whole genome shuffling feasible as an approach to be used alternately with classical random mutagenesis in industrial strain improvement programs.

During a strain improvement program, spontaneous mutants with single or combined resistance to streptomycin (Strr), gentamycin (Genr) or rifamycin (Rifr) were selected from the industrial strain of Planobispora rosea, which is the producer of thiazolylpeptide GE2270. Among the mutants resistant to each single antibiotic, higher producers occurred more frequently (60%) among Genr than in Rifr (10%) and Strr (24%) populations. Two Genr mutants showed up to 1.5-fold improvement in GE2270 production while single resistant mutants Strr and Rifr produced slightly more than the parental strains. The combination of Strr and Rifr in the same strain improved GE2270 yield up to 1.7-fold. Finally, a higher GE2270 producing strain (1.8-fold improvement with respect to the parental strain) was selected among those mutants with triple resistance to streptomycin, rifamycin and gentamycin. A hierarchical increase in aerial mycelium and spore formation was observed which paralleled GE2270 production improvement.

In the course of a microbial product screening aimed at the discovery of novel antibiotics acting on bacterial protein synthesis, a complex of three structurally related tetrapeptides, namely, GE81112 factors A, B, and B1, was isolated from a Streptomyces sp. The screening was based on a cell-free assay of bacterial protein synthesis driven by a model mRNA containing natural initiation signals. In this study we report the production, isolation, and structure determination of these novel, potent and selective inhibitors of cell-free bacterial protein synthesis, which stably bind the 30S ribosomal subunit and inhibit the formation of fMet-puromycin. They did not inhibit translation by yeast ribosomes in vitro. Spectroscopic analyses revealed that they are tetrapeptides constituted by uncommon amino acids. While GE81112 factors A, B, and B1 were effective in inhibiting bacterial protein synthesis in vitro, they were less active against Gram-positive and Gram-negative bacterial cells. Cells grown in minimal medium were more susceptible to the compounds than those grown in rich medium, and this is most likely due to competition or regulation by medium components during peptide uptake. The novelty of the chemical structure and of the specific mode of action on the initiation phase of bacterial protein synthesis makes GE81112 a unique scaffold for designing new drugs.

GE 23077 factors A1, A2, B1 and B2 are novel antibiotics isolated from fermentation broths of an Actinomadura sp. strain. GE23077 antibiotics are cyclic peptides, which inhibit Escherichia coli RNA polymerase at nM concentrations. Both rifampicin-sensitive and rifampicin-resistant polymerases are inhibited, whereas E. coli DNA polymerase and wheat germ RNA polymerase are substantially not affected. In spite of the potent activity on the enzyme, the antibiotics generally show poor activity against whole cell bacteria. The spectrum of activity is restricted to Moraxella catarrhalis, including clinical isolates, with partial activity against Neisseria gonorrhoeae and Mycobacterium smegmatis.

GE23077, a novel microbial metabolite recently isolated from Actinomadura sp. culture media, is a potent and selective inhibitor of bacterial RNA polymerase (RNAP). It inhibits Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) RNAPs with IC50 values (i.e. the concentration at which the enzyme activity is inhibited by 50%) in the 10(-8) m range, whereas it is not active on E. coli DNA polymerase or on eukaryotic (wheat germ) RNAP II (IC50 values > 10(-4) m in both cases). In spite of its potent activity on purified bacterial RNAPs, GE23077 shows a narrow spectrum of antimicrobial activity on Gram-positive and Gram-negative bacteria. To investigate the molecular basis of this behaviour, the effects of GE23077 on macromolecular biosynthesis were tested in E. coli cells permeabilized under different conditions. The addition of GE23077 to plasmolyzed cells resulted in an immediate and specific inhibition of intracellular RNA biosynthesis, in a dose-response manner, strongly suggesting that cell penetration is the main obstacle for effective antimicrobial activity of the antibiotic. Biochemical studies were also conducted with purified enzymes to obtain further insights into the mode of action of GE23077. Interestingly, the compound displays a behaviour similar to that of rifampicin, an antibiotic structurally unrelated to GE23077: both compounds act at the level of transcription initiation, but not on the sigma subunit and not on the formation of the promoter DNA-RNAP complex. Tests on different rifampicin-resistant E. coli RNAPs did not show any cross-resistance between the two compounds, indicating distinct binding sites on the target enzyme. In conclusion, GE23077 is an interesting new molecule for future mechanistic studies on bacterial RNAP and for its potential in anti-infective drug discovery.

In actinomycetes the catabolism products of branched chain amino acids provide biosynthetic precursors for the formation of several lipid-containing antibiotics. We have determined in Nonomuraea sp. ATCC 39727 the effect of valine on production of glycopeptide antibiotic A40926, which is a complex of factors structurally differing in fatty acid moieties. Addition of valine to minimal medium increased A40926 production and modified complex composition towards a mono-component. Similar results were also obtained in a rich production medium.

Complejo de antibiotico 107891 que comprende factor A1 y factor A2 que es un polvo blanco que tiene las siguientes caracteristicas: (A) espectro de masas registrado a partir de una disolucion 0, 2 mg/ml en metanol:agua 80/20 (v/v) con un 0, 1% de acido trifluoroacetico (figura 1A y 1B) sobre un instrumento Thermofinnigan LCQ deca equipado con una fuente de electropulverizacion, usando una mezcla de calibracion Thermofinnigan en las siguientes condiciones de electropulverizacion: potencial de pulverizacion: 4, 7 kV; temperatura capilar: 220o C; potencial capilar: 3V; modo de infusion 10 mul/min, que muestra dos iones dobles protonados am/z 1124 ym/z 1116, que corresponden a la composicion de isotopos inferiores de factor A1 y A2; respectivamente. (B) espectro infrarrojo (figura 2) registrado en KBr con un espectrofotometro Bruker FT-IR modelo IFS 48, que muestra maximos de absorcion a (cm-1): 3263; 2929; 1661; 1533; 1402; 1114; 1026. (C) espectro de UV (figura 3); realizado en metanol:H2O 80:20 (v/v) con un espectrofotometro Perkin-Elmer Lambda 16, que muestra dos hombros a 226 y 267 nm. (D) espectro de 1H-RMN (figura 4) registrado a 600 MHz en la mezcla metanol-d4:H2O (pH 4, 3 HCl) 40:10 (v/v) a 40oC sobre un espectrometro Bruker AMX 600 aplicando una secuencia de supresion de agua usando como patron interno la senal residual de metanol-d4 a 3, 31 ppm, que muestra las siguientes senales [delta = ppm multiplicidad; (atribucion)]: 0, 93 d (CH3), 0, 98 d (CH3), 1, 07 t (CH3 superpuestos), 1, 18 t (CH3 superpuestos), 1, 26 s (CH3), 1, 30 t (CH3 superpuestos), 1, 62-1, 74 m (CH2), 1, 78 d (CH3), 1, 80 d (CH3), 2, 03 m (CH2), 2, 24 m (CH), 2, 36 m (CH2), 2, 72-3, 8 m(CH alfa peptidicos), 3, 8-5, 2 m (CH alfa peptidicos), 5, 53-6, 08 s (CH2), 5, 62 d (doble enlace de CH), 6, 42 m (CH), 6, 92 d (doble enlace de CH), 7, 0-7, 55 m (CH aromaticos), 7, 62-10, 4 dym (NH aromatico y peptidico). (E) espectro de 13C-RMN (figura 5) registrado en la mezcla metanol-d4:H2O (pH 4, 3 HCl) 40:10 (v/v) a 40oC sobre un espectrometro Bruker AMX 600, usando como patron interno la senal residual de metanol-d4 a 49, 15 ppm, que muestra las siguientes senales: [delta = ppm; (atribucion)]: 13:6-23, 2 (CH3 alifaticos), 26, 16-73 (CH2 alifaticos y CH alfa peptidicos), 105-136 (CH aromaticos y de dobles enlaces y carbonos cuaternarios), 164, 3-176, 3 (carbonilos peptidicos); y sus sales con acidos.

AbstractFor Abstract see ChemInform Abstract in Full Text.

L'invention concerne une substance antibiotique d'origine microbienne, appelee arbitrairement antibiotique 107891, produit par fermentation de Microbispora sp. ATCC PTA-5024, ses sels pharmaceutiquement acceptables et ses compositions. L'invention concerne egalement son utilisation en tant qu'agent antibacterien presentant une activite inhibitrice versus des microbes susceptibles. L'antibiotique 107891, qui est un complexe comprenant deux facteurs, appeles A1 et A2, comprend une structure peptidique contenant de la lanthionine et de la methyllanthionine comme constituants, ces dernieres etant des caracteristiques typiques des antibiotiques des groupes lantibiotiques. L'antibiotique 107891 et ses facteurs A1 et A2 presentent une bonne activite antibacterienne contre des bacteries Gram positif comprenant des chaines resistant a la methicilline et a la vancomycine, et il est egalement actif contre des bacteries Gram negatif, telles que M. catharralis, des especes de Neisseria et H.influenzae et des mycobacteries.

Chemical and biological modifications of glycopeptides have long been performed in order to obtain derivatives with improved antimicrobial activity or better pharmacokinetics1). Deglycosilated derivatives of several different glycopeptides were produced and used either for biosynthetic or for antimicrobial activity studies1). Deglycosilated derivatives were mainly obtained by chemical treatment or by biotransformation with yields of up to 80%1). Particularly interesting is the biotransformation process for the yields of transformation and the selectivity of the process. However, this requires a second fermentation step and a second downstream purification. Nonomuraea sp. ATCC 39727 (formerly Actinomadura sp. ATCC 39727) is the producer of the lipoglycopeptide antibiotic complex A409262,3). A40926 is the natural precursor of the semi-synthetic derivative dalbavancin, which is currently under clinical development at Biosearch Italia SpA. Dalbavancin shows superior activity against Staphylococci and Enterococci, including some VanB isolates, and has excellent pharmacokinetic characteristics4). Antibiotic A40926 possesses the typical heptapeptide structure of the D-alanyl-D-alanine binding glycopeptides and is characterized by a Nacylaminoglucuronic acid and a mannose moiety bound to the aminoacids number 4 and 7 respectively (Figure 1). Several different related molecules have been identified in the A40926 complex3,5). The two major components of the complex are named B0 (formerly B) and B1 and are characterized respectively by an iso-C12:0 and a n-C12:0 acylic moiety bound to the aminoglucuronic acid of the antibiotic (Figure 1). A40926 aglycone and pseudoaglycones were obtained by chemical hydrolysis and subsequent purification6). The A40926 demannosyl derivative showed improved activity against some classes of Staphylococci and was also used as the starting material for the synthesis of several semisynthetic A40926 derivatives6,7). It is thus of primary importance the development of a rapid and convenient process for the production of demannosyl-A40926. In the course of a screening program for Nonomuraea sp. ATCC 39727 mutants with desirable characteristics (such as high productivity), we have isolated a clone showing a noteworthy activity in an overlay antimicrobial test. This mutant was able to directly produce demannosyl-A40926 with an abundance of ca 95% with respect to the other components of the antibiotic complex. The isolation of this strain allowed demannosyl-A40926 production by a single fermentation/purification process.

Nonomuraea sp. ATCC 39727 is the producer of the A40926 complex of lipoglycopeptide antibiotics which contain chlorine atoms in amino acids 3 and 6 of the peptide backbone. Using a classical mutagenesis and selection approach we have isolated a Nonomuraea sp. ATCC 39727 mutant strain able to direct production towards new A40926 analogues dechloro-A40926 (DDC) lacking two chlorine atoms and the two monochloro-A40926 (MDC1 and MDC2) that are not produced fermenting the wild type strain. Dechlorinated A40926 derivatives were obtained in considerable amount in a standard fermentation process and were purified and chemically characterized. The dechlorinated A40926 derivatives DDC and MDC2 showed improved antimicrobial activity against coagulase negative staphylococci strains in respect to A40926 complex. Dechlorinated derivatives of the related antibiotic teicoplanin are also reported in the literature and are generally less active than the parental products.