Development of Novel Antimicrobial Agents: Emerging Strategies Chapter Abstracts

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Karl Lohner and Erich Staudegger

Abstract

At the end of the 20^th century we face a world-wide rapid increase in pathogenic bacteria which are multi-resistant to antibiotics. This alarming situation has its origin in the excessive and often inappropriate use of antibiotics in human and animal health care for the treatment and prevention of bacterial infections. Since the development of the first commercially available antibiotic penicillin in the 1940s, the high expectations by man in the healing power of these "wonder drugs" has not been fulfilled, as resistance - which is not a new problem - is a vital part of the survival strategy of bacteria. Some stategies for the containment of antimicrobial resistance are discussed, of which the continuous research and development of new classes of antibiotics with novel mechanisms of action, the topic of this book, and the awareness of a more sophisticated and prudent use of antibiotics are the most important ones.

Chapter 2. Origins and Evolution of Antibiotic and Multiple Antibiotic Resistance in Bacteria

Ruth M. Hall and Christina M. Collis

Abstract

The selective pressure of antibiotic use has brought to prominance strains of many important pathogens that are resistant to more than one antibiotic. Acquisition of antibiotic resistance genes is the predominant factor in the emergence, evolution and spread of multiply antibiotic resistant bacteria. Two processes are important. Horizontal gene transfer enables genes to move from one bacterium to another, and translocation enables them to move from a location on one DNA molecule to another. The accumulation of genes conferring resistance to different antibiotics on the same horizontal gene transfer vehicle leads to the ability to simultaneously transfer multiple antibiotic resistance determinants to further bacterial strains, species or genera. Since genes for toxins and other pathogenicity and virulence determinants, genes conferring resistance to heavy metals and many others use the same highways and byways, the emergence of antibiotic resistance cannot be viewed in isolation from many other selective forces.

Chapter 3. Antimicrobial Peptide Resistance Mechanisms in Bacteria

Arden Aspedon and Eduardo A. Groisman

Abstract

Small cationic peptides with broad-spectrum antimicrobial activity are produced by a wide variety of plants and animals and represent a nonspecific arm of the immune systems of these organisms. Antimicrobial peptides are believed to kill bacteria by damaging the cytoplasmic membrane by a mechanism that does not appear to involve interaction of the peptide with discrete protein targets. Bacteria employ several mechanisms to resist the toxic effects of these agents: exclusion of the peptide from its target (membrane), inactivation of the peptide, modification of the target, and the ability of the cell to overcome peptide-induced sublethal damage. The expression of resistance determinants is often under transcriptional control in a manner that presumably enhances the chances of survival of the bacterial cell in a given environment. The propensity of bacteria to acquire antibiotic-resistance genes suggests a potential for the evolution of peptide hyperresistant strains analogous to the multiantibiotic resistant forms present today.

Chapter 4. Genetic Engineering of Novel Macrolide Antibiotics

Robert McDaniel and Leonard Katz

Abstract

Macrolides, such as erythromycin and tylosin, are a class of antibiotics that are used widely in human and veterinary medicine belonging to the family of natural products known as polyketides. These compounds are synthesized by large multienzyme complexes, polyketide synthases (PKSs), which contain a program at the genetic level encoding the structure of the compound. Recent advances in our understanding of PKSs as well as the development of molecular biology tools for the genetic engineering of PKSs has brought forth a number of strategies for creating novel polyketides by gene manipulation. Much of this development has been performed with the PKS that produces erythromycin and is reviewed here. Since the potential for creating structural diversity with PKSs is great, it is hoped that next-generation macrolides can be developed which overcome the increasing problem of resistant pathogens.

Chapter 5. Granulysin, A Novel Mediator of Antimicrobial Activity of Cytolytic T-cells

Steffen Stenger, Alan M. Krensky and Robert L. Modlin

Abstract

Cytolytic T-cells play an important role in immunity against many intracellular pathogens. One effector mechanism of CTL is the lysis of infected target cells, thereby exposing the microbes to the hostile extracellular environment. Here, we characterize a novel effector mechanism of CTL which not only results in lysis of the target cell but simultaneously to the death of the intracellular invader. The lethal hit is delivered by the combined action of two components of cytolytic granules of CD8+ CTL. One is the well known lytic protein perforin; the other is the recently discovered antimicrobial protein granulysin. In this review we will summarize current knowledge about granulysin with special focus on its functional role as an antibacterial effector molecule of the adaptive immune response.

Chapter 6. The Immunostimulatory Properties of Bacterial DNA

David S. Pisetsky

Abstract

Depending on base sequence, DNA can cause powerful immunostimulation and serve as a "danger signal" to activate host defense. In bacterial DNA, immunostimulation results from sequences of 6 bases that occur much more commonly in prokaryotic than eukaryotic DNA. These sequences, known as CpG motifs or immunostimulatory sequences (ISS), center on an unmethylated CpG dinucleotide and lead to the activation of B cells and macrophage as well as production cytokines such as IL-12, TNF-a and IFN-a/b. These cytokines can lead to Th1 cell predominance. In addition to CpG motifs, other DNA sequences such as runs of dG have immunostimulatory activity. The properties of DNA as an immunomodulator are also influenced by backbone chemistry since phosphorothioate oligonucleotides can cause potent immune stimulation. The immunostimulatory properties of DNA allow the design of novel therapeutic agents that can augment host defense during infection as well as influence the balance of TH1/TH2 responses.

Chapter 7. Bacteria-Mediated DNA Transfer for Gene Therapy and Genetic Vaccination

Siegfried Weiss and Trinad Chakraborty

Abstract

Transfer of eukaryotic expression plasmids to mammalian cells has recently been achieved using live attenuated bacteria. These successes have encouraged the use and generation of bacterial vector delivery systems that use local, mucosal and systemic routes of infection to deliver the desired gene directly to the cell type or organ of interest. Intrinsic properties of invasive bacteria such as their tropism for cell types or cell to cell spread are currently understood in great detail and provide the necessary basis for the design of novel vehicles. Finally, the ability of bacteria to harbor very large plasmids makes them very attractive as vehicles for gene therapy.

Escherichia coli rendered artificially invasive and Listeria monocytogenes were used in vitro to transfer reporter genes into various types of cells from different species. Transfer could also be observed in vivo with L. monocytogenes albeit at low frequency. Shigella flexneri, Salmonella typhimurium and Salmonella thyphi were used as vehicles to transfer plasmids for genetic immunisation in vivo. Immune responses against the antigen encoded by the expression plasmid could be detected in all cases. Thus, bacteria represent a simple and versatile carrier system for genetic immunisation that should provide the flexibility required for the various vaccination problems. The low production cost of such vaccines makes them attractive candidates for mass application.

Chapter 8. Potential Mucosal Adjuvants For Human Use

Mari Ohmura , Raymond J. Jackson , Yoshifumi Takeda and Jerry R. McGhee

Abstract

There have been no effective and safe adjuvants for use in humans since aluminium compounds were approved by the US Federal Drug Administration (FDA). At present new adjuvants approved for use such as MDP and ISCOMs are limited to veterinary vaccines. Recent advances at the cellular and molecular levels of the immune system have led to the clinical application of certain cytokines for both immunotherapeutic and conventional vaccines. While not yet in widespread use, the cytokines IL-2 and IL-12 hold potential promise for human adjuvant applications. A purified saponin, QS-21 is another promising adjuvant candidate that has proven safe in Phase I and Phase II human clinical trials. Finally, considerable effort has been focused on developing detoxified derivatives of bacterial enterotoxins. Thus, mutants of cholera toxin (CT) produced by Vibrio cholerae and of the heat labile enterotoxin (labile toxin; LT) produced by enterotoxigenic Escherichia coli, which are non-toxic but which retain adjuvanticity have been constructed. Among these are CT E112K and S61F, which harbor single amino acid substitutions in the enzymatically active A subunit and are promising and excellent adjuvant candidates for mucosal vaccination. These molecules are currently undergoing pre-clinical evaluation as potential mucosal adjuvants for use in humans. Thus, several potentially safe and effective mucosal adjuvants with the ability to redirect the immune system to a Th1 or Th2-type response are on the horizon.

Chapter 9. Attenuated Bacteria as Delivery Vehicles for H. pylori Vaccines

Thomas G. Blanchard and Steven J. Czinn

Abstract

Helicobacter pylori is the etiologic agent of gastritis and most peptic ulcers. Antibiotic therapy is complicated and prohibitively expensive in developing nations where the prevalence of infection is greater than 80%. Helicobacter vaccine development has shown great promise in rodent models but not in large animals or humans, largely owing to lack of a suitable means of stimulating effective immunity by mucosal routes. Several live attenuated bacterial vaccines have been developed for use in humans and others are under development including Vibrio cholerae, Mycobacterium bovis, Listeria monocytogenes, Shigella flexneri, and Salmonella typhi. The success of these vaccines and the advantages conferred by using live bacterial vaccine vectors has generated an interest in exploiting this technology to deliver recombinant proteins to vaccinate against heterologous pathogens. Attenuated recombinant S. typhimurium has now been employed for the development of a vaccine against Helicobacter pylori infection of mice. These initial studies indicate recombinant bacterial vaccine vectors may prove to be an effective solution to the lack of suitable mucosal adjuvants for Helicobacter immunization

Chapter 10. Development of an Adhesin Vaccine to Prevent Urinary Tract Infection

Matthew A. Mulvey, Scott J. Hultgren and Solomon Langermann

Abstract

Colonization of the mucosal epithelium is a critical step in the early stages of bacterial infection. Adherence to host cells usually involves a specific interaction between bacterial surface proteins called adhesins and surface structures, often glycoconjugates, on the target cell. Interactions between pathogenic bacteria and host cells may also induce signaling cascades, both in the bacteria as well as in the host cells, resulting in local inflammation, invasive disease and triggering of innate host defenses. Recent advances in our understanding of the biological basis for bacterial binding, coupled with the ability to purify adhesins away from whole bacteria, allow for new approaches to prophylaxis and therapy. By using purified adhesins as candidate vaccines, it has been possible to show that such proteins elicit protective antibody responses that prevent the interaction of the bacteria with the host and subsequent infection. This chapter will focus on studies with uropathogenic Escherichia coli (E. coli) and their pilus-associated FimH adhesin proteins. These studies clearly demonstrate both that adhesins play a crucial role in bacterial attachment and that an adhesin-based vaccine protects against colonization and infection. Furthermore, FimH is also critical for invasion of the bladder epithelium that may result in chronic infection, hence a FimH vaccine might also protect against recurrent infections. Development of a FimH vaccine to prevent urinary tract infections (UTIs) addresses an important medical disorder of significant morbidity especially in women. More broadly these studies point to the potential for developing adhesin-based vaccines that could target a wide range of pilus-associated adhesins ubiquitous among Gram negative bacteria.

Chapter 11. Antimicrobial Peptides in Innate Immunity

Tomas Ganz and Robert I. Lehrer

Abstract

Antibiotic peptides encoded by genes are increasingly recognized as effector molecules of host defense in plants and animals. In higher animals, the peptides are particularly abundant on epithelial surfaces and in the storage granules of phagocytic cells. In these settings, antimicrobial peptides are positioned to act during the earliest stages of the interaction between the host and its microbial invaders. The most abundant antimicrobial peptides of mammals include defensins, characterized by six conserved disulfide-bridged cysteines and cathelicidins, structurally heterogenous peptides that share a conserved precursor domain, cathelin. A rich array of additional antimicrobial peptides are also expressed by vertebrates and invertebrates. Studies of antimicrobial peptides are providing new insights into the dynamic interactions between microbes and their hosts, and generating new paradigms for the pathogenesis and treatment of diseases.

Chapter 12. The Role of Membrane Lipid Composition in Cell Targeting of Antimicrobial Peptides

Karl Lohner

Abstract

Host defense peptides, which evolved in nature, show high specificity in their action towards their target cells, e.g. some exhibit toxicity towards bacteria. Most of these antimicrobial peptides kill bacteria by permeation or destruction of their cytoplasmic membrane, although alternative mechanism such as binding to DNA after translocation across the membrane may exist. In order to understand the molecular basis of the specific interaction of antimicrobial peptides with bacterial membranes, it is important to consider the membrane architecture of eukaryotic and prokaryotic cell membranes, which differ markedly in their complexity and lipid composition. Research in this field showed that antimicrobial peptides can discriminate between the major lipid classes of mammalian and bacterial cell membranes. Besides of electrostatic interactions between the cationic, amphipatic peptides and the negatively charged bacterial lipids, intrinsic membrane properties, like membrane curvature strain, modulate the mode of action and efficiency of these peptides. Understanding the mutual dependence of these lipid-peptide interaction is a key for the rational design of novel peptide antibiotics. This is likely to be accomplished only if the molecular basis of the action of these peptides is known.

Chapter 13. Molecular Mechanisms of Membrane Perturbation by Antimicrobial Peptides

Katsumi Matsuzaki

Abstract

Recently, a large number of antimicrobial peptides have been discovered from animals as well as plants. These peptides are recognized as important components of innate defense mechanisms. Many of these molecules form cationic amphipathic secondary structures that can interact with anionic bacterial membranes. Peptide-induced membrane permeabilization is an effective mechanism of antimicrobial action, which enables rapid and broad-spectrum bacterial killing. In this chapter, the various modes of membrane perturbation reported to date will be critically reviewed, emphasizing that the mode is strongly dependent on the physicochemical properties not only of the peptide but also of the target membrane.

Chapter 14 Molecular Mechanism of Cell Selectivity by Linear Amphipatic a-helical and Diasteriomeric Antimicrobial Peptides

Ziv Oren and Yechiel Shai

Abstract

Studies described in the last two decades have demonstrated the essential role of antimicrobial peptides in the first line of defense against invading pathogens and their uncontrolled proliferation. Despite numerous studies on the structure and activity of antimicrobial peptides, our knowledge of their mode of action is incomplete and controversial. The most studied group includes the linear, mostly a-helical peptides. Although developed by distant and diverse species such as plants, insects, amphibians and humans, linear antimicrobial peptides share two properties: a net positive charge, and a high propensity to adopt amphipatic a-helical conformation in hydrophobic environments. Numerous studies have shown that peptide-lipid interactions leading to membrane permeation play a major role in their activity. Membrane permeation by amphipatic a-helical peptides has been proposed to occur via one of two general mechanisms: (i) transmembrane pore formation via a "barrel-stave" mechanism; and (ii) membrane destruction/solubilization via a "carpet" mechanism. Critical evaluation of recent studies on linear a-helical antimicrobial peptides is presented in light of these two proposed mechanisms. This chapter, which is focused on representatives of the amphipatic a-helical antimicrobial peptides, supports the "carpet" rather than the "barrel-stave" mechanism. In addition, the different stages of membrane disintegration by antimicrobial peptide will be evaluated based on the recent studies with a novel group of diasteriomeric antimicrobial peptides.

Chapter 15. Molecular Interactions Involved in Bactericidal Activities of Lantibiotics

Ulrike Pag and Hans-Georg Sahl

Abstract

Lantibiotics are produced by a wide range of and mainly act on Gram-positive bacteria. Based on their structures and their mode of action they are currently divided into two distinct groups. The elongated amphiphilic, screw-shaped peptides of the type A-lantibiotics act primarily by pore formation in the bacterial membrane. Additional effects such as inhibition of spore outgrowth and triggering of autolytic enzymes have been observed. The small, globular type-B lantibiotics can be further subdivided: the peptides of the cinnamycin-subtype bind to specific phospholipids and thereby inhibit phospholipases and other enzyme functions; mersacidin and actagardine inhibit the bacterial cell wall biosynthesis by trapping the membrane-bound peptidoglycan precursor lipid II. Recent results indicate that some pore-forming lantibiotics, e.g. nisin and epidermin, also bind to lipid II, using it as a docking molecule for subsequent pore formation. These results demonstrate that at least some lantibiotics, in contrast to many defense peptides, bind with high specificity to particular membrane components. On the other hand, binding to the same membrane target (lipid II) may result in completely different modes of action, i.e. pore formation with nisin and inhibition of cell wall biosynthesis with mersacidin.

Chapter 16. Cyclic Cystine-Knot ß-Stranded Antimicrobial Peptides: Occurrence, Design and Synthesis

James P. Tam, Yi-An Lu, Jin-Long Yang and Qitao Yu

Abstract

Amphipathicity of antimicrobial peptides is an important attribute to their membranolytic actions. However, the relationship of amphipathicity to membranolytic selectivity that dissociates cytotoxicity from antimicrobial activity remains poorly understood. Analog study using rigid preorganized amphipathic structures may provide insight for selective interactions with microbial rather than eukaryotic membrane. Cyclic cystine-knot peptides with two or three strands, referred as cc₃2 and cc₃3 peptides respectively, represent novel and highly constrained scaffoldings of antimicrobial peptides containing 18 to 33 amino acid residues. This report describes their natural occurrence in higher organisms as well as our efforts in designing and developing new synthetic methods for cc₃2, cc₃3 peptides and their analogs. The rigidity imparted by the close-ended amide backbone and the tricystine constraints of cc₃2 and cc₃3 peptides also facilitates developing therapeutic useful peptide antibiotics of -stranded defensins, tachyplesins and protegrins that are membrane-selective, salt-insensitive and low cytoxicity.

Chapter 17. Synthetic Combinatorial Libraries: An Emerging Approach Toward the Identification of Novel Antibiotics

Sylvie E. Blondelle

Abstract

Development of novel therapeutics for the treatment of bacterial infection has become a clinical imperative. The greatest threat to current antibiotic coverage is the rapid evolution and spread of drug-resistance, which has now been reported against every currently available antibiotics. A solution to this dilemma is to develop a broad range of lead compounds available for clinical trials. Synthetic combinatorial libraries made up of hundreds to millions of small organic molecules, peptidomimetic compounds, and peptides have been successfully developed and used to discover new antimicrobial leads. The strength of combinatorial libraries relies on: 1) the rapid identification of highly active compounds from large pools or arrays of individual compounds, 2) the ongoing development of solid-phase approaches for the generation of small molecule libraries in an efficient and reproducible manner, and 3) the generation of many non-support bound libraries, which allow the ready performance of cell-based assays. Although not thoroughly exploited in the antibiotic research area, combinatorial chemistry is anticipated to greatly decrease the time it takes to develop novel antibiotic leads.

Chapter 18. The Commercial Development of the Antimicrobial Peptide Pexiganan

Michael A. Zasloff

Abstract

The development of an antimicrobial peptide from its discovery to its realization as a therapeutic is the subject of this personal account. The story spans at least 12 years and has involved the efforts of hundreds of people, including both scientists and business people, involving disciplines ranging from peptide chemistry to banking, at a cost of about $100,000,000. As yet the antimicrobial peptide remains unavailable for human therapeutic applications.

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