Antibacterial Properties of D-Amino Acid Oxidase: Impact on the Food Industry

Food quality is also related to safety and prevention of spoilage. Biological antimicrobial agents represent suitable alternatives to clinical preservatives in food industry to increase both safety and stability of aliments. Here, we focused on the enzyme D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis, a well-studied protein for biotechnological use based on its stability, high activity, and easy recombinant production. DAAO catalyzes the O-2-dependent oxidative deamination of D-enantiomer of amino acids generating alpha -keto acids, ammonia, and hydrogen peroxide. DAAO shows antibacterial activity on both Gram-positive and Gram-negative bacteria in the presence of D-alanine when tested on plates and reduced by half their growth when tested on liquid cultures. Control experiments performed with alternative amino acid-specific flavoenzymes (able or not to generate H2O2 acting on amino acids), a DAAO inactive variant, catalase (H2O2 scavenger), and L-amino acids instead of D-alanine identified H2O2 as the antibacterial agent. DAAO showed a good ability to decrease the bacterial growth on various food stuffs: e.g., 10-fold less colonies were formed on grated cheese incubated for 16 h at 37 degrees C when a tiny amount (0.01 mg corresponding to 1.2 units) of DAAO was added. No exogenous D-amino acids were added since DAAO used the ones naturally occurring or the ones generated during ripening. Notably, simultaneously to H2O2 generation, DAAO also acts as O-2-scavenger thus further hampering food deterioration

Direct chromatographic methods for enantioresolution of amino acids: recent developments

\u3b1-Amino acids are present in two opposite configurations due to the presence of a central carbon atom which is a chiral center. While l-amino acids are present in large amount in nature, only tiny quantities of their d-enantiomers exist. For a long time, d-amino acids have been considered of bacterial origin only, but recently we realized that they are present in all living organisms: notably, d-amino acids play specific and relevant functions in the different organisms. Detection and quantification of d-amino acids are mandatory to shed light on their physiological roles, especially related to foods and human health. Chromatographic techniques are among the most commonly used analytical methods for the enantioseparation of amino acids. Here, we revised the latest improvements in chromatographic direct methodologies based on chiral selectors and aimed to improve analytical speed, sensitivity, robustness, and reproducibility. While current methods are well suited for d-amino acid analysis in foodstuffs and pharmaceuticals, further improvements seem required for their simultaneous, fast and sensitive detection in biological fluids, an emerging field since d-amino acids have been proposed as biomarkers of different and relevant human pathologic states

Characterization of GE82832, a peptide inhibitor of traslocation intercacting with bacterial 30S ribosomal subunits

GE82832, a secondary metabolite produced by Streptosporangium cinnabarinum (strain GE82832), has been identified as a translational inhibitor by in vitro screening of a library of natural products. Secondary functional tests specific for individual steps of the translational pathway demonstrated that translocation is the specific target of GE82832. Chemical probing in situ demonstrated that this antibiotic protects bases A1324 and A1333 and exposes C1336 of 16S rRNA, thereby indicating that its binding site is located on the head of the 30S ribosomal subunit. The ribosomal location of GE82832, near ribosomal protein S13 and G1338, two elements of the small subunit that are part of or close to the B1a intrasubunit bridge, suggests that translocation inhibition results from an altered dynamics of 30S-50S ribosomal subunit interaction

The IN SIGNO project: Identification of novel molecules supporting the impact of β-lactams against clinically-relevant Gram-negative multidrug resistant organisms

The spread of antimicrobial resistance represents an enormous global health crisis, with infections caused by multi-drug resistant bacteria contributing to >1 million deaths per year. A prompt action to limit the impact of this largely unmet medical need is mandatory, and it includes both the preservation of existing antibiotics and the identification of novel molecules. The aim of the IN SIGNO project, recently funded by Fondazione Regionale per la Ricerca Biomedica (Regione Lombardia), is discovering -through a biological activity-guided screening- novel antibiotic adjuvants, which could be used in combination with β-lactams against multidrug-resistant Gram-negative (MDR-GN) bacteria. To this purpose, we first selected five clinically-relevant models of β-lactam resistance in MDR-GN: an extended-spectrum β-lactamase-producing Escherichia coli strain (CTX-M type), three carbapenemase-producing Klebsiella pneumoniae strains (KPC-1, KPC-31, and VIM-1 types), and a VIM-2-producing Pseudomonas aeruginosa. Then, we proceed with the screening of a filamentous actinomycetes/fungi-based microbial library (39,000 crude extracts) and of a chemical library (9,500 pure compounds), to select molecules able to restore the activity of β-lactams against the resistant isolates previously described. The three most promising candidates thus far selected are natural products, since no hits have been identified yet from chemical library. If their activity is confirmed, we will progress in elucidating the chemical structure of the putative inhibitors, test their in vitro and in silico interaction with β-lactamases to better define their mode of action, evaluate activity on a wider panel of MDR-GN clinical isolates, and assess their cytotoxicity on different eukaryotic cells

Novel tetrapeptide inhibitors of bacterial protein synthesis produced by a Streptomyces sp.

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

Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic

Many known antibiotics target the translational apparatus, but none of them can selectively inhibit initiation of protein synthesis and/or is prokaryotic-specific. This article describes the properties of GE81112, an effective and prokaryotic-specific initiation inhibitor. GE81112 is a natural tetrapeptide produced by a Streptomyces sp. identified by an in vitro high-throughput screening test developed to find inhibitors of the prokaryotic translational apparatus preferentially acting on steps other than elongation. In vivo GE81112 inhibits protein synthesis but not other cell functions such as DNA duplication, transcription, and cell wall synthesis. In vitro GE81112 was found to target the 30S ribosomal subunit and to interfere with both coded and noncoded P-site binding of fMet-tRNA, thereby selectively inhibiting formation of the 30S initiation complex