Elisa Binda | Università degli Studi dell'Insubria (original) (raw)

Papers by Elisa Binda

Journal of Biotechnology, 2010

Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes ... more Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes as lipases and esterases are often used to resolve racemic mixture of esters. We propose a enantiospecific enzymatic resolution in order to produce enatiopure synthons precursor of pharmaceutical active substances.

VanY n is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 397... more VanY n is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727, which produces the glycopeptide antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase III of clinical development. VanY n (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanY n was successfully expressed as a soluble and active protein in Escherichia coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn 2+ and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanY n as in VanY, VanX and VanXY Zn 2+ -dependent D,D-carboxypeptidases and D-Ala-D-Ala dipeptidases acting on peptidoglycan maturation and involved in glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanY n shows D,D-carboxypeptidase and D,D-dipeptidase activity, but lacks D,D-carboxyesterase ability on D-Ala-D-Lac-terminating peptides. VanY n belongs to the metallo-D,D-carboxypeptidase family, but it is inhibited by b-lactams.

Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic ... more Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic dalbavancin), was isolated from a soil sample collected in India, and it was originally classified as a member of the genus Actinomadura on the base of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicates that the strain forms a distinct clade within the genus Nonomuraea, and it is most closely related to Nonomuraea angiospora DSM 43173 T (98.72 % similarity) and Nonomuraea jabiensis A4036 T (98.69 %). The strain forms an extensively branched substrate mycelium and aerial hyphae that form spiral chains of spores with ridged surfaces. The cell wall contains meso-diaminopimelic acid and the whole-cell sugars are glucose, ribose, galactose, mannose and madurose (madurose as the diagnostic sugar). The N-acyl type of muramic acid is acetyl. The predominant menaquinone is MK-9(H 4), with minor amounts of MK-9(H 2), MK-9(H 6) and MK-9(H 0). The polar-lipid profile includes diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylmethylethanolamine, phosphatidylinositol and a series of uncharacterized phospholipids, glycolipids and phosphoglycolipids. The major cellular fatty acids are iso-C 16 : 0 and 10-methyl C 17 : 0. The genomic DNA G+C content is 71.2 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA–DNA relatedness between strain ATCC 39727 and closely related type strains, clearly demonstrated that strain ATCC 39727 represents a novel species of the genus Nonomuraea, for which the name Nonomuraea gerenzanensis sp. nov. is proposed. The type strain is ATCC 39727 T (5DSM 100948 T).

This work contributes to the understanding of cell wall modifications during sporulation and germ... more This work contributes to the understanding of cell wall modifications during sporulation and germination in Streptomyces by assessing the biological function and biochemical properties of SCO4439, a D-alanyl-D-alanine carboxypeptidase (DD-CPase) constitutively expressed during development. SCO4439 harbors a DD-CPase domain and a putative transcriptional regulator domain, separated by a putative transmembrane region. The recombinant protein shows that DD-CPase activity is inhibited by penicillin G. The spores of the SCO4439::Tn5062 mutant are affected in their resistance to heat and acid and showed a dramatic increase in swelling during germination. The mycelium of the SCO4439::Tn5062 mutant is more sensitive to glycopeptide antibiotics (vancomycin and teicoplanin). The DD-CPase domain and the hydrophobic transmembrane region are highly conserved in Streptomyces, and both are essential for complementing the wild type phenotypes in the mutant. A model for the biological mechanism behind the observed phenotypes is proposed, in which SCO4439 DD-CPase releases D-Ala from peptidoglycan (PG) precursors, thereby reducing the substrate pool for PG crosslinking (transpeptidation). PG crosslinking regulates spore physical resistance and germination, and modulates mycelium resistance to glycopeptides. This study is the first demonstration of the role of a DD-CPase in the maturation of the spore cell wall. Streptomycetes are mycelial microorganisms characterized by their complex developmental cycles, including programmed cell death (PCD) and hyphae differentiation, which leads to aerial mycelium formation and sporulation 1,2. Streptomycetes are important industrial bacteria producing approximately two-thirds of clinical antibiotics, as well as a large number of eukaryotic cell differentiation inducers and inhibitors 3. Most of these bioactive compounds are specialized metabolites 4 , the production of which is regulated, at least in part, by hyphal differentiation 5. Streptomyces development, is activated by extracellular signals, including nutritional stimuli or cell density (quorum sensing), and is regulated by complex signaling pathways that are only partially known 5–7. The best-characterized stages of Streptomyces development are those related to aerial mycelium and sporulation. Several key regulatory networks controlling these stages have been characterised (bald, sky or white pathways, reviewed in Flärdh and Buttner 1). Despite this, the regulation of aerial mycelium and sporulation remains incompletely understood, and new genes and proteins regulating these important processes, are still being discovered 8 .

Antimicrobial agents and chemotherapy, 2014

Glycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; ... more Glycopeptides and β-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 β-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 fro...

Microbial Cell Factories, 2013

Background: A number of valuable candidates as tuberculosis vaccine have been reported, some of w... more Background: A number of valuable candidates as tuberculosis vaccine have been reported, some of which have already entered clinical trials. The new vaccines, especially subunit vaccines, need multiple administrations in order to maintain adequate life-long immune memory: this demands for high production levels and degree of purity. Results: In this study, TB10.4, Ag85B and a TB10.4-Ag85B chimeric protein (here-after referred as full) -immunodominant antigens of Mycobacterium tuberculosis -were expressed in Escherichia coli and purified to homogeneity. The rational design of expression constructs and optimization of fermentation and purification conditions allowed a marked increase in solubility and yield of the recombinant antigens. Indeed, scaling up of the process guaranteed mass production of all these three antigens (2.5-25 mg of pure protein/L cultivation broth). Quality of produced soluble proteins was evaluated both by mass spectrometry to assess the purity of final preparations, and by circular dichroism spectroscopy to ascertain the protein conformation. Immunological tests of the different protein products demonstrated that when TB10.4 was fused to Ag85B, the chimeric protein was more immunoreactive than either of the immunogenic protein alone.

Journal of Industrial Microbiology & Biotechnology, 2010

Nonomuraea sp. ATCC 39727 belongs to the Streptosporangiaceae family of filamentous actinomycetes... more Nonomuraea sp. ATCC 39727 belongs to the Streptosporangiaceae family of filamentous actinomycetes. This microorganism produces the teicoplanin-like glycopeptide A40926, which is the starting material for the synthesis of the second-generation glycopeptide dalbavancin. Notwithstanding the strain's pharmaceutical relevance, the lack or poor efficiency of genetic tools to manipulate Nonomuraea sp. ATCC 39727 has hampered strain and product improvement. Here we report the development of gene transfer systems based on protoplast transformation and intergeneric conjugation from Escherichia coli. Efficiency of transformation and conjugation, followed by site specific or homologous recombination with the Nonomuraea sp. genome, were determined using the integrative plasmid pSET152 (5.7 kb), and the Super-cos1 derivative cosmid A40DY (30 kb). To our knowledge, this is the first report of the transformation of protoplasts of Nonomuraea sp. ATCC 39727, even though the improved procedure for intergeneric conjugation makes it the method of choice for introducing large segments of DNA into Nonomuraea sp. ATCC 39727.

Journal of Biotechnology, 2010

Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes ... more Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes as lipases and esterases are often used to resolve racemic mixture of esters. We propose a enantiospecific enzymatic resolution in order to produce enatiopure synthons precursor of pharmaceutical active substances.

BMC Biotechnology, 2013

Background: VanY n , encoded by the dbv7 gene (also known as vanY n ) of the biosynthetic cluster... more Background: VanY n , encoded by the dbv7 gene (also known as vanY n ) of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727. This filamentous actinomycete is an uncommon microorganism, difficult-to-handle but biotechnologically valuable since it produces the glycopeptide antibiotic A40926, which is the precursor of the second-generation dalbavancin in phase III of clinical development. In order to investigate VanY n role in glycopeptide resistance in the producer actinomycete an appropriate host-vector expression system is required. Results: The cloning strategy of vanY n gene (G-C ratio 73.3%) in the expression vector pIJ86 yielded a recombinant protein with a tag encoding for a histidine hexamer added at the C-terminus (C-His 6 -vanY n ) or at the N-terminus (N-His 6 -vanY n ). These plasmids were used to transform three Streptomyces spp., which are genetically-treatable high G-C content Gram-positive bacteria taxonomically related to the homologous producer Nonomuraea sp.. Highest yield of protein expression and purification (12 mg of protein per liter of culture at 3 L bioreactor-scale) was achieved in Streptomyces venezuelae ATCC 10595, that is a fast growing streptomyces susceptible to glycopeptides. VanY n is a transmembrane protein which was easily detached and recovered from the cell wall fraction. Purified C-His 6 -VanY n showed D,D-carboxypeptidase and D,D-dipeptidase activities on synthetic analogs of bacterial peptidoglycan (PG) precursors. C-His 6 -VanY n over-expression conferred glycopeptide resistance to S. venezuelae. On the contrary, the addition of His 6 -tag at the N-terminus of the protein abolished its biological activity either in vitro or in vivo assays. Conclusions: Heterologous expression of vanY n from Nonomuraea sp. ATCC 39727 in S. venezuelae was successfully achieved and conferred the host an increased level of glycopeptide resistance. Cellular localization of recombinant VanY n together with its enzymatic activity as a D,D-peptidase/D,D-carboxypeptidase agree with its role in removing the last D-Ala from the pentapeptide PG precursors and reprogramming cell wall biosynthesis, as previously reported in glycopeptide resistant pathogens.

Antimicrobial Agents and Chemotherapy, 2010

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by rep... more 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 g/ml, but this increased to ca. 20 g/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

Antibiotics, 2014

Glycopeptides are considered antibiotics of last resort for the treatment of life-threatening inf... more Glycopeptides are considered antibiotics of last resort for the treatment of life-threatening infections caused by relevant Gram-positive human pathogens, such as Staphylococcus aureus, Enterococcus spp. and Clostridium difficile. The emergence of glycopeptide-resistant clinical isolates, first among enterococci and then in staphylococci, has prompted research for second generation glycopeptides and a flurry of activity aimed at understanding resistance mechanisms and their evolution. Glycopeptides are glycosylated non-ribosomal peptides produced by a diverse group of soil actinomycetes. They target Gram-positive bacteria by binding to the acyl-D-alanyl-D-alanine (D-Ala-D-Ala) terminus of the growing peptidoglycan on the outer surface of the cytoplasmatic membrane. Glycopeptide-resistant organisms avoid such a fate by replacing the D-Ala-D-Ala terminus with D-alanyl-D-lactate (D-Ala-D-Lac) or D-alanyl-D-serine (D-Ala-D-Ser), thus markedly reducing antibiotic affinity for the cellular target. Resistance has manifested itself in enterococci and staphylococci largely through the expression of genes (named van) encoding proteins that reprogram cell wall biosynthesis and, thus, evade the action of the antibiotic. These resistance mechanisms were most likely co-opted from the glycopeptide producing actinomycetes, which use them to avoid suicide during antibiotic production, rather than being orchestrated by pathogen bacteria upon continued treatment. van-like gene clusters, similar to those described in enterococci, were in fact identified in many glycopeptideproducing actinomycetes, such as Actinoplanes teichomyceticus, which produces teicoplanin,

Journal of Biotechnology, 2010

Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes ... more Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes as lipases and esterases are often used to resolve racemic mixture of esters. We propose a enantiospecific enzymatic resolution in order to produce enatiopure synthons precursor of pharmaceutical active substances.

VanY n is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 397... more VanY n is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727, which produces the glycopeptide antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase III of clinical development. VanY n (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanY n was successfully expressed as a soluble and active protein in Escherichia coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn 2+ and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanY n as in VanY, VanX and VanXY Zn 2+ -dependent D,D-carboxypeptidases and D-Ala-D-Ala dipeptidases acting on peptidoglycan maturation and involved in glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanY n shows D,D-carboxypeptidase and D,D-dipeptidase activity, but lacks D,D-carboxyesterase ability on D-Ala-D-Lac-terminating peptides. VanY n belongs to the metallo-D,D-carboxypeptidase family, but it is inhibited by b-lactams.

Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic ... more Strain ATCC 39727, which produces the antibiotic A40926 (the natural precursor of the antibiotic dalbavancin), was isolated from a soil sample collected in India, and it was originally classified as a member of the genus Actinomadura on the base of morphology and cell-wall composition. A phylogenetic analysis based on 16S rRNA gene sequences indicates that the strain forms a distinct clade within the genus Nonomuraea, and it is most closely related to Nonomuraea angiospora DSM 43173 T (98.72 % similarity) and Nonomuraea jabiensis A4036 T (98.69 %). The strain forms an extensively branched substrate mycelium and aerial hyphae that form spiral chains of spores with ridged surfaces. The cell wall contains meso-diaminopimelic acid and the whole-cell sugars are glucose, ribose, galactose, mannose and madurose (madurose as the diagnostic sugar). The N-acyl type of muramic acid is acetyl. The predominant menaquinone is MK-9(H 4), with minor amounts of MK-9(H 2), MK-9(H 6) and MK-9(H 0). The polar-lipid profile includes diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxyphosphatidylmethylethanolamine, phosphatidylinositol and a series of uncharacterized phospholipids, glycolipids and phosphoglycolipids. The major cellular fatty acids are iso-C 16 : 0 and 10-methyl C 17 : 0. The genomic DNA G+C content is 71.2 mol%. Significant differences in the morphological, chemotaxonomic and biochemical data, together with DNA–DNA relatedness between strain ATCC 39727 and closely related type strains, clearly demonstrated that strain ATCC 39727 represents a novel species of the genus Nonomuraea, for which the name Nonomuraea gerenzanensis sp. nov. is proposed. The type strain is ATCC 39727 T (5DSM 100948 T).

This work contributes to the understanding of cell wall modifications during sporulation and germ... more This work contributes to the understanding of cell wall modifications during sporulation and germination in Streptomyces by assessing the biological function and biochemical properties of SCO4439, a D-alanyl-D-alanine carboxypeptidase (DD-CPase) constitutively expressed during development. SCO4439 harbors a DD-CPase domain and a putative transcriptional regulator domain, separated by a putative transmembrane region. The recombinant protein shows that DD-CPase activity is inhibited by penicillin G. The spores of the SCO4439::Tn5062 mutant are affected in their resistance to heat and acid and showed a dramatic increase in swelling during germination. The mycelium of the SCO4439::Tn5062 mutant is more sensitive to glycopeptide antibiotics (vancomycin and teicoplanin). The DD-CPase domain and the hydrophobic transmembrane region are highly conserved in Streptomyces, and both are essential for complementing the wild type phenotypes in the mutant. A model for the biological mechanism behind the observed phenotypes is proposed, in which SCO4439 DD-CPase releases D-Ala from peptidoglycan (PG) precursors, thereby reducing the substrate pool for PG crosslinking (transpeptidation). PG crosslinking regulates spore physical resistance and germination, and modulates mycelium resistance to glycopeptides. This study is the first demonstration of the role of a DD-CPase in the maturation of the spore cell wall. Streptomycetes are mycelial microorganisms characterized by their complex developmental cycles, including programmed cell death (PCD) and hyphae differentiation, which leads to aerial mycelium formation and sporulation 1,2. Streptomycetes are important industrial bacteria producing approximately two-thirds of clinical antibiotics, as well as a large number of eukaryotic cell differentiation inducers and inhibitors 3. Most of these bioactive compounds are specialized metabolites 4 , the production of which is regulated, at least in part, by hyphal differentiation 5. Streptomyces development, is activated by extracellular signals, including nutritional stimuli or cell density (quorum sensing), and is regulated by complex signaling pathways that are only partially known 5–7. The best-characterized stages of Streptomyces development are those related to aerial mycelium and sporulation. Several key regulatory networks controlling these stages have been characterised (bald, sky or white pathways, reviewed in Flärdh and Buttner 1). Despite this, the regulation of aerial mycelium and sporulation remains incompletely understood, and new genes and proteins regulating these important processes, are still being discovered 8 .

Antimicrobial agents and chemotherapy, 2014

Glycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; ... more Glycopeptides and β-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 β-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 fro...

Microbial Cell Factories, 2013

Background: A number of valuable candidates as tuberculosis vaccine have been reported, some of w... more Background: A number of valuable candidates as tuberculosis vaccine have been reported, some of which have already entered clinical trials. The new vaccines, especially subunit vaccines, need multiple administrations in order to maintain adequate life-long immune memory: this demands for high production levels and degree of purity. Results: In this study, TB10.4, Ag85B and a TB10.4-Ag85B chimeric protein (here-after referred as full) -immunodominant antigens of Mycobacterium tuberculosis -were expressed in Escherichia coli and purified to homogeneity. The rational design of expression constructs and optimization of fermentation and purification conditions allowed a marked increase in solubility and yield of the recombinant antigens. Indeed, scaling up of the process guaranteed mass production of all these three antigens (2.5-25 mg of pure protein/L cultivation broth). Quality of produced soluble proteins was evaluated both by mass spectrometry to assess the purity of final preparations, and by circular dichroism spectroscopy to ascertain the protein conformation. Immunological tests of the different protein products demonstrated that when TB10.4 was fused to Ag85B, the chimeric protein was more immunoreactive than either of the immunogenic protein alone.

Journal of Industrial Microbiology & Biotechnology, 2010

Nonomuraea sp. ATCC 39727 belongs to the Streptosporangiaceae family of filamentous actinomycetes... more Nonomuraea sp. ATCC 39727 belongs to the Streptosporangiaceae family of filamentous actinomycetes. This microorganism produces the teicoplanin-like glycopeptide A40926, which is the starting material for the synthesis of the second-generation glycopeptide dalbavancin. Notwithstanding the strain's pharmaceutical relevance, the lack or poor efficiency of genetic tools to manipulate Nonomuraea sp. ATCC 39727 has hampered strain and product improvement. Here we report the development of gene transfer systems based on protoplast transformation and intergeneric conjugation from Escherichia coli. Efficiency of transformation and conjugation, followed by site specific or homologous recombination with the Nonomuraea sp. genome, were determined using the integrative plasmid pSET152 (5.7 kb), and the Super-cos1 derivative cosmid A40DY (30 kb). To our knowledge, this is the first report of the transformation of protoplasts of Nonomuraea sp. ATCC 39727, even though the improved procedure for intergeneric conjugation makes it the method of choice for introducing large segments of DNA into Nonomuraea sp. ATCC 39727.

Journal of Biotechnology, 2010

Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes ... more Biocatalysis has become recently a promising tool for chemical synthesis. In particular, enzymes as lipases and esterases are often used to resolve racemic mixture of esters. We propose a enantiospecific enzymatic resolution in order to produce enatiopure synthons precursor of pharmaceutical active substances.

BMC Biotechnology, 2013

Background: VanY n , encoded by the dbv7 gene (also known as vanY n ) of the biosynthetic cluster... more Background: VanY n , encoded by the dbv7 gene (also known as vanY n ) of the biosynthetic cluster devoted to A40926 production, is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727. This filamentous actinomycete is an uncommon microorganism, difficult-to-handle but biotechnologically valuable since it produces the glycopeptide antibiotic A40926, which is the precursor of the second-generation dalbavancin in phase III of clinical development. In order to investigate VanY n role in glycopeptide resistance in the producer actinomycete an appropriate host-vector expression system is required. Results: The cloning strategy of vanY n gene (G-C ratio 73.3%) in the expression vector pIJ86 yielded a recombinant protein with a tag encoding for a histidine hexamer added at the C-terminus (C-His 6 -vanY n ) or at the N-terminus (N-His 6 -vanY n ). These plasmids were used to transform three Streptomyces spp., which are genetically-treatable high G-C content Gram-positive bacteria taxonomically related to the homologous producer Nonomuraea sp.. Highest yield of protein expression and purification (12 mg of protein per liter of culture at 3 L bioreactor-scale) was achieved in Streptomyces venezuelae ATCC 10595, that is a fast growing streptomyces susceptible to glycopeptides. VanY n is a transmembrane protein which was easily detached and recovered from the cell wall fraction. Purified C-His 6 -VanY n showed D,D-carboxypeptidase and D,D-dipeptidase activities on synthetic analogs of bacterial peptidoglycan (PG) precursors. C-His 6 -VanY n over-expression conferred glycopeptide resistance to S. venezuelae. On the contrary, the addition of His 6 -tag at the N-terminus of the protein abolished its biological activity either in vitro or in vivo assays. Conclusions: Heterologous expression of vanY n from Nonomuraea sp. ATCC 39727 in S. venezuelae was successfully achieved and conferred the host an increased level of glycopeptide resistance. Cellular localization of recombinant VanY n together with its enzymatic activity as a D,D-peptidase/D,D-carboxypeptidase agree with its role in removing the last D-Ala from the pentapeptide PG precursors and reprogramming cell wall biosynthesis, as previously reported in glycopeptide resistant pathogens.

Antimicrobial Agents and Chemotherapy, 2010

In glycopeptide-resistant enterococci and staphylococci, high-level resistance is achieved by rep... more 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 g/ml, but this increased to ca. 20 g/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

Antibiotics, 2014

Glycopeptides are considered antibiotics of last resort for the treatment of life-threatening inf... more Glycopeptides are considered antibiotics of last resort for the treatment of life-threatening infections caused by relevant Gram-positive human pathogens, such as Staphylococcus aureus, Enterococcus spp. and Clostridium difficile. The emergence of glycopeptide-resistant clinical isolates, first among enterococci and then in staphylococci, has prompted research for second generation glycopeptides and a flurry of activity aimed at understanding resistance mechanisms and their evolution. Glycopeptides are glycosylated non-ribosomal peptides produced by a diverse group of soil actinomycetes. They target Gram-positive bacteria by binding to the acyl-D-alanyl-D-alanine (D-Ala-D-Ala) terminus of the growing peptidoglycan on the outer surface of the cytoplasmatic membrane. Glycopeptide-resistant organisms avoid such a fate by replacing the D-Ala-D-Ala terminus with D-alanyl-D-lactate (D-Ala-D-Lac) or D-alanyl-D-serine (D-Ala-D-Ser), thus markedly reducing antibiotic affinity for the cellular target. Resistance has manifested itself in enterococci and staphylococci largely through the expression of genes (named van) encoding proteins that reprogram cell wall biosynthesis and, thus, evade the action of the antibiotic. These resistance mechanisms were most likely co-opted from the glycopeptide producing actinomycetes, which use them to avoid suicide during antibiotic production, rather than being orchestrated by pathogen bacteria upon continued treatment. van-like gene clusters, similar to those described in enterococci, were in fact identified in many glycopeptideproducing actinomycetes, such as Actinoplanes teichomyceticus, which produces teicoplanin,