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Biomolecules as Host Defense Weapons Against Microbial Pathogens

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Biomolecules as Host Defense Weapons Against Microbial Pathogens
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   Recent Patents on DNA & Gene Sequences 2008,  2,  000-000   1   1872-2156/08 $100.00+.00 © 2008 Bentham Science Publishers Ltd.   Biomolecules as Host Defense Weapons Against Microbial Pathogens Marco Dalla Rizza 1, * ,  Paola Díaz Dellavalle 1 , Rafael Narancio 1 , Andrea Cabrera 1  and Fernando Ferreira 2   1  Biotechnology Unit, National Institute of Agricultural Research, INIA Las Brujas, Ruta 48 Km 10, Canelones, Uruguay, 2 Faculty of Chemistry, University of Uruguay (UdelaR), Montevideo, Uruguay  Received: April 21, 2008; Accepted: April 29, 2008; Revised: April 29, 2008    Abstract:  Antimicrobial peptides have been considered a new source of biomolecules in several fields of research/innovative applications: they would adjust to an ideal behavior seeking to overcome clinicians, microbiological, human-animal-plant-environmental concerns. Antimicrobial peptides can be considered as ancient weapons found in living organisms suggesting they have played a fundamental role in his successful co-evolution with pathogens. Acting on microorganism membrane or having intracellular targets, they can also act as effectors of the innate immune response resulting on non-specific mechanisms of action. Two elements have speeded the research on pathogen control alternatives: a verified increase of antibiotic resistance and the relevance of finding amenable environmental compounds in plant health. As a result of its importance, great efforts have been accomplished to find, characterize, combine and synthesize effective antimicrobial peptides. This review intends to emphasize the generation of biomolecules, whether native or synthetic analogues, that have been matter of recent patents. Developments of biomolecules suitable for therapeutic scopes and agricultural use have several challenges such as intrinsic toxicity, in vivo  stability and suitable formulation contemplating the cost of production. Thus, biotechnological procedures using microbial systems or transgenic crop as  plant factories might help to solve these challenges. Keywords:  Antimicrobial, peptides, modes of action, biocontrol agents. INTRODUCTION It is widely accepted among professionals of several fields that antibiotic resistance will be promptly a global social concern in the fight against pathogen infections [1]. Two of the major contributors to these infections are widespread over prescription and misuse of antibiotic drugs,  practices that have promoted the dissemination of a series of  particularly harmful bacterial strains that resist to conventional antimicrobial treatments. Another potential main reason for the developed bacterial resistance in humans, is the massive use of preventive antibiotics in animal food. Because of a fast growth rate, the frequencies of genetic mutations and selections, and the ability of  bacteria to rapidly exchange genes, bacterial resistance to antibiotics seems to take place swiftly in the evolution of the  bacterial development. [2] On plants, several diseases caused  by pathogens (viruses, bacteria and fungi) affect plant crops resulting in yield losses and decreasing the quality and safety of agricultural products. Their control relies on finding plant genetic resistance and/or chemical pesticides, currently subjected to restrictions and regulatory requirements. [3] It is likely that many of the new antibiotics currently in development will not be approved by the US Food and Drug Administration. Most of these drugs in development are analogs of previous antibiotics which work on a select number of bacterial targets. [4] Consequently, the priority for the next decades should be focused in the development of alternative drugs and/or the recovery of natural molecules *Address correspondence to this author at the Biotechnology Unit, National Institute of Agricultural Research, INIA Las Brujas, Ruta 48 Km 10, Canelones, Uruguay; Tel: +598 2 3677641; Fax: +598 2 3677609; E-mail: mdallarizza@lb.inia.org.uy that would allow the consistent and proper control of  pathogen-caused diseases. Ideally, these molecules should be as natural as possible, with a wide range of action over several pathogens, characterized by different ways of action, easy to produce, and not prone to induce resistance [1]. In addition to the natural peptides, thousands of synthetic variant peptides have been produced, which also share similar structures [5]. The new generation of native and synthetic peptide mole-cules, also known as AntiMicrobial Peptides (AMPs), isolated from a full range of organisms and species from  bacteria to man, seem to fit this description. As a consequence, they have been named as ‘natural antibiotics’,  because they are active against a large spectrum of microorganisms, including bacteria and filamentous fungi, in addition to protozoan and metazoan parasites. All of these molecules are key elements directly implicated in the innate immune response of their hosts, which includes the expression of fluid phase proteins that recognize pathogen-associated molecular patterns, instead of specific features of a given agent to promote their destruction. As a result, the response is very fast, highly efficient and applicable to a wide range of infective organisms. [1] In plant protection there is a need of new compounds in plant health that fit into the new regulations (compounds being more selective, with lower intrinsic toxicity and reduced environmental impact); in fact, several countries have banned many of them due to regulatory changes in pesticides registration requirements, generating difficulties on plant diseases control. Therefore, AMPs are being considered as candidates for plant protec-tion products. [3] The knowledge acquired in the past two decades and the discovery of new groups of antimicrobial peptides, make  2  Recent Patents on DNA & Gene Sequences 2008  , Vol. 2, No. 2 Rizza et al. natural antibiotics the basic element of a novel generation of drugs for the treatment of bacterial and fungal infections [1]. In addition, the wide spectrum of antimicrobial activities reported for these molecules suggests they potential benefit in the treatment of cancer and viral or parasitic infections. Different therapeutic applications of these compounds, from topical administration to systemic treatment of infections, have been developed by several biotechnological companies (http://www.inimexpharma.com; http://biotech.deep13. com/Alpha/alpha.html; http://www.geniconsciences.com/). [1] Interestingly, to date, clinical Phase I and II trials have demonstrated a limited resistance for the bacterial strains tested. These features make the antibiotic peptides a power-ful arsenal of molecules that could be the antimicrobial drugs of the new century as an innovative response to the increa-sing problem of multi-drug resistance (MDR) (http://www. multi-drug-resistance.org; http://www.demegen.com). [1] The antibiotics peptides are divided into two classes: the non-ribosomally synthetized peptides and the ribosomally synthetized (natural) peptides. The former are often drastically modified and are produced by bacteria, fungi, and streptomycetes. The latter instead, of wider distribution, are  produced by all species of life (including bacteria) as a major component on the natural host defense molecules of these species. [6] Antimicrobial peptides are evolutionary ancient weapons that can be found in organisms ranging from bacteria to  plants, invertebrates and vertebrates species, including mammals. [7] Their widespread distribution throughout the animal and plant kingdoms, suggests that antimicrobial  peptides have served a fundamental role in the successful evolution of complex multicellular organisms; being part of the ancient, nonspecific innate immune system, which is the  principal defense system for the majority of living organisms [7,8]. These small and diverse peptides were initially isolated in the 1980s, from frogs and insects and in the former example, they have been found to play a vital role in their survival in bacteria infested swamps [9]. Since then, a large number of additional antimicrobial peptides has been found virtually everywhere in nature and it is difficult to categorize, except broadly on the basis of their net charge and/or their secondary structure [10]. Several hundred different antimicrobial peptides, from many different organisms, have been characterized to date; they are already listed in the publicly available databases including Swissprot and TrEMBL (http://www.expasy.org/sprot/sprot-top.html), RCSB Protein Databank (http://www.rcsb.org/pdb/home/ home.do), Antimicrobial Peptide Database (http://aps.unmc. edu/AP/main.html), Antimicrobial Sequences Database (http://www.bbcm.units.it/~tossi/antimic.html), and there are indications of the fact that this number will continue to grow rapidly. [2,11,12] Collectively, the antimicrobial peptides display direct microbicidal activities toward Gram-positive and Gram-negative bacteria, fungi [13-19], some protozoan parasites [20] and viruses [21]. The importance of these activities in contributing to host defense may vary between different sites within a particular organism and also between different types of organism. [7] The expression of the antimicrobial peptides can be constitutive or can be inducible by ‘therapeutic’-inducing substance such as sodium butyrate [12], infectious and/or inflammatory stimuli, such as proinflammatory cytokines,  bacteria, or bacterial molecules that induce innate immunity, e.g., lipopolysaccharides (LPS). In multicellular animals, they may be expressed systemically (for example, in insect haemolymph or vertebrate immune cells) and/or localized to specific cell or tissue types in the body most to infection, such as mucosal epithelia and the skin. [7,22]  DIVERSITY AND BIOLOGICAL ACTIVITY OF CATIONIC PEPTIDES Cationic peptides is the largest group and the first to be reported; to date, hundreds of such peptides have been identified and over 50% of them have been isolated from insects. [1,7] They are typically relatively short (12 to 100 amino acids), and positively charged (net charge of +2 to +9) due to lysine and arginine residues and a substantial portion (around 50%) of hydrophobic residues. [6,23] They are found in all species of life, ranging from plants and insects to animals, including mollusc species, crustaceans, amphibians,  birds, fish, mammals and humans [6,24]. The cationic pep-tides have a broad spectrum of antimicrobial activity including activity against both Gram-negative and Gram- positive bacteria, fungi, eukaryotic parasites and viruses. [4] The fundamental structural principle of these peptides is the ability of the molecule to adopt a shape in which clusters of hydrophobic and cationic amino acids are spatially organized in discrete sectors of the molecule (termed ‘amphipathic’ design). [8] This feature allows the peptides to interact well with membranes that are composed of amphi- pathic molecules, especially negatively charged bacterial membranes. For the most part, animal cells tend to have membranes with no net charge so they are unaffected by cationic peptides. [4] All cationic peptides are derived from larges precursors, including a signal sequence. Post-translational modifications include proteolytic processing, and in some cases glycosy-lation, carboxi-terminal amidation, amino-acid isomeri-zation, and halogenation. The diversity of sequences is such that the same peptide sequence is rarely recovered from two different species of animal, even those closely related. However, both within the antimicrobial peptides from a single species, and even between certain classes of different  peptides from diverse species, significant conservation of amino acid sequences can be recognized in the preproregion of the precursor molecules; the design suggests that constraints exist on the sequences involved in the translation, secretion or intracellular trafficking of this class of membrane-disruptive peptide. [4,8] The diversity of cationic peptides discovered is so great that it is difficult to categorize them except broadly on the  basis of their secondary structure [25]. Basically, this  peptides can be classified in four major classes:  -helices,  -sheet (peptides with two to four  -strands stabilized by disulphide bonds), loop structures, and extended peptides [26], the first two classes being the most common in nature.   Biomolecules as Host Defense Weapons Recent Patents on DNA & Gene Sequences 2008  , Vol. 2, No. 2 3   The NMR solution structures of a list of some well-studied AMPs of the major classes are shown in Table  1 . FROM PROKARYOTES Antimicrobial peptides produced by bacteria were among the first to be isolated and characterized [7]. While they do not offer protection against infection in the classical sense, they contribute to the survival of individual bacterial cells by killing other bacteria that might compete for nutrients in the same environment [7]. As shown in Table  2  [27-33] investigators have been  patenting aminoacidic sequences of bacterial antimicrobial  peptides, also called bacteriocins, produced by many or most  bacteria. This AMPs are generally extremely potent compared with most of their eukaryotic counterparts, their activities may be either narrow or broad spectrum [7], capable of targeting bacteria within the same species or from different genera (see Table 2  US7238515). The bacteriocins constitute a structurally diverse group of  peptides and the classification into two broad categories has  been recently proposed: lanthionine containing (lantibiotics) and non-lanthionine containing [7]. Lantibiotics are charac-terized by the inclusion of the unusual amino acid lanthionine and the necessity for postranslational processing to acquire their active forms. The most extensively studied lantibiotic is nisin, produced by  Lactococcus lactis ; which has been the center of attention because of their application as food preservatives without significant development of resistance. [1,7] A high number of lantibiotic aminoacidic sequences has been described and patented in the last years (see EP1169340 [34] and US0196900 [35]; these patents and others are referenced in Table  2 ). FROM EUKARYOTES PLANTS In plants, it is widely believed that antimicrobial peptides  play an important and fundamental role in defense against infection by bacteria and fungi [3,7,36]. Observations to support this role include the presence and expression of genes encoding antimicrobial peptides in a wide variety of  plant species (see some relevant patents in Table 3 [37-50]). There are many demonstrations of their bactericidal and fungicidal activity in vitro , and correlations between expression levels of peptides and susceptibility to a given  pathogen or the extent of resistance of a particular bacterium to plant-derived peptides and its virulence [7,36]. The two major and best-studied groups were thionins and defensins. The thionins with a molecular weight of 5 kDa, are generally  basic and contain 6 or 8 conserved cysteine residues [51,52]. Physiologically relevant concentrations of thionins are active against bacteria and fungi in vitro , and studies utilizing transgenic plants have shown that heterologous expression of thionins can confer protection against bacterial challenge. The plant defensins, is a group of small AMPs (45-54 amino acids), highly basic cysteine-rich peptides that are apparently ubiquitous throughout the plant kingdom and display antibacterial and antifungal activities. To date, sequences of more than 80 different plant defensin genes from different  plant species are available [36,53-56] and isolation of these has been recently patented (Table  3 , US7238781, US6911577, US6770750 and EP1849868). Consistent with a defensive role, they are particularly abundant in seeds, but have also been described in leaves, pods, tubers, fruit and floral tissues [17,57]. In the Defensins Knowledgebase (http://defensins.bii.a-star.edu.sg/) there are lists of patents referred to plant defensins (Table 3 , e.g. WO009174 and US037100). A new family of antimicrobial peptides has been described from  Macadamia integrifolia  and the first purified member has been termed MiAMP2c (Table 3 , US7067624). The peptide, active against a number of plant pathogens in vitro,  derives from a precursor protein similar to vicilins 7S globulin proteins, suspected of a putative participation in defense during seed germination [1].  INVERTEBRATES Since invertebrates lack the adaptive immune system found in vertebrate species, they are reliant solely upon their innate immune systems to counteract invading pathogens. Considering the extraordinary evolutionary success of this group of organisms, it is evident that invertebrate innate immune mechanisms are extremely effective. [7] One component of the defense weapons, developed by inverteb-rates to rapidly eliminate invading pathogens, is the fast and massive production of potent AMPs [2]. To demonstrate the effectiveness of invertebrates antimicrobial peptides the researchers have isolated and evaluated in vitro  and in vivo  the AMPs; in Table  4 [58-68] are listed some patents related. They are found in the haemolymph (plasma and hemocy-tes), in phagocytic cells, and in certain epithelial cells of invertebrates. They can be expressed constitutively, for example, in the hemocytes of marine arthropods such as shrimps, oysters, and horseshoe crabs (Table  4 , US5861378), or induced in response to pathogen recognition, such as antifungal peptides in  Drosophila . [7] Although usually cationic, the primary structures of insects AMPs vary markedly. Members of the most frequent AMP families adopt an  -helical conformation in memb-rane-mimetic environments [11]; these are the  -helical cecropins. This is a family of 3 – 4 kDa linear peptides described in the haemolymph of insects in the early 1980s. Patenting of isolation and amino acid sequentiation of these  peptides has also been done in 1982 (Table  4 , US4355104). Cecropins were the first animal inducible AMPs to be isolated and fully characterized; these molecules are devoid of cysteine residues and contain two distinctive helical segments, a strongly basic N-terminal domain and a long hydrophobic C-terminal helix, linked by a short hinge. [1,2] The cecropins have a broad spectrum of activity [69] (see  patent US5962410 [70]), some cecropins are capable of inhibiting cell-associated production of HIV-1 by suppres-sing HIV-1 gene expression [71]. Some C-terminus modifi-cations (cecropin-like peptides) exhibit increased potency and broader spectrum of antimicrobial activity than the cecropins, this was claimed in patent (Table  4 , US5166321). Another family, that is commonly found in insects is represented by peptides with high content in one or two  particular amino acids, most frequently proline and/or glycine residues [2,72]. However, the most abundant group of antimicrobial peptides in invertebrates is the defensins,  4  Recent Patents on DNA & Gene Sequences 2008  , Vol. 2, No. 2 Rizza et al. Table 1. Description of Some Antimicrobial Peptides Isolated from Different Sources, Selected as Representative Examples of their Structural Class. High-Resolution Images of Peptide Backbones were Obtained from RCSB PDB (http://www.rcsb.org/pdb/home/home.do) and PDBsum (http://www.ebi.ac.uk/) Melittin PDB code:  1bh1 Class:    -helical Source:    Apis mellifera  (Honeybee venom) Amino acid sequence:  GIGAVLKVLTTGLPALISWIKRKRQQ Activity:  Gram +, Gram -, Virus, Fungi, Mammalian cells, Cancer cells Authors:  Barnham, K.J., Hewish, D., Werkmeister, J., Curtain, C., Kirkpatrick, A., Bartone, N., Liu, S.T., Norton, R., Rivett, D. Ref.:  [27] Magainin 2 PDB code:  2mag Class:    -helical Source:    Xenopus laevis (Epithelial tissue of the African frog) Amino acid sequence:  GIGKFLHSAKKFGKAFVGEIMNS Activity:  Gram +, Gram -, Fungi, Cancer cells Authors:  Gesell, J.J., Zasloff, M., Opella, S.J. Ref.:  [28] Lactoferricin B PDB code:  1lfc Class:    -hairpin Source:    Bos taurus  (Bovine) Amino acid sequence:  FKCRRWQWRMKKLGAPSITCVRRAF Activity:  Gram +, Gram -, Virus, Fungi, Cancer cells Authors:  Hwang, P.M., Zhou, N., Shan, X., Arrowsmith, C.H., Vogel, H.J. Ref.:  [29] Human -defensin-1 (hBD-1) PDB code:  1e4s Class:    -sheet Source:    Homo sapiens (Human, extracellular protein) Amino acid sequence:  DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK Activity:  Gram +, Gram -, Fungi, Virus, Chemotactic Authors:  Bauer, F., Schweimer, K., Kluver, E., Adermann, K., Forssmann, W.G., Roesch, P., Sticht, H. Ref.:  [30] Antifungal protein 1 (RS-AFP1) PDB code:  1ayj Class:  Alpha Beta Source:    Raphanus sativus var. Niger (Radish seeds) Amino acid sequence:  EKLCERPSGTWSGVCGNNNACKNQCINLEKARHGSCNYVFPAHKCICYFPC Activity:  Gram +, Fungi Authors:  Fant, F., Borremans, F.A.M. Ref.: [31]    Biomolecules as Host Defense Weapons Recent Patents on DNA & Gene Sequences 2008  , Vol. 2, No. 2 5   (Table 1) Contd…. Thanatin PDB code:  8tfv Class:  Loop Source: Podisus maculiventris  (Insect, haemolymph tissue) Amino acid sequence:  GSKKPVPIIYCNRRTGKCQRM Activity:  Gram +, Gram -, Fungi Authors:  Mandard, N., Sodano, P., Labbe, H., Bonmatin, J.M., Bulet, P., Hetru, C., Ptak, M., Vovelle, F. Ref.: [32] Indolicidin PDB code:  1g89 Class:  Extended Source:  Synthetic construct based on  Bos taurus  sequence (Bovine neutrophils) Amino acid sequence:  ILPWKWPWWPWRR Activity:  Gram +, Gram -, Fungi, Virus Authors:  Rozek, A., Friedrich, C.L., Hancock, R.E. Ref.:  [33] Table 2. Recent Patents Related to AMPs Isolated from Prokaryotes Publication number Title Inventors Publication date Source Ref. No. US7238515 Anti-Listeria bacteriocin Berjeud, J.M., Fremaux, C., Cenatiempo, Y., Simon, L 2007/07/03  Lactobacillus sakei  [34] US7166468 Production of the lantibiotic cinnamycin with genes isolated from Streptomyces cinnamoneus  Bibb, M.J., Widdick, D. 2007/01/23 Streptomyces cinnamoneus [35] US5594103 Lantibiotic similar to a nisin A De Vos, W.M., Roelant, J.S., Kuipers, O.P. 1997/01/14  Lactococcus lactis [36] US7247306 Bacteria strain and bacteriocin  produced therefrom Fliss, I., Desbiens, M., Lacroix, L., Tahiri, I., Benech, R., Kheadr, E. 2007/07/24 Carnobacterium divergens [37] US6541607 Sublancin lantibiotic produced by  Bacillus subtilis 168 Hansen, N. 2003/01/04  Bacillus subtilis [38] EP1908774 Antibacterial and antiviral  peptides from  Actinomadura namibiensis  The designation of the inventors has not yet been filled (Not designed) 2008/04/09  Actinomadura namibiensis  [39] US6989370 Bacteriocins and novel bacterial strains Stern, N.J., Svetoch, E.A., Urakov, N.N., Eruslanov, B.V., Volodina, L.I., Kovalev, Y.N., Kudryavtseva, T.Y., Perelygin, V.V., Pokhilenko, D., Levchuk, V.P., Borzenkov, V.N., Svetoch, O.E., Mitsevich E.V., Mitsevich I.P. 2008/01/22 Paenibacillus  and  Bacillus  species   [40]
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