Current Pharmaceutical Biotechnology - Volume 10, Issue 1, 2009

The spread of microbial resistance leads to a growing demand for novel antimicrobial drugs. However, despite the significant efforts in academia and the pharmaceutical industry, no genuinely new class of antimicrobial compounds has reached the market in the last years. However, a large variety of substances with potential antimicrobial activity is widely distributed in nature being produced by both prokaryotes and eukaryotes. Therefore, in this special issue of Current Pharmaceutical Biotechnology, we attempt to provide the reader with an overview on antimicrobial substances produced by bacteria, marine sponges, and tropical plants. Concerning prokaryotes, the reviews will focus on bacteriocins, antimicrobial peptides produced by bacteria. Bierbaum and co-workers focus their review on the current developments in the lantibiotic (class I bacteriocins) field and on recent findings concerning mode of action, biosynthesis and engineering of lantibiotics produced by Gram-positive bacteria, while the review written by Nissen-Meyer and co-workers focuses on the structure and mode of action of class II bacteriocins. Structure-function analysis of bacteriocins is particularly relevant for illuminating how these potent bactericidal agents function at a molecular level. Such knowledge is fundamental when considering the rational design of new bacteriocin variants with improved properties that make them especially useful for medical and biotechnological applications. Bastos and co-workers describe the relevant features of staphylococcins, bacteriocins produced by staphylococci, discussing examples of their potential biotechnological applications mainly as antibacterial agents. Diep and co-workers discuss some well-characterised quorum sensing networks involved in regulation of bacteriocin production in lactobacilli, with special focus on the use of the regulatory components in gene expression and on lactobacilli as potential delivery vehicle for therapeutic and vaccine purposes. The review of Lagos and co-workers is focused on microcin E492 features, a bacteriocin that, besides an antimicrobial activity, also displays a cytotoxic effect on tumor cell lines involving apoptosis induction. This trait makes this bacteriocin also suitable for cancer treatment. Laport and co-workers describe an overwhelming number of bioactive substances that have been discovered in sponges and their associated microorganisms. Sponges are considered the most prolific marine producers of novel substances exhibiting antimicrobial activity with a variety of biotechnologically relevant properties.

Lantibiotics are gene-encoded peptides that contain intramolecular ring structures, introduced through the thioether containing lanthionine and methyllanthionine residues. The overwhelming majority of the lantibiotics shows antibacterial activity. Some lantibiotics, e. g. nisin, are characterized by a dual mode of action. These peptides form a complex with the ultimate cell wall precursor lipid II, thereby inhibiting cell wall biosynthesis. The complexes then aggregate, incorporate further peptides and form a pore in the bacterial membrane. Recent results show that complexing of lipid II is widespread among lantibiotics; however, pore formation depends on the overall length of the peptide and the lipid composition of the test strain membrane. In the two-component system of lacticin 3147, the two functions are performed by the two different peptides. The genetic information for production of lantibiotics is organized in gene clusters which contain a structural gene (lanA) for the lantibiotic prepeptide. The modifications are introduced by one biosynthetic enzyme (LanM) or a combination of a dehydratase (LanB) and a cyclase (LanC). These enzymes have been in the focus of recent bioengineering studies: The structure of NisC has been resolved, the reaction mechanism of LctM was elucidated and the active site residues were characterized by mutagenesis studies. In vitro modification systems have successfully been used to introduce thioether rings into other biologically active peptides. Furthermore, variant lantibiotics with enhanced properties have been engineered and at least one promising new lantibiotic with strong activity against multiresistant pathogens has been described.

This review focuses on the structure and mode-of-action of non-lanthionine-containing peptide bacteriocins produced by Gram-positive bacteria. These bacteriocins may be divided into four groups: (i) the anti-listerial one-peptide pediocin-like bacteriocins that have very similar amino acid sequences, (ii) the two-peptide bacteriocins that consist of two different peptides, (iii) the cyclic bacteriocins, and (iv) the linear non-pediocin-like onepeptide bacteriocins. These bacteriocins are largely cationic, contain 20 to 70 residues, and kill cells through membrane- permeabilization. The pediocin-like bacteriocins are the ones that are best characterized. Upon contact with target membranes, their cationic N-terminal half forms a β-sheet-like structure that binds to the target cell surface, while their more hydrophobic helical-containing C-terminal half penetrates into the hydrophobic core of target-cell membranes and apparently binds to the mannose phosphotransferase permease in a manner that results in membrane leakage. Immunity proteins that protect cells from being killed by pediocin-like bacteriocins bind to the bacteriocin- permease complex and prevent bacteriocin-induced membrane-leakage. Recent structural analyses of two-peptide bacteriocins indicate that they form a helix-helix structure that penetrates into cell membranes. Also these bacteriocins may act by binding to integrated membrane proteins. It is proposed that many membrane-active peptide bacteriocins kill target-cells through basically the same mechanism; the common theme being that a membranepenetrating part of bacteriocins bind to a membrane embedded region of an integrated membrane protein, thereby causing conformational alterations in the protein that in turn lead to membrane-leakage and cell death.

Bacteriocins are bacterial antimicrobial peptides with bactericidal activity against other bacteria. Staphylococcins are bacteriocins produced by staphylococci, which are Gram-positive bacteria with medical and veterinary importance. Most bacteriocins produced by staphylococci are either lantibiotics (e.g., Pep5, epidermin, epilancin K7, epicidin 280, staphylococcin C55/BacR1, and nukacin ISK-1) or class II bacteriocins (e.g., aureocins A70 and 53). Only one staphylococcin belonging to class III, lysostaphin, has been described so far. Production of staphylococcins is a selfprotection mechanism that helps staphylococci to survive in their natural habitats. However, since these substances generally have a broad spectrum of activity, inhibiting several human and animal pathogens, they have potential biotechnological applications either as food preservatives or therapeutic agents. Due to the increasing consumer awareness of the risks derived not only from food-borne pathogens, but also from the artificial chemical preservatives used to control them, the interest in the discovery of natural food preservatives has increased considerably. The emergence and dissemination of antibiotic resistance among human and animal pathogens and their association with the use of antibiotics constitute a serious problem worldwide requiring effective measures for controlling their spread. Staphylococcins may be used, solely or in combination with other chemical agents, to avoid food contamination or spoilage and to prevent or treat bacterial infectious diseases. The use of combinations of antimicrobials is common in the clinical setting and expands the spectrum of organisms that can be targeted, prevents the emergence of resistant organisms, decreases toxicity by allowing lower doses of both agents and can result in synergistic inhibition.

Lactobacilli are common microorganisms in diverse vegetables and meat products and several of these are also indigenous inhabitants in the gastro-intestinal (GI) tract of humans and animals where they are believed to have health promoting effects on the host. One of the highly appreciated probiotic effects is their ability to inhibit the growth of pathogens by producing antimicrobial peptides, so-called bacteriocins. Production of some bacteriocins has been shown to be strictly regulated through a quorum-sensing based mechanism mediated by a secreted peptide-pheromone (also called induction peptide; IP), a membrane-located sensor (histidine protein kinase; HPK) and a cytoplasmic response regulator (RR). The interaction between an IP and its sensor, which is highly specific, leads to activation of the cognate RR which in turn binds to regulated promoters and activates gene expression. The HPKs and RRs are built up by conserved modules, and the signalling between them within a network is efficient and directional, and can easily be activated by exogenously added synthetic IPs. Consequently, components from such regulatory networks have successfully been exploited in construction of a number of inducible gene expression systems. In this review, we discuss some well-characterised quorum sensing networks involved in bacteriocin production in lactobacilli, with special focus on the use of the regulatory components in gene expression and on lactobacilli as potential delivery vehicle for therapeutic and vaccine purposes.

Microcins are a family of low-molecular weight bacteriocins produced and secreted by Gram-negative bacteria. This review is focused on microcin E492, a pore-forming bacteriocin produced by Klebsiella pneumoniae RYC492 that exerts its antibacterial action on related strains. The steps necessary for the production of active microcin E492 involve post-translational modification with a catechol-type siderophore at the C-terminal and proteolytic processing during export to the extracellular space. This bacteriocin has a modular structure, with a toxic domain at the N-terminal and an uptake domain at the C-terminal of the mature protein. The mechanism by which the C-terminal of microcin E492 is recognized by catecholate siderophore receptors is called the “Trojan horse” strategy, because the C-terminal structure mimics essential bacterial elements, which are recognized by the respective receptors and translocated across the outer membrane to exert antibacterial action. The C-terminal uptake module can be exchanged and used with other toxic domains. Microcin E492 also has a cytotoxic effect on malignant human cell lines. The cytotoxic mechanism is through apoptosis, a desired mechanism for cancer therapy. The ability of microcin E492 to form amyloid-like fibrils constitutes a property that can be exploited in the formulation of this bacteriocin as an antitumoral agent, because these fibrils can behave as stable depots to ensure the sustained release of a biologically active molecule. Alternatively, live bacteria can be used as a continuous source of microcin E492 production in specific tumors.

Sponges (phylum Porifera) are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms. Marine sponges are among the richest sources of pharmacologically-active chemicals from marine organisms. It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms. More than 5,300 different products are known from sponges and their associated microorganisms, and more than 200 new metabolites from sponges are reported each year. As infectious microorganisms evolve and develop resistance to existing pharmaceuticals, the marine sponge provides novel leads against bacterial, viral, fungal and parasitic diseases. Many marine natural products have successfully advanced to the late stages of clinical trials, as for example ara-A (vidarabine), an anti-viral drug used against the herpes simplex encephalitis virus. This substance is in clinical use for many years. Moreover, a growing number of candidates have been selected as promising leads for extended preclinical assessment, including manzamine A (activity against malaria, tuberculosis, HIV, and others), lasonolides (antifungal activity) and psammaplin A (antibacterial activity). In this review we have surveyed the discoveries of products derived from marine sponges and associated bacteria that have shown in vivo efficacy or potent in vitro activity against infectious and parasitic diseases, including bacterial, viral, fungal and protozoan infections. Our objective was to highlight the susbtances that have the greatest potential to lead to clinically useful treatments.

Medicinal plants constitute the base of health care systems in many societies. The recovery of the knowledge and practices associated with these plant resources are part of an important strategy linked to the conservation of biodiversity, discovery of new medicines, and the bettering of the quality of life of poor rural communities. Research in phytosciences, an emerging multidisciplinary science, is almost unlimited, with several aspects to be discussed. Therefore, the focus of the present review is mainly on the antimicrobial and antioxidant properties of bioactive phytocompounds resultant of our research with crude plant extracts and essential oils of medicinal plants belonging to different families, used in various infectious disorders. The results obtained in the last years warrant the present review, discussing not only the use of several medicinal plants against bacteria, yeast, filamentous fungi and protozoa, but also their mechanisms of action, interactions with macromolecules and potential for toxicity in mammalian cells. Problems related to the efficacy of the isolation techniques and stability of bioactive compounds are also commented on. In addition, this review aims to emphasize the greatest importance to investigate plant species that have not been the subject of pharmacological studies, although their popular uses have been reported.

Drug delivery strategies for peptide pharmaceuticals have incorporated a wide range of structure activity relationships, analog generation to impart protease resistance and increased bioavailability, novel formulations, and delivery systems to target optimal therapeutic dosing requirements. Advances in peptide pharmaceuticals have provided products for the treatment of diabetes, obesity, Crohn's disease, osteoporosis, cancer, cardiovascular disease, immunotherapy, acromegaly, enuresis, pain, and antimicrobials. Here we review these marketed peptides and new peptidomimetic therapies currently in clinical trials.

Glycan-protein interactions play important biological roles in biological processes. But there is a lack of simple high-throughput methods to elucidate recognition events between carbohydrates and protein. Although, there have been a number of glycan arrays developed in recent years utilizing different strategies and for different purposes, the method presented in this paper, a direct covalent immobilization of sugars to hydroxyl-modified glass surface, can be a very useful general method. Here, two strategies have been developed for the production of carbohydrate microarrays by the underivatized sugars that efficiently immobilized on hydroxyl-functionalized glass surface by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. Firstly, untreated glass slides were amino- and epoxy-silanized, respectively. Then, they were further hydroxyl functionalized with different surface chemistries. The carbohydrate microarrays were fabricated on hydroxyl-functionalized glass by robotic arrayer. Additionally, experiments for the characterization of carbohydrates-protein interaction were performed to compare these strategies. Overall best results in terms of conveniency and sensitivity were obtained with hydroxyl-functionalization on epoxysilanized surfaces. The hydroxyl-functionalized slide was used to detect the amount of mannose immobilized on the glass surface. The limit of detection of the fabricated mannose microarray was 100 nM.