Abstract
Toxic molecules have provided both defence and attack strategies for many species across the biological kingdom. Whether it is a hemolytic protein secreted by a bacterium, poly-peptides in the venom glands of snakes or scorpions, or small organic molecules secreted from the skin of a fish or frog, these molecules have evolved as vital elements in the survival of the species that play their host.
Many natural toxins are lethal by virtue of interfering with crucial aspects of signaling processes in nerve and muscle cells—for example, blocking ion channels or competing with native agonists for binding sites on neurotransmitter receptors. These toxins are highly selective and their ability to distinguish, for example, between a sodium and a potassium channel, or an acetylcholine receptor in the brain as opposed to skeletal muscle, have made them extremely valuable tools for identifying receptors, dissecting ionic currents, and characterizing signaling pathways. This book discusses a group of toxins that are equally effective in compromising the viability of a cell—but by a much more general mechanism—by puncturing holes in cell membranes and reeking havoc on the homeostasis of that cell. Although this group of toxins, known as pore-forming toxins, have no common structural thread, they all share a broad feature in their mechanism of action, namely each toxin (inactive as a monomer in aqueous solution) forms an oligomeric structure upon interaction with a membrane target. These oligomers then insert into the membrane in such a manner as to create a central pore. As distinct from a highly selective ion channel, these pores are promiscuous and will allow a wide variety of small molecules to exchange between the cytoplasm of the cell and its extracellular environment.
The Editors of this monograph have succeeded in bringing together authors with experience of a wide range of pore-forming toxins, emphasising the diversity of targets that these molecules have. The Editors should also be congratulated for choosing a group of authors who, when their efforts have been assembled together, provide a remarkable multidisciplinary view of the field. The reader can find, for example, structural details of both toxin monomers and oligomeric pores, using nuclear magnetic resonance (NMR) and X-ray diffraction techniques, a variety of electrophysiological and fluorescence energy transfer studies to monitor the mechanism of oligomerisation and pore formation in artificial bilayers, single-channel analyses of currents generated by ions traversing the pore, and cell biology studies of the consequences of pore formation. This multidisciplinary approach has not been sacrificed at the expense of superficiality; all contributions are in sufficient detail to satisfy the individual specialist, who is also provided with extensive reference to the primary literature. The mechanism of pore formation is a modeller’s dream and the book provides an extensive collection of models, albeit with varying degrees of experimental evidence. There is a good collection of 17 high quality color plates that adds both to the understanding and visual presentation of the authors’ data.
This book is divided into two sections, the first discussing proteins of (primarily) prokaryotic origin, whereas the second discusses peptides of (primarily) eukaryotic origin. The Editors have judiciously chosen studies of Staphylococcus aureus for their first chapter and this will certainly grab the attention of many nonspecialists. S. aureus (in the form of methicillinresistant S. aureus, or MRSA) has achieved notoriety in the popular press as a superbug and one of the most frequently isolated bacteria in hospital environments, being responsible for up to 15% of nosocmial infections. Prevost and colleagues (Strasbourg and Trento) review the genetics, physicochemical properties, and structural features of two pore-forming leucotoxin families (S- and F-toxin or LukS-PV and LukF-PV) isolated from S. aureus. The two families of toxins are synergistic in their mechanism of action and share common antigenic epitopes. The authors then go on to discuss their biological activities and human diseases associated with the toxins, as well as inflammatory mediators generated by toxin action.
The second chapter by Benz (Würzburg) provides a more focussed experimental discussion on the formation of channels in artificial bilayer membranes, by the RTX group of toxins. RTX toxins are unusual in that the pores formed are transient structures and can be formed from either monomers or oligomers, resulting in pores of inconsistent diameter. This group of bacterial toxins has a common feature of a calcium-binding domain, consisting of repeats in a glycine-rich nonapeptide (L/I/F)-x-G-G-x-G-(N/D)-D-x. The unfortunate name RTX toxins (standing for
The two subsequent chapters discuss pore-forming toxins that have been implicated in human pathological conditions—Pseudomonas toxin (cystic fibrosis) and Helicobacter pylori vacuolating toxin (gastric ulcer). The brief chapter by Sliwinski-Korell and colleagues (Giessen) describes biochemical experiments (sodium dodecyl sulfate–polyacrylamide gel electrophoresis [SDS-PAGE] and tryptic digests) analyzing the structural changes involved in the oligomerization of Pseudomonas cytotoxin and mutants deficient in pore formation. H. pylori vacuolating toxin (VacA) oligomerizes to form an anion-selective channel and the multifaceted chapter by Zoratti and colleagues (Padova) describes the physicochemical properties of the VacA channel and information on pore structure, gained from biophysical analysis of anion channel blockers, using both planar lipid bilayers and the apical membranes of polarized cells, permeabilized by the toxin.
Baccillus thuringienus produces insect-specific δ-endotoxins, belonging to the crystal insecticidal protein (Cry) gene family. Cry proteins bind to a receptor on the surface of midgut epithelial cells, insert into the membrane, oligomerize and form ionic channels that lead to osmotic cell lysis and death of the insect. The tertiary structure of δ-endotoxins reveals three domains and the contribution by Gerber and Shai (Rehovot) discusses the use of synthetic peptides to determine the membrane binding affinity of various segments, as well as their assembly and interaction, using fluorescence energy transfer techniques. The next chapter by Soberon and colleagues (Cuernavaca) describes complementary functional studies, analyzing the functional complementation of Cry mutants, deficient in either receptor binding or pore formation. Direct binding studies and fluorescence measurements of membrane potential are used, as well as direct toxicity testing on insect larve.
There follows one of the highlights of the book, a fascinating chapter by Slatin and Kienker (New York) on the paradoxical mechanism of action of pore-forming colicins. Colicins are toxic proteins found in one third of Escherichia coli, in partnership with a cognate immunity protein, which serves to protect the cell from its toxic partner. Not all colicins are pore-forming molecules—some are enzymes that act in the target cell cytoplasm and enter the cell by an as yet unexplained mechanism. Slatin and Kienker focus on pore forming colicins that form a voltage-gated channel and here we come to the conundrum—the pore has a large diameter, with cis- and trans- entrance of 18 Å and 10 Å, respectively, narrowing to 7 Å at its most restricted point. However, there is much evidence to show that the channel is formed by a monomer, although there is not enough protein in the membrane to form a pore (one of the largest colicins, colicin Ia, has 626 amino acids or a molecular weight of approximately 60 kDa). Another mysterious feat is that colicins are capable of protein translocation, although the amount of colicin protein that forms the protein translocation pathway is demonstrably larger than the ion-conducting pore. The chapter concludes with a review of diphtheria toxin–induced channels and speculation that colicin and diphtheria toxin may promote protein translocation by a similar mechanism.
The final chapter of the first section, written by Anderluh and Macek (Ljubljana), makes a break from all of the bacterial toxins described earlier and discusses structural and functional features of actinoporins, pore-forming cytolytic peptides from sea anemones (Actinaria). The role of actinoporins is intriguing—they are highly toxic to fish and crustaceans and it has been suggested that they are secreted in their underwater habitat, to act as a repellent against potential predators. Actinoporins form rectifiable, cation-selective pores in a multistep process by oligomerization of a soluble protein into a membrane aggregate consisting of three to four molecules.
The second half of the book is devoted to pore-forming peptides. These peptides found in animals, plants, and microorganisms have attracted widespread attention in the pharmaceutical industry as potential novel antibiotics, because of the seeming inability of bacteria to develop resistance against them. The peptides play a crucial role in innate immunity, the first-line defence mechanism of all species that do not possess the adaptive immunity of higher vertebrates.
Antimicrobial peptides fall into several diverse structural groups; the common thread to their activity is thought to be the spatial arrangement of cationic and hydrophobic domains within their structure. The first chapter in this half of the book, by Zhao and colleagues (Helsinki, L’Aquila, and Cagliari) provides a brief overview of the structural and charge requirements for biological activity. This is followed by an authoritative account by Lazarovici (Jerusalem) of the properties of pardaxins, toxic pore-forming peptides secreted from specialized epithelial glands of several species of sole, which the fish uses to repel shark predators. Pardaxins are also presynaptic neurotoxins and Lazarovici and his colleagues have exploited this specialized property of pardaxins to dissect molecular events in exocytosis. This chapter discusses a range of topics, from the structural and biophysical properties of the oligomeric pardaxin “pore” to model pharmacological studies using PC12 cells, investigating the roles of calcium, the arachadonic acid cascade, and mitogenactivated protein (MAP) kinases on pardaxin-stimulated neurotransmitter release.
Rivas and Andreu (Madrid and Barcelona) go on to discuss cecropins, linear pore-forming peptides from insects that are one of the most widely used templates in the design of antimicrobial agents and membrane-active peptides. The review exhaustively details the structural and biophysical properties of cecropin analogues that have been synthesized, including chimeras of cecropin and mellitin. The antibacterial, antifungal, and antiparasitic properties of cecropinmellitin hybrids are also detailed, as well as their inhibitory effects on viruses and tumour cell lines.
The plant bacterium Pseudomonas syringae produces two related groups of pore-forming antifungal peptides. The syringomycins are cyclic amphiphilic peptides of nine amino acids, containing a common dehydroaminobutanoic acid/C-terminal chlorothreonine motif and an N-terminal serine residue modified with various lipid chains. The chapter by Takemoto and colleagues (Logan, Philadelphia, and Budapest) briefly discusses the factors influencing pore formation, using biophysical and molecular genetic studies. The syringopeptins (22 to 25 amino acids) share some common features with syringomycins, namely a cyclic structure, several unusual amino acids and a hydroxy fatty acyl modified N-terminus. The chapter by Dalla Serra and colleagues (Trento, Genoa, Naples, and Rome) discusses the structure of syringopeptins, their biological role, and physicochemical and biophysical studies of pore formation, using artificial lipid membranes, plant vacuole, and erythrocytes.
The final chapter of the book by Hirakura and colleagues (Tokyo and Los Angeles) is a little out of place in these surroundings. It provides a cursory survey of cationic channels formed by amyloid peptides and their role in human disorders, such as Alzheimer’s disease, prion diseases, and type II diabetes.
My only (minor) gripe to the Editors is that they have provided a rather arbitrary and uneven glossary—for example, I question the necessity to define “antigen,” “GABA,” or “sea anemone.” On the other hand, many important keywords are omitted from the index; for example, the preface mentions the use of pore-forming toxins as biosensors but this keyword is not to be found in the index. Many pore-forming peptides have been tested for antiparasitic activity (for example, against malaria) and this is extensively discussed in the chapter on cecropinmellitin hybrids; however, “malaria” is not to be found in the index.
In conclusion, I unreservedly recommend this book for addition to the institutional library of any research group interested in natural toxins.