Recent application of metagenomic approaches toward the discovery of antimicrobials and other bioactive small molecules

Bacteria grown in pure culture have been the starting point for the discovery of many of the antibacterials now in use. Metagenomics, which utilizes culture-independent methods to access the collective genomes of natural bacterial populations, provides a means of exploring the antimicrobials produced by the large collections of bacteria that are known to be present in the environment but remain recalcitrant to culturing. Both novel small molecule antibiotics and new antibacterially active proteins have been identified using metagenomic approaches. The recent application of metagenomics to the discovery of bioactive small molecules, small molecule biosynthetic gene clusters and antibacterially active enzymes is discussed here.     

Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules

Culture-independent studies on environmental samples indicate that most bacteria are not readily cultured in the laboratory. The small fraction of bacteria that have been successfully cultured from environmental samples have been a very rewarding source of novel biologically active natural products. The introduction of DNA extracted directly from environmental samples into easily cultured bacteria and the screening of these large libraries for clones that produce biologically active small molecules is one means to access natural products encoded by the genomes of previously uncultured bacteria. This protocol provides detailed procedures for cloning DNA directly from environmental samples and screening these clones for the production of antibacterially active natural products. The entire protocol, from soil sample to the identification of antibacterially active environmental DNA clones, will take approximately 2 weeks.     

Antibacterial properties of the Vietnamese cajeput oil and ocimum oil in combination with antibacterial agents.

Main antibacterially active agents obtained from plants-Cajeput essential oil--1,8 cineol, linalool, alpha-terpineol and terpinen-4-ol, for example from Melalleuce leucadendron (Myrtaceae) as well as essential oil from Ocimum gratissimum (Labiatae) were combined in tests in vitro with selected antibiotics. Above mentioned plant products were found to be effective medicaments for local application in modern medical practice. Combinations with antibiotics potentiated their therapeutical action. On the basis of tests in vitro the synergistic action of these two kinds of medicaments, i.e., preparations traditionally used for a few last decades--antibiotics--might be well applied for therapeutical needs.     

The production, isolation, and characterization of a grisein-like sideromycin complex

Streplomyces griscus var. X-2455 produces an antibiotic complex which is active in vitro against a number of gram-positive and gram-negative bacteria and in mice against systemic infections caused by K, pneumoniae and D, pneumoniac. In view of the favorable chemotherapeutic index and the broad in vitro spectrum of crude concentrates, isolation of the pure antibiotic complex and the individual constituents was undertaken. The antibiotics referred to as Ho 5-2667, Ro 7-7730, and Ho 7-7731 can be differentiated by tle, ultraviolet light absorption spectra, and in vitro antibacterial activities. They all contain iron and may be classified as sideromycins. From antibiotic concentrates an antibacterially inactive substance was isolated and identified as N-acetyltyramine.     

Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124

Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L. gasseri JCM 2124. The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced. Gassericin B2 did not show any sequence similarities to other known bacteriocins. The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus. However, synergistic activity upon complementation of B1 and B3 was not observed. Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3).     

Antimicrobial hydantoin-containing polyesters

A new N-hydantoin-containing biocompatible and enzymatically degradable polyester with antibacterial properties is presented. Different polyesters of dimethyl succinate, 1,4-butanediol, and 3-[N,N-di(β-hydroxyethyl)aminoethyl]-5,5-dimethylhydantoin in varying molar ratios are prepared via two-step melt polycondensation. The antibacterially active N-halamine form is obtained by subsequent chlorination of the polyesters with sodium hypochlorite. Chemical structures, thermal properties, and spherulitic morphologies of the copolymers are studied adopting FT-IR, NMR, TGA, DSC, WAXD, and POM. The polyesters exhibit antibacterial activity against Escherichia coli. The adopted synthetic approach can be transferred to other polyesters in a straightforward manner.     

Penicillin-bound polyacrylate nanoparticles: restoring the activity of beta-lactam antibiotics against MRSA

This report describes the preparation of antibacterially active emulsified polyacrylate nanoparticles in which a penicillin antibiotic is covalently conjugated onto the polymeric framework. These nanoparticles were prepared in water by emulsion polymerization of an acrylated penicillin analogue pre-dissolved in a 7:3 (w:w) mixture of butyl acrylate and styrene in the presence of sodium dodecyl sulfate (surfactant) and potassium persulfate (radical initiator). Dynamic light scattering analysis and atomic force microscopy images show that the emulsions contain nanoparticles of approximately 40 nm in diameter. The nanoparticles have equipotent in vitro antibacterial properties against methicillin-susceptible and methicillin-resistant forms of Staphylococcus aureus and indefinite stability toward beta-lactamase.     

Long-chain N-acyltyrosine synthases from environmental DNA

The heterologous expression of DNA extracted directly from environmental samples (environmental DNA [eDNA]) in easily cultured hosts provides access to natural products produced by previously inaccessible microorganisms. When eDNA cosmid libraries were screened in Escherichia coli for antibacterially active clones, long-chain N-acyltyrosine-producing clones were found in every eDNA library. These apparently common natural products have not been previously described from screening extracts of cultured bacteria for biologically active natural products. Of the 11 long-chain N-acyl amino acid synthases (NASs) that were characterized, 10 are unique sequences. A predicted protein of previously unknown function from Nitrosomonas europaea, a gram-negative nitrifying beta-proteobacterium, is 14 to 37% identical to eDNA NASs. When cloned into E. coli, this open reading frame confers the production of long-chain N-acyltyrosines to the host and is therefore the first NAS from a cultured bacterium to be functionally characterized. Understanding the role that long-chain N-acyl amino acids play in soil microbial communities should now be feasible with the identification of a cultured organism that has the genetic capacity to produce these compounds.     

Screening for biologically active metabolites with endosymbiotic bacilli isolated from arthropods

Endosymbiotic bacteria from the genus Bacillus were isolated from different compartments of the gut of various members of insects (Hexapoda) and millipedes (Diplopoda). They were grown in submerged culture and investigated by biological assays and HPLC-diode array analysis regarding their production of bioactive metabolites, which were isolated and determined in structure. Known compounds and yet unknown derivatives from the primary metabolism were detected, as well as antibacterially and antifungally acting peptide antibiotics.     

Long-Chain N-Acyltyrosine Synthases from Environmental DNA

The heterologous expression of DNA extracted directly from environmental samples (environmental DNA [eDNA]) in easily cultured hosts provides access to natural products produced by previously inaccessible microorganisms. When eDNA cosmid libraries were screened in Escherichia coli for antibacterially active clones, long-chain N-acyltyrosine-producing clones were found in every eDNA library. These apparently common natural products have not been previously described from screening extracts of cultured bacteria for biologically active natural products. Of the 11 long-chain N-acyl amino acid synthases (NASs) that were characterized, 10 are unique sequences. A predicted protein of previously unknown function from Nitrosomonas europaea, a gram-negative nitrifying beta-proteobacterium, is 14 to 37% identical to eDNA NASs. When cloned into E. coli, this open reading frame confers the production of long-chain N-acyltyrosines to the host and is therefore the first NAS from a cultured bacterium to be functionally characterized. Understanding the role that long-chain N-acyl amino acids play in soil microbial communities should now be feasible with the identification of a cultured organism that has the genetic capacity to produce these compounds.     

Insect immunity. Attacins,a family of antibacterial proteins from Hyalophora cecropia.

Six closely related antibacterial proteins, attacins A-F, were isolated from the hemolymph of immunized pupae of the Cecropia moth, Hyalophora cecropia. Chromatofocusing separated attacins A-F, with isoelectric points between 5.7 and 8.3. Immunological experiments show that the attacins constitute antibacterially active forms of the previously isolated inducible immune protein P5. Their mol. wts., 20-23 K, are similar to that of protein P5, but significantly lower than 28 K found for preP5 synthesized in vitro (see accompanying paper). The six attacins can be divided into two groups according to their amino acid composition and amino-terminal sequences, attacins A-D constitute a basic group and attacins E and F an acidic one. Within each group the forms are very similar. The attacins efficiently killed Escherichia coli and two other Gram-negative bacteria isolated from the gut of a silk worm but they did not act on other Gram-positive and Gram-negative bacteria tested. Only growing cells of E. coli were attacked; cells suspended in phosphate buffer were inert. Besides the cecropins and lysozyme, the attacins represent a third class of antibacterial proteins in the humoral immune system of H. cecropia.     

甘草根茎乙醇提取物抗菌活性研究

本实验采用琼脂扩散法和微量肉汤稀释法,研究了甘草根茎乙醇提取物对5种细菌(表皮葡萄球菌、金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌和绿脓杆菌)和2种真菌(白色念珠菌和黑曲霉)的抗菌活性。结果表明,甘草根茎乙醇提取物对革兰氏阳性菌非常敏感,而对革兰氏阴性菌和真菌不敏感,80%乙醇提取物对革兰氏阳性菌的MIC范围为0.156～0.312 mg·mL^-1,而10%乙醇提取物对革兰氏阳性菌的MIC范围为0.625～1.250 mg·mL^-1,表明甘草根茎抗菌活性成分在高浓度乙醇中溶解度较大,为临床上应用甘草根茎醇提物作为抗菌制剂提供了科学依据。     

Acidic protease from human seminal plasma. Purification and some properties of active enzyme and of proenzyme.

A procedure to purify to homogeneity the active form as well as the proenzyme form of the acidic protease of human seminal plasma is described. This involved precipitation with ammonium sulfate, chromatography on diethylaminoethylcellulose, Sephadex G-200, and Sephadex G-100. The molecular weights of the active form and of the proenzyme were determined by electrophoresis and gel filtration to be 35,000 and 42,000, respectively. The proenzyme was more stable than the active form in alkaline solution and can be converted into the active enzyme under acidic conditions. The active form of the acidic protease can hydrolyze hemoglobin, N,N'-dimethylcasein, N-acetyl-L-phenylalanyl-L-diiodotyrosine, and N-benzyloxycarbonyl-L-glutamyl-L-phenylalanine, but cannot hydrolyze bovine serum albumin, ovalbumin, N-benzyloxycarbonyl-L-glutamyl-L-tyrosine. The active form was also inhibited by p-bromophenacyl bromide and 1,2-epoxy-3-(p-nitrophenoxy)propane.     

Bacterial Viability and Physical Properties of Antibacterially Modified Experimental Dental Resin Composites

Purpose

To investigate the antibacterial effect and the effect on the material properties of a novel delivery system with Irgasan as active agent and methacrylated polymerizable Irgasan when added to experimental dental resin composites.

Materials and Methods

A delivery system based on novel polymeric hollow beads, loaded with Irgasan and methacrylated polymerizable Irgasan as active agents were used to manufacture three commonly formulated experimental resin composites. The non-modified resin was used as standard (ST). Material A contained the delivery system providing 4 % (m/m) Irgasan, material B contained 4 % (m/m) methacrylated Irgasan and material C 8 % (m/m) methacrylated Irgasan. Flexural strength (FS), flexural modulus (FM), water sorption (WS), solubility (SL), surface roughness R_a, polymerization shrinkage, contact angle Θ, total surface free energy γ_S and its apolar γ_S ^LW, polar γ_S ^AB, Lewis acid γ_S ⁺and base γ_S ^- term as well as bacterial viability were determined. Significance was p < 0.05.

Results

The materials A to C were not unacceptably influenced by the modifications and achieved the minimum values for FS, WS and SL as requested by EN ISO 4049 and did not differ from ST what was also found for R_a. Only A had lower FM than ST. Θ of A and C was higher and γ_S ^AB of A and B was lower than of ST. Materials A to C had higher γ_S ⁺ than ST. The antibacterial effect of materials A to C was significantly increased when compared with ST meaning that significantly less vital cells were found.

Conclusion

Dental resin composites with small quantities of a novel antibacterially doped delivery system or with an antibacterial monomer provided acceptable physical properties and good antibacterial effectiveness. The sorption material being part of the delivery system can be used as a vehicle for any other active agent.     

Diffuse reflectance infrared Fourier transform spectroscopic characterization of a silica-immobilized N-hydroxysuccinimide active ester crosslinking agent and its precursors

Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy was used to characterize the product of each step in the preparation of a silica-immobilized N-hydroxysuccinimide (NHS) active ester. The preparation of this NHS active ester linkage was based on a literature procedure for the immobilization of proteins. The DRIFT method was used to guide modification of this literature procedure. The DRIFT method also was used to indicate an impurity entrapped in the 60-A diameter pores of the silica support during the formation of the immobilized active ester. Degradation of the immobilized NHS active ester, stored under either argon or dioxane, can be followed by the DRIFT method. Myoglobin and glycine were allowed to react with the active ester, and the result for this silica support was evaluated by the DRIFT method. Elemental analysis was used to provide information on the loading of the silica-immobilized moieties that were presented for DRIFT analysis.     

Effect of rare earth cations on bactericidal activity of anionic surfactants

Summary Rare earth metal cations are antibacterially synergistic with anionic surfactants, yielding mixtures that have bactericidal activity against a variety of gram-positive and gram-negative bacteria at minimum concentrations ranging from 16 to 125 g/ml. Uptake of surfactant byEscherichia coli increases in the presence of lanthanum, suggesting that the role of rare earth metal cations is to reduce the net negative surface charge on the bacteria, thereby increasing the affinity between the negatively charged surfactant and the bacterial surface.     

Absence of in vitro innate immunomodulation by insect‐derived short proline‐rich antimicrobial peptides points to direct antibacterial action in vivo

Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well‐known immunomodulatory activities of murine cathelicidin‐related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro‐inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS‐mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Large-scale preparation and biological activity of recombinant human parathyroid hormone

Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-β-galactosidase-hPTH fusion protein. We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step Chromatographie procedure. We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active.     

Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach to the homology modeling of matrix metalloproteinases, exemplified by the modeling of MMP2, MMP9, MMP12 and MMP14 is described. The models were refined using an energy minimization procedure developed for matrix metalloproteinases. This procedure includes incorporation of parameters for zinc and calcium ions in the AMBER 4.1 force field, applying a non-bonded approach and a full ion charge representation. Energy minimization of the apoenzymes yielded structures with distorted active sites, while reliable three-dimensional structures of the enzymes containing a substrate in active site were obtained. The structural differences between the eight enzyme-substrate complexes were studied with particular emphasis on the active site, and possible sites for obtaining selectivity among the MMP's are discussed. Differences in the P1' pocket are well-documented and have been extensively exploited in inhibitor design. The present work indicates that selectivity could be further improved by considering the P2 pocket as well.     

Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome

We developed an aqueous spreading procedure that permits simultaneous analysis of human chromosomes by Q-banding and indirect immunofluorescence. Using this methodology and anticentromere antibodies from an autoimmune patient we compared the active and inactive centromeres of an isodicentric X chromosome. We show that a family of structurally related human centromere proteins (CENP-A, CENP-B, and CENP-C) is detectable only at the active centromere. These antigens therefore may be regarded both as morphological and functional markers for active centromeres.