ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 18, No. 1 Copyright © 1988, Institute for Clinical Science, Inc. Bacterial Toxins MICHAEL M. LUBRAN, M.D., Ph.D. Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA 90509 ABSTRACT Many bacterial toxins are proteins, encoded by the bacterial chromo­ somal genes, plasmids or phages. Lysogenic phages form part of the chro­ mosome. The toxins are usually liberated from the organism by lysis, but some are shed with outer membrane proteins in outer membrane vesicles. An important non-protein toxin is lipopolysaccharide or endotoxin, which is a constituent of the cell wall of gram negative bacteria. Toxins may dam­ age the eukaryotic cell membrane by combining with some structural component, or otherwise alter its function. Many toxins combine with specific receptors on the surface membrane, frequently glycoproteins or gangliosides, and penetrate the cell to reach their intracellular target. A common mechanism of entry is absorptive endocytosis. Many protein toxins have an A-B structure, B being a polypeptide which binds to the receptor and A being an enzyme. Many toxins are activated, either when produced by the bacterium or when bound to the membrane receptor, by proteases (nicking). An enzymatic process common to many toxins is adenosine diphosphate (ADP)-ribosylation of the adenylate cyclase regula­ tory proteins, leading to an increase in intracellular cyclic adenosine monophosphate (eAMP). This is the mechanism of action of cholera toxin. Diphtheria toxin catalyzes the transfer of ADP-ribose to elongation factor- 2, inhibiting protein synthesis. Most toxins act on the target cells to which they bind, but tetanus toxin, and, to a lesser degree, botulinum toxin, ascend axons and affect more distant structures. Although many toxin effects caused by bacteria have been described, only a few toxins have been identified, characterized, and their mode of action determined at the molecular level. The best known of these are discussed. Introduction some intracellular target. Occasionally, they are the sole cause of disease; in Toxins produced by pathogenic bacte­ most cases, they act in concert with ria interfere with the physiological func­ other virulence factors which enable the tions of cells and are sometimes lethal. bacteria to establish themselves in the They act on the cell membrane28 (e.g., host and resist or evade its defensive hemolysins, lysins, phospholipases) or mechanisms. Although bacterial toxins 58 0091-7370/88/0100-0058 $02.00 © Institute for Clinical Science, Inc. BACTERIAL TOXINS 59 have been studied for many years, only a TABLE II few have been fully characterized and Principal Toxins Produced by Pathogenic Bacteria their mode of action determined at a molecular level. Many of the toxins are Organism Toxins proteins produced within the organisms. Mycobacteriurn Mycosides, cord factor, sulfatides tuberculosis Exotoxins are exported through the bac­ Neisseria Endotoxin terial membranes or released by lysis of meningitides Pseudomonas Exotoxin A the organisms. Gram negative bacteria aeruginosa Salmonella Enterotoxin, cytoxin have an outer membrane containing a (strains) lipopolysaccharide endotoxin. This toxin Shigella Cytotoxin (Shiga) Staphylococcus Enterotoxins, pyrogens, toxic escapes from the organism in vesicles aureus shock toxin (TST), exfoliatin, leukocidins, a-, 3-, y-, 6-toxins shed from the outer membrane. Toxins Streptococcus Streptolysins, erythrogenic toxin (group A, are also classified by their target organs ß-hemolytic) or cells, for example, neurotoxins, S. pneumoniae Pneumolysin V. cholera Enterotoxin enterotoxins, and cytotoxins; however, V. mimicus Enterotoxin V. parahemolyticus Kanagawa hemolysin one toxin may act on different target V. vulnificus Enterotoxin cells. The effects of toxins have been Yersinia Enterotoxin studied in vivo in intact animals and in enterocolitica vitro on cells or cell fractions, using whole bacteria, bacterial fractions or cell-free extracts. The sensitivity of dif­ Protein Toxin Export in Bacteria42 ferent animals and cells to toxins varies considerably, and it is often uncertain The protein toxins are synthesized on which effects observed experimentally ribosomes as single polypeptide chains can be extrapolated to human disease. with a signal aminoacid sequence com­ Bacteria and their toxins are listed in prising about ten hydrophobic residues tables I and II. Many protein toxins have at its NH 2 terminal. In gram positive similar or related properties, which will organisms, which have a single mem­ be discussed in general and in more brane, the protein traverses the mem­ detail for the different toxins: these prop­ brane by diffusion or by creating a pore. erties are export, surface binding, inter­ The signal peptide is removed by a pro­ nalization (or translocation), action on tease synthesized concurrently with the target structures and genetic control. toxin, acting at the membrane. In many T A B L E I Principal Toxins Produced by Pathogenic Bacteria Organism Toxins Bacillus anthracis Edema factor, lethal factor, protective antigen B. cereus Enterotoxins Bordetella pertussis Pertussigen, dermonecrotic toxin, tracheal toxin, lipopolysaccharide, adenylate cyclase toxin Campylobacter jejuni Enterotoxin Escherichia coli Lipopolysaccharide, enterotoxins, cytotoxin, Shiga-like cytotoxin Clostridium botulinum Neurotoxins C. perfringens Enterotoxin C. tetani Neurotoxin C. dificile Enterotoxin, cytoxin Corynebacterium diphtheriae Diphtheria toxin Klebsiella (species) Enterotoxins Listeria monocytogenes Endotoxin 60 LUBRAN cases, further mild proteolysis occurs as cholera toxin, can affect a wide range outside the organism (nicking), resulting of cells in vitro. In human cholera, how­ in two polypeptide chains held together ever, the toxin acts mainly as an entero- by one or more disulfide bonds. toxin. Specificity is related to the pres­ Protein export is more complicated in ence on the cell surface of membrane gram negative bacteria. The cell enve­ receptors specific for a particular toxin. lope consists of three layers or compart­ The receptors may be glycoproteins, ments:6,46 an inner, cytoplasmic mem­ gangliosides, which may also act as brane, an outer membrane, and a receptors of neurotransmitters and other periplasmic space between them. The physiological substances, sterols, and outer membrane is about 75 A thick and other molecules of unknown composi­ is a bilayer consisting of two leaflets, an tion. The receptors are usually randomly outer one of lipopolysaccharide and an distributed on the surface membrane. In inner one made of phospholipids. The some cases, the ligand-receptor com­ periplasmic space contains a peptidogly- plexes of toxins acting intracellularly ean membrane which gives the organism cluster after binding into specialized its rigid shape. The outer membrane areas of the membrane termed “coated contains porous areas, filled with pro­ pits”, which are lined with clathrin, a fil­ teins (porins), which allow the passage of amentous membrane protein. Clustering small molecules but not proteins. Some into coated pits is aided by a cross-link- toxins are released by cell lysis; others ing enzyme, transglutaminase. This pro­ pass into the lipopolysaccharide of the cess results in the concentration of toxin outer membrane and reach the exterior molecules from a dilute solution and in outer membrane vesicles as a protein- their localization to specific regions of lipopolysaccharide complex. True secre­ the surface membrane. tion of proteins by gram negative bacte­ Some protein toxins of high molecular ria is unusual, although it may occur weight are made up of two protomers, A with some hemolysins. and B. The active part of the toxin mole­ The complexity of protein export is cule, A, is responsible for its toxicity. It well illustrated by the excretion of heat- is usually an enzyme, acting on an intra­ labile toxin (LT) by Escherichia coli.23 cellular target; B may be composed of This toxin, as detailed, is composed of A smaller subunits. It is responsible for and B protomers. Both LT-A and LT-B binding the toxin molecule to the recep­ are synthesized with signal peptides by tor and may aid in releasing A from com­ cytoplasmic membrane-bound ribo­ bination, so that it may enter the cell; B somes. The LT-B is elongated rapidly, remains on the surface. Some toxins, processed by a signal peptidase and such as cholera toxin, bind to specific immediately assembled into B5-pen- receptors and are translocated as the tamers and transferred to the periplas­ complete toxin, not as subunits. The mic space. The LT-A is formed more mode of binding of many toxins is not slowly and remains associated externally known. with the cytoplasmic membrane until it meets and combines with a B5-pentamer, forming the soluble toxin molecule, T ranslocation o f P r o te in T o x in s28,35 which is shed in outer membrane vesi­ cles. Small molecular weight toxins proba­ Cell Surface Binding of Protein Toxins28 bly enter the cell by simple pinocytosis. Most toxins have a high affinity for A small amount of surrounding fluid specific cells, although a few toxins, such containing the toxin is enclosed in a pino- BACTERIAL TOXINS 61 cytic vesicle and moves into the cyto­ inhibiting its action and disrupting pro­ plasm. This is an inefficient process, and tein synthesis and the cell dies. its success depends on the presence of a Adenylate cyclase activity is increased high concentration of toxin in the neigh­ by cholera toxin,