Antimicrobial peptides: A natural alternative to chemical antibiotics and a potential for applied biotechnology
A large group of low molecular weight natural compounds that exhibit antimicrobial activity has been isolated from animals and plants during the past two decades. Among them, cationic peptides are the most widespread. Interestingly, the variety and diversity of these peptides seem to be much wider than suspected. In fact, novel classes of peptides with varying chemical propertiescontinue to be isolated from different vertebrate and invertebrate species, as well as from bacteria. To the early characterized peptides, mostly cationic in nature, anionic peptides, aromatic dipeptides, processed forms of oxygen-binding proteins and processed forms of natural structural and functional proteins can now be added, just to name a few. In spite of the astonishing diversity in structure and chemical nature displayed by these molecules, all of them present antimicrobial activity, a condition that has led researchers to consider them as "natural antibiotics" and as such a new and innovative alternative to chemical antibiotics with a promising future as biotechnological tools. A resulting new generation of anti microbial peptides (AMPs) with higher specific activity and wider microbe-range of action could be constructed, and hopefully endogenously expressed in genetically-modified organisms.
The continuous use of antibiotics has resulted in multi-resistant bacterial strains all over the world and as expected, hospitals have become breeding grounds for human-associated micro organisms (Mainous and Pomeroy, 2001). Nonetheless, the same time-bomb effect is slowly developing with animal-associated pathogens in commercially driven activities, such as aquaculture and confined poultry breeding, where the indiscriminate use of antibiotics is perceivedasessential for industries survival. Consequently, there is an urgent need to search for alternatives to synthetic antibiotics. The discovery of two classes of antimicrobial peptides, non-ribosomally synthesized (Hancock and Chapple, 1999) - present in bacteria - lower eukaryotes and plants - and ribosomally-synthesized peptides, of wider distribution (Boman, 1995; Broekaert et al. 1997; Hancock and Lehrer, 1998; Hoffmann et al. 1999; Thevissen et al. 1999; Zasloff, 2002; Ezekowitz and Hoffmann, 2003), provided a new therapeutic strategy to fight micro organisms. Recent studies show that several cationic and non-cationic peptides expressed in many vertebrate, invertebrate and bacterial species (Lüders et al. 2003) act synergistically to improve immune responses.
The knowledge acquired in the past two decades and the discovery of new groups of antimicrobial peptides make natural antibiotics the basic element of a novel generation of drugs for the treatment of bacterial and fungal infections (De Lucca, 2000; Hancock, 2000; Welling et al. 2000; Selitrennikoff, 2001). In addition, the wide spectrum of antimicrobial activities reported for these molecules suggests they potential benefit in the treatment of cancer (Tanaka, 2001) and viral (Chinchar et al. 2001; Andersen et al. 2001; Chernysh et al. 2002) or parasitic infections (Vizioli and Salzet, 2003). Different therapeutic applications of these compounds, from topical administration to systemic treatment of infections, have been developed by several biotechnological companies (Hancock, 2000; http://www.inimexpharma.com; http://biotech.deep13.com/Alpha/alpha.html; http://www.geniconsciences.com/) Interestingly, to date, clinical Phase I and II trials have shown a limited resistance for the bacterial strains tested (Zasloff, 2002). These features make the antibiotic peptides a powerful arsenal of molecules that could be the antimicrobial drugs of the new century as an innovative response to the increasing problem of MDR (http://www.multi-drug-resistance.org; http://www.multi-drug-resistenz.de; http://www.demegen.com.)
It is widely accepted among clinicians, medical researchers, microbiologists and pharmacologists, that antibiotic resistance will, in the very near future,leave healthcare professionals without effective therapies for bacterial infections. As an example, it is now estimated that about half of all Staphylococcus aureus strains found in many medical institutions are resistant to antibiotics such as methicillin (Roder et al. 1999). The emergence among enterococci of resistance to another useful and widely effective antibiotic, vancomycin (Novak et al. 1999), might accelerate the spread of vancomycin-resistant genes, via plasmids, throughout other species, eventually limiting the efficacy of this drug. Consequently, the priority for the next decades should be focused in the development of alternative drugs and/or the recovery of natural molecules 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, easy to produce, and not prone to induce resistance.
The new generation of native peptide molecules, also known as Anti Microbial 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 termed "natural antibiotics", because they are active against a large spectrum of microorganisms, including bacteria and filamentous fungi - in addition to protozoan and metazoan parasites (Liu et al. 2000; Vizioli and Salzet, 2003). All of these molecules are key elements directly implicated in the innate immune response of their hosts, which includesthe 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 (Hoffmann and Reichhart, 2002). Additionally, the effect of AMPs can go beyondisolated bacterial cells, as shown by the inhibition they can exert over clusters of pathogenic bacteria, such in biofilm development (Singh et al. 2002).
In order to survive in a world laden with microorganisms, most multi-cellular organisms ought to depend on a network of host defense mechanisms which in most cases, involves several levels of interacting systems. Since the initial contact of fastidious microorganismswith the host usually occurs at inner or outer body surfaces, they should be the primary site for an immune reaction to occur. Thus, innate immune responses refer to the first line of host defense, which acts within a few hours after microbial exposure to mucosal surfaces. Upon recognition of conserved molecular microbial patterns such as PAMs or Pathogen-Associated Molecular Patterns (e.g. LPS and cell wall components) and Toll-like receptors (TLR) (Hoffman et al. 1999; Aderem and Ulevitch, 2000; Akira et al. 2001) initiate the immune responses of the host. Using the urinary and gastro-intestinal tract as model systems, information has been obtained on how organ- and cell-specific expression patterns of TLR on epithelial cells correlate to the ability of an organ to rapidly respond to bacterial infections has been obtained(BSckhed et al. 2003). It has become clear now that understanding the innate response to pathogens will certainly provide insights to host defenses as well as the strategies used by pathogens to circumvent these defense mechanisms. Remarkably, the pattern-specific recognition system already acknowledged in animals, has also been reported in plants (Dangl and Jones, 2001).
In complex system suchas humans, an invading microorganism can simply be eliminated by this primary reaction - the innate response - without requiring an activation of the adaptive immunity, the next step in this complex cascade (Bals, 2000). If the invading microbe outgrows the innate host defence, endogenous effector mechanisms of the innate system are up-regulated and have direct antimicrobial activity and mediator function to attract inflammatory cells and cells of the adaptive immune system. In lower eukaryotes, mostly invertebrates, the adaptive system is nonexistent, thus accounting for the versatile and effective role the innate system has in order to control, by itself, the invasiveness of a given pathogen (reviewed by Otvos, 2000).
Memberss of the major groups of antimicrobial peptides have been classified mainly on the basis of their biochemical (net charge) and/or structural features (linear/circular/amino acid composition), looking for common patterns that might help to distinguish them. (Tossi and Sandri, 2002; Zasloff, 2002). The resulting most important groups are the following:
Cationic peptides: This is the largest group and the first to be reported, being widely distributed in animals and plants. So far, more than a thousand of such peptides have been characterized and over 50 % of them have been isolated from insects (Bulet et al. 1999; Andreu and Rivas, 1998; http://www.bbcm.univ.trieste.it/~tossi/antimic.html). On the basis of their structural features, cationic peptides can be divided as well into three different classes: (1) linear peptides forming-helical structures; (2) cysteine-rich open-ended peptides containing single or several disulfide bridges; and (3) molecules rich in specific amino acids such as proline, glycine, histidine and tryptophan.
Important subfamilies of cationic peptides include:
Two other forms of precursor-derived peptides are represented by cathelicidins and thrombocidins. The formers are quite abundant in mammals and generated from precursor proteins bearing an amino-terminal cathepsin L inhibitor domain (cathelin) (Lehrer and Ganz, 2002). The latters are compounds released from platelets and arise from deletions of the CXC chemokines neutrophil-activating peptide 2 and connective tissue-activating III in humans (Krijgsveld et al. 2000).
In plants, a similar picture is slowly emerging. A new family of antimicrobial peptides has been described from Macadamia integrifolia of which the first purified member has been termed MiAMP2c (Marcus et al. 1997). 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 (Marcus et al. 1997). The novel peptide is inserted in the highly hydrophilic N-proximal region of the precursor, where three additional cysteine-containing MiAMP2c-like patterns exist, suggestive of three additional peptide isoforms, a pattern already described for fish AMPs (Lauth et al. 2002).
In spite of the fact that the mechanism of action is not satisfactory established for all cationic peptides, the structural model established by Shai-Matzusaki-Huang (Matzusaki, 1999) provides a reasonable explanation for most antimicrobial activities of these compounds (Zasloff, 2002). The model proposes that these linear amphipatic-helical peptides interact with bacterial membranes and increase their permeability, either by the effect of their positive charges with anionic lipids of the target membrane or by membrane destabilization through lipid displacements due to the drastic changes in the net charge of the composed system. A similar mechanism has been proposed for the cysteine-rich peptides such as defensins, which are suggested to form ion-permeable channels in the lipid bilayer. In contrast, some peptides penetrate into cells to exert their action over target molecules (Kragol et al. 2001). Several additional hypotheses have been proposed to explain the mechanisms by which peptides kill target cells; such hypotheses include induction of hydrolases which degrade the cell wall, disturbance of membrane functions and damage to crucial intracellular targets after internalization of the peptide (Zasloff, 2002).
The antimicrobial peptides produced by bacteria have been grouped into different classes based upon the producer organisms, molecular size, chemical structure and mode of action, which resulted in different names for putative compounds which turned out to be identical: (thiolbiotics, lantibiotic microcin, colicin, bacteriocin, to name a few) (Kolter and Moreno, 1992). The most relevant active-membrane peptides among them are produced by gram-positive bacteria and classified taxonomically as bacteriocins (Oscáriz and Pisabarro, 2001). Some of them have been the center of attention because of their application as food preservatives (Schillinger et al. 1996).
Bacteriocins, cationic, neutral and anionic in chemical nature, are all in the range of 1.9 (Actagardine) and 5.8 (Lactococcin B) kDa in molecular mass (Jack et al. 1995), cationic, neutral and anionic in chemical nature (Oscáriz and Pisabarro, 2001). The most thoroughly studied bacteriocins are those produced by lactic-acid bacteria, of which sakacins seem to be most unique (Jack et al. 1995; Simon et al. 2002), and the lantibiotics, which contain modified amino acid residues (Oscáriz and Pisabarro, 2001). Another representative, pediocins, are usually co-transcribed with a gene encoding a cognate-immunity protein (Fimland et al. 2002). The 44-amino acid pediocin produced by Pediococcus acidilactici strains is encoded in an 8.9 kb plasmid.The importance of AMPs in humans
Peptides of the defensin, cathelicidin, thrombocidin and histatin classes are found in humans protecting epithelia against invading microorganims and assisting neuthrophils and platelets (Peschel, 2002). In the airways, α-and-β-defensins and the cathelicidin LL-37/hCAP-18 are produced by the respiratory epithelium and alveolar macrophages and then secreted into the airway surface fluid (Wang et al. 1999). Beyond their antimicrobial function, these peptides are known to be multi-functional. In fact, it has been demonstrated their multiple roles as mediators of inflammation with effects on epithelial and inflammatory cells, and the impact these roles have over such diverse processes as proliferation, immune induction, wound healing, cytokine release, chemotaxis, protease-antiprotease balance, and redox homeostasis (Ganz, 2002; Cole et al. 2003; Com et al. 2003; Liu et al. 2003).
Considering that AMPs are natural barriers to bacterial infections, pathogens ought to have developeda variety of strategies that render them resistant to antimicrobial host defenses. The only currently available structural model explaining the mechanism of action of AMPs (Shai-Matzusaki-Huang) (Matzusaki, 1999), the action of these peptides is from the outside and over the pathogen's membrane either by increasing their permeability or by destabilizing membranes by changing the net charge of the composed system. Since biological membranes are indeed dynamic fluids, the generation of resistance appears to be less likely to occur. Nonetheless, pathogens have evolved countermeasures not to resist, but at least to limit AMPs' effectiveness, such as chemical modifications and/or alternation of energy-dependent pumps at the membrane level (Peschel, 2002). The same is true for intracellular bacterial pathogens, in which resistance-limitation is less effective against mostly cationic peptide-driven antimicrobial activity existing in the phagosomes of circulating monocytes, neutrophils and some mucosal epithelial cells (Ernst et al. 1999). Additionally, the fact that the common features for most peptides are a net positive charge and an amphipathic nature, allows them to persist at water-lipid interfaces and then to disturb microbial membrane components (Ruissen et al. 2001).AMPs and biotechnology: Is there a promising future?
Good progress has been achieved with respect to defining the rules by which the immune system works and its complexity and interconnections are being slowly understood. In this perspective, the innate immune response has been neglected, but the consolidation of new discoveries in the field is slowly repositioning it (Fearon, 2000; Nathan, 2002). Nonetheless, the potential massive use of these natural compounds is hampered by the limited amount that can be extracted in vivo as well as non-optimal specific activities, which would require huge amounts for clinical and therapeutical application. This is the point where biotechnology should play a pivotal role in the near future, independent that chemical synthesis of peptides could also be a non exclusive alternative. Classically, these peptides are encoded by small genes, with conserved sequences and patterns that make their cloning easy, and should allow easy expression and both small- and large scale purification (Uteng et al. 2002). From a more innovative point of view, gene amplification and transgenesis seem like feasible ways to increase production and enhance specific activity of selected molecules. But, is this possible to achieve in vivo? The answer is, once again, yes. Biosynthetic and preparative production of AMPs have been successfully reported (Haught et al. 1998; Martemyanov et al. 2001), as have synthetic forms of AMP analogues displaying enhanced antimicrobial activity (Cudic et al. 2003). There are some additional examples: Since AMPs were first characterized in insects, a great deal of complementary work comes from that area of applied research. One of the most notable pieces of work deals with Drosophila mutants not expressing any known endogenous AMP genes and, as a consequence, highly susceptible to bacterial infections. Genetic manipulation of these mutants complemented with a single constitutively expressed AMP gene can rescue susceptibility to infections (Tzou et al. 2002). In plants, as expected, tobacco has been thetarget for successful engineered-production of mammalian AMPs (Morassutti et al. 2002), as well as amphibian anti microbial peptides, where vertical transmission of resistance occurs (Ponti et al. 2003). In addition, AMPs from other origins have been added to confer disease resistance in transgenic tobacco and banana (Chakrabarti et al. 2003) and potato (Osuky et al. 2000), thus opening unsuspected alternatives to provide agronomically relevant levels of disease control worldwide (Van der Biesen, 2001).
Although at present AMPs are believed to exerttheir primary activity on bacterial membranes, new evidence is suggesting that AMP activity might be broader, including selective inhibition of intracellular targets (Cudic and Otvos, 2002). It is thought that cationic peptides might induce genomic responses in bacteria treated with AMPs, in addition to any lethal effect on the bacterial membrane. This appears to be the case, as recently demonstrated (Hong et al. 2003). These authors have shown that the transcription profiles of at least 26 Escherichia coli genes change specifically and significantly after exposure to lethal and sub lethal concentrations of Cecropin A, an emblematic cationic peptide. Moreover, half of these transcripts corresponded to proteins of unknown function, which makes these observations quite intriguing.
Now, regarding the wide variety and diverse classes of natural peptides, we must add necessarily, the processing alternatives, which are slowly being reported that might make these molecules incommensurable, approaching the diversity of immunoglobulins. The case of lactoferricin-C, generated as a functional internal domain of caprine lactoferrin in a manner mimicking the generation of inteins (selfish DNA elements inserted in-frame and translated together with their host proteins: http://bioinfo.weizmann.ac.il/~pietro/inteins), opens a new and broad area of research. Something similar occurs with milk-derived compounds, where it is clear that milk contains a group of proteins, which perform a protective function. These proteins harbor in their primary sequence, peptides that are inactive in the parent protein and that are released during gastrointestinal digestion or food processing (Yamauchi, 1992). In contrast, the generation of thrombocidin, arising from carboxy-terminal deletions of key neuthrophil- and connective tissue-activating peptides in humans, broadens the spectrum of alternative for processing associated with the generation of AMPs. Additionally, slight variations in the structure of preexisting peptides might broaden their potential as AMPs. A good example is that of histatin-5, a naturally occurring anti-fungal peptide in human saliva, which presents at least two variants (dhvar4 and dhvar5) displaying increased anti-microbial activity by subtle changes in their amphipathicity, a good indicator of membrane destroying activity, which allows them to be internalised showing a more destructive effect on mitochondria than on external membranes (Ruissen et al. 2001). Therefore, it is reasonable to think that a number of existing functional proteins, unrelated to immune responses, might contain potential and fully active AMPs This is a complementary strategy to that of natural anti-microbial peptides, which by themselves might adjust to potential bacterial adaptations to counteract their pathogenicity. This is only the tip of the iceberg in this appealing topic. The recent proposalthat antibody multi specificity can be mediated by conformational diversity of pre existing isomers to increase the effective size of the antibody repertoire (James et al. 2003), is perfectly applicable to understand diversity of existing AMPs as well as the potential of those derived from multiple and heterogeneous type of precursors.Only time will verify these assumptions.
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