Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century

Toxicon 39 (2001) 1793±1803

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Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century R.J. Collier* Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA

Abstract Diphtheria toxin is one of the most extensively studied and well understood bacterial toxins. Ever since its discovery in the late 1800's this toxin has occupied a central focus in the ®eld of toxinology. In this review, I present a chronology of major discoveries that led to our current understanding of the structure and activity of diphtheria toxin. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Toxin; ADP-Ribosylation; Translocation; Receptor

1. Introduction At the beginning of the 20th century, bacterial toxins had only recently been discovered, and concepts about their structures and actions were amorphous. Now, a century later, we have atomic-level structures for many of the toxins associated with major diseases; we know the fundamental biochemical mechanisms by which they act; and we have a general, albeit imprecise, understanding of how the biochemical lesions lead to overt symptoms. Diphtheria toxin (DT) is one of the most extensively studied and well understood bacterial toxins. Ever since its discovery in the late 1800s, it has occupied a central focus of the ®eld of toxinology. There are several reasons for this. One derives from the fact that the causative agent of diphtheria, Corynebacterium diphtheriae, was one of the ®rst bacterial pathogens to be isolated and grown in pure culture, and DT among the ®rst toxins discovered. Another relates to the fact that diphtheria is a relatively simple disease, one the few in which the major symptoms are attributable to the action of a single toxin. This fact, once realized, raised hopes that understanding the activity of this single molecule could hold the answer to how, in biochemical terms, a bacterium killed a person. Finally, several properties of DTÐe.g. its general cytotoxicity, it ability to inhibit protein synthesis, and its property of crossing membranesÐhave placed it in the midst of some of the * Tel.: 11-617-432-1930; fax: 11-617-432-0115. E-mail address: [email protected] (R.J. Collier).

most active and exciting ®elds of biological science in recent decades. In this review, I present a chronology of major discoveries in the progression of our fundamental understanding of the structure and activity of DT from the time of its discovery through the year 2000. Following this, I present a brief review of our knowledge of Pseudomonas aeruginosa exotoxin A (ETA), the closest relative of DT in terms of intracellular mode of action. The discussion of both toxins will be restricted to fundamental research on structure and mode of action, although there have been important applications of both of these toxins in developing targeted toxins for therapy. 2. Diphtheria toxin After Loef¯er isolated C. diphtheriae in pure culture in 1884 (Loef¯er, 1884), he found that intratracheal inocculation of the organism into rabbits and pigeons led to localized infection and formation of the characteristic pseudomembrane seen in human disease. He also observed lesions in internal organs of these animals, and showed that they were sterile. This suggested that the bacteria produced a diffusible toxic substance that caused tissue damage at sites distant from the localized infection. Direct evidence for such a toxin came in 1888, when Roux and Yersin reported that injection of animals with sterile ®ltrates of liquid cultures of C. diphtheriae caused death with a pattern of lesions characteristic of diphtheria (Roux and Yersin, 1888).

0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0041-010 1(01)00165-9

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Fig. 1. ADP-ribosylation of EF-2. A, adenine moiety; N, nicotinamide moiety; R, ribosyl moiety; P, phosphate moiety, of NAD.

These seminal ®ndings, together with the almost simultaneous discovery of tetanus toxin, led rapidly to the discovery of humoral immunity. Sublethal doses of either of these toxins induced mammals to form substances (antibodies) in the blood stream that would neutralize the activity of the speci®c toxin injected (Behring, 1890). It was but a short step from this to the use of immune serum (containing antitoxin) to treat patients with diphtheria. The limited duration of the immunity effected by passive immunization limited its use to epidemics, however. In 1923 Ramon discovered that formaldehyde-treatment of DT produced a form (toxoid) that was entirely nontoxic, but could still induce the formation of toxin-neutralizing antibodies (Ramon, 1923). This provided the basis for widespread immunization, which has made diphtheria a rare disease in countries with high levels of medical care. Early attempts to determine the chemical nature of DT were frustrated by the lack of adequate methods for isolating it from the complex medium required to grow C. diphtheriae. By 1937, however, both Pappenheimer (1937) and Eaton (1936) had isolated the toxin in suf®ciently pure form to establish that it was a protein. Progress on understanding the structure and mode of action of DT was interrupted by World War II. Also, the advent of antibiotic therapy in the 1940s, and the erroneous assumption that antibiotics could eliminate the threat of bacterial diseases, shifted the focus of research away from such diseases for an extended period. A few stalwart individuals maintained a strong interest in bacterial toxins throughout this period, however, including A.M. Pappenheimer, Jr., who devoted a major portion of his career to understanding the mode of action of DT. For more than three decades Pappenheimer inspired those who passed through his laboratory to perform research in this area (Pappenheimer, 1993). 2.1. Biochemical mode of action The early 1950s saw major advances in methodology and fundamental understanding of cellular processes that formed the foundation for seminal discoveries on DT action later in the decade. Protocols were developed for maintaining mammalian cells continuously in culture; radioisotopically labeled compounds (e.g. amino acids, nucleotides) came into widespread use; and there were major advances in knowledge about the mechanism by which cells synthesize proteins. In 1957, two groups (Lennox and Kaplan, 1957; Placido Sousa and Evans, 1957) reported that low concentrations of DT were lethal for many mammalian cell lines, causing cell

lysis within a few days. Later it was found that cell lines from rats or mice were relatively resistant to the toxin, a ®nding that correlated with the resistance of these animals to DT. This work established cultured mammalian cells as an excellent platform for the study of DT action. Strauss and Hendee, working with Pappenheimer, were the ®rst to probe the action of DT on metabolic functions of cultured cells (Strauss and Hendee, 1959). In 1958, these investigators reported that DT, at saturating toxin concentrations, caused a complete cessation of protein synthesis in HeLa cells after a lag period of ,1.5 h. Effects on glycolysis, respiration, nucleic acid synthesis, and morphology, were seen only long after this lag period (Strauss, 1960; Strauss and Hendee, 1959). This elegant study represented the ®rst indication that DT might act speci®cally on the proein synthesis. The 1960s witnessed elucidation of the fundamental biochemical reaction by underlying the action of DT. Early in the decade, Collier and Pappenheimer showed that DT blocked incorporation of radiolabeled amino acids into protein in cell-free systems from HeLa cells and rabbit reticulocytes (Collier and Pappenheimer, 1964). Further, they found that this inhibition was dependent on a low molecular mass component of cells, identi®ed as nicotinamide adenine dinucleotide (NAD). Collier then identi®ed the DTsensitive component of the eukaryotic protein synthesis machinery as elongation factor-2 (EF-2), a 100 kDa protein that functions in polypeptide chain elongation on ribosomes (Collier, 1967). Building on this knowledge, Honjo et al., in the laboratory of Osamu Hayaishi, used radiolabeled preparations of NAD to demonstrate that DT catalyzed transfer of the ADP-ribose moiety of NAD to EF-2 (Fig. 1) (Honjo et al., 1968). Independent evidence for this mechanism came from Gill, working with Pappenheimer (Gill et al., 1969). Thus, by the end of the 1960s it had been established that DT was an enzyme that catalyzed the ADP-ribosylation of EF-2. Con®rmation that this reaction underlay the toxicity of the molecule, and thus the pathogenesis of diphtheria, came shortly through mutational analysis of the toxin. DT was thus the ®rst member to be identi®ed of the class of bacterial toxins that act by ADP-ribosylation of a target protein. More generally, it was the ®rst recognized member of the large class of toxins that enzymically modify substrates within the cytosol. In 1975 Iglewski and Kabat reported that ETA inhibited protein synthesis by the same biochemical mechanism as DT (Iglewski and Kabat, 1975). Later in the decade cholera toxin and the related protein, heat-labile toxin from E. coli were also found to exhibit ADP-ribosyltransferase activity, but modi®ed a different protein acceptor (Beckner and

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Fig. 2. Activation of DT by proteolytic processing and reduction. Cleavage at the furin site may be accomplished by mild trypsinolysis in solution.

Blecher, 1978; Gill and Meren, 1978; Moss and Richardson, 1978). Since then a wide variety of other toxins, including ones from Bordetella pertussis and various Clostridium species, have been found to use this same fundamental reaction to disrupt various metabolic processes within host cells. Concomitantly the range of catalytic mechanisms used by the broader class of intracellularly acting toxins has expanded greatly, to include members that act as proteases, as N-glycohydrolases, deamidases, glucosyltransferases, and other mechanisms. Bacteria have thus been remarkably resourceful in subverting a variety of enzymic mechanisms to nefarious ends. 2.2. Basic structure±function relationships Collier and Cole reported in 1969 that ADP-ribosylation activity of DT was a property not of the holotoxin, but rather of a subunit or fragment of the molecule (Collier and Cole, 1969). Subsequent studies in the Collier laboratory (Collier and Kandel, 1971; Drazin et al., 1971) and by Gill et al. in Pappenheimer's laboratory (Gill and Dinius, 1971; Gill and Pappenheimer, 1971) showed that the holotoxin was a proenzyme, which must be cleaved into two fragments for enzymatic activity to be expressed (Fig. 2). The 193-residue N-terminal fragment, fragment A, carried the ADP-ribosyltransferase function of the toxin, and the 342-residue Cterminal fragment, fragment B, was assumed to bind the toxin to its receptor on cells. DT thereby became the ®rst example of an A±B toxin, in which the catalytic and receptorbinding functions are partitioned onto separate polypeptides. The A±B motif is now known to be almost universal among intracellularly acting toxins. In DT the A and B moieties are connected by both a peptide bond and a disul®de bridge. Both proteolytic nicking of the polypeptide and reduction of the disul®de are required for the A and B fragments to separate and for enzymic activity of fragment A to be expressed. Similar dual linkages between A and B moieties were later found in other toxins, which thus require both proteolysis and reduction for expression of the enzymic function.

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Another major advance in the early 1970s, was the isolation by Uchida, Gill, and Pappenheimer of the ®rst mutant forms of DT (Uchida et al., 1971). Freeman reported in 1951 that only strains of C. diphtheriae that were lysogenic for certain bacteriophages were capable of producing DT (Freeman, 1951), and evidence accumulated over the ensuing two decades had suggested that the structural gene for DT lay on the phage genome. From a mutagenized lysogenic strain of C. diphtheriae, C7(b ), Uchida et al. isolated mutant phages that speci®ed the formation of nontoxic forms of DT when they lysogenized C7(2). This was the ®rst unequivocal evidence that the structural gene for DT resides within the genome of bacteriophage b . Toxins produced by many other bacterial pathogens are now known to reside on mobile genetic elements, including phage genomes. Analysis of various DT mutants complemented the biochemical studies of toxin structure and helped generate a map of the toxin's major functional sites (Uchida et al., 1972, 1973a,b,c). The inability of CRM45, a nontoxic chain-termination mutant of DT lacking the C-terminal region, to bind to cells gave indication that this missing region was involved in receptor binding. Another nontoxic mutant, CRM197, contained a lesion that blocked ADPribosylation activity. When CRM45 and CRM197 were nicked and reduced, and the preparations were mixed with each other and allowed to reoxidize, it was found that toxicity was regenerated. Through exchange of subunits the active A chain of CRM45 had joined with the active B chain of CRM197. This showed unequivocally that both the A and the B fragments of DT were required for toxicity, consistent with the results of biochemical studies. 2.3. Membrane interactions: insertion, pore-formation, and translocation The discovery that puri®ed DT catalyzed the ADPribosylation of EF-2, a cytosolic protein, implied that the toxin (or, as we later learned, an enzymically active piece of it) crossed a cytosol±contiguous membrane. This idea ran counter to the conventional notion that membranes were impermeable to proteins, but then DT was no ordinary protein and might have evolved ways to translocate across a phospholipid bilayer. In 1976, Boquet, Pappenheimer and coworkers reported that the N-terminal domain of fragment B (that portion of B within CRM45), and molecules containing this domain, bound the nonionic detergent, Triton X-100, above the detergent's critical micelle concentration (Boquet and Pappenheimer, 1976; Boquet et al., 1976). This property was known to be associated with membrane proteins and suggested that this hydrophobic domain of the B chain might enable the toxin to insert into a phospholipid bilayer and facilitate transfer of the enzymatic A chain across it. This hydrophobic domain was cryptic in the holotoxin, in

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that prior denaturation of the protein in sodium dodecyl sulfate was required for it to bind Triton X-100. The early 1980s saw two major discoveries relevant to the translocation of DT across membranes. In 1980, Sandvig and Olsnes (Sandvig and Olsnes, 1980, 1981), and independently Draper and Simon (Draper and Simon, 1980), reported that low pH triggered translocation of the A-chain of cell surfacebound DT across the plasma membrane. This followed on the heels of the discovery by Helenius that acidic endosomal pH (pH , 5) triggered entry of Semliki Forest virus into cells (Helenius et al., 1980; White and Helenius, 1980); and it correlated with prior indications that DT was endocytosed after binding to its receptor on cells. The ®nding also explained the puzzling observation of Kim and Groman in the 1960s that ammonium chloride (a lysosomotropic agent) blocked the action of DT in cell culture (Kim and Groman, 1965). The implication of the results of Sandvig and Olsnes, and Draper and Simon, was that receptor-bound DT is endocytosed and traf®cked to an acidic membrane-bound compartment within the cell, where translocation to the cytosol occurs in response to the low pH. Ammonium chloride and other lysosomotropic agents raised the pH of the acidic compartment above the threshold necessary for translocation, thereby inhibiting toxin action. This model has borne the test of time. The second major discovery relevant to translocation was the ®nding in 1981 by Kagan, Finkelstein, and Colombini (Kagan et al., 1981) that the N-terminal domain of fragment B chain formed ion-conductive channels in arti®cial membranes (planar phospholipid bilayers) under acidic conditions. Donovan et al. made a similar observation with whole DT (Donovan et al., 1981). Roa et al. (1985) later reported evidence that the fragment A moiety of DT is translocated into phospholipid vesicles under acidic conditions. These results correlated with the pH-dependent translocation demonstrated in cell culture and provided evidence that the B chain of DT could indeed convert from a soluble to an integral membrane form as a step in toxin action. Thus by the mid-1980s, a rough outline of the major events occurring in the entry of DT into a cell and the translocation of its A fragment to the cytosol had been established. 2.4. Enzymatic properties: diphthamide and the active site of DT The 1980s also witnessed interesting developments relevant to the mechanism by which DT inactivated EF-2. In 1980, Bodley and coworkers added a peculiar twist to the DT story by showing that the amino acid residue of EF-2 that was ADP-ribosylated is, in fact, a post-translationally modi®ed histidine (Van Ness et al., 1980a,b). They dubbed this residue diphthamide. Diphthamide was found to be unique to EF-2 and was present in this protein from all eukaryotes. In later studies Bodley et al. (Chen and Bodley, 1988), and Moehring and Moehring (1983, 1984), demonstrated that diphthamide is generated by a multienzyme reaction sequence. The physiological function of this post-

translational modi®cation of EF-2 remains unknown to this day, and the evolutionary pathway that led DT to modify a post-translationally modi®ed site on EF-2 remains speculative. It has been suggested that ADP-ribosylation of the diphthamide moiety of EF-2 may occur as a (presumably reversible) regulatory event in normal cellular physiology, and there is some evidence for this (Fendrick and Iglewski, 1989; Fendrick et al., 1992). In another advance relevant to how DT inactivates EF-2, the identity of a crucial active-site residue in DT was revealed through discovery of an ef®cient photolabeling reaction. Many years earlier, Kandel, Collier, and Chung had shown that fragment A binds a single molecule of NAD (Kd , 8 mM) (Kandel et al., 1974). Carroll and Collier reported in 1984 that under the in¯uence of UV radiation the nicotinamide moiety of NAD was covalently transferred to a speci®c residue of DT, Glu-148 (Carroll and Collier, 1984; Carroll et al., 1985). By directed mutagenesis Glu-148 was demonstrated to be essential for catalysis of ADP-ribosylation (Tweten et al., 1985). Later this Glu residue was revealed to be the only universally conserved active-site residue of the broad ADP-ribosyltransferase family of enzymes (Bazan and Koch-Nolte, 1997). Montecucco, Papini, and coworkers have presented evidence that His-21 is important in NAD binding (Papini et al., 1990, 1989). The functions of these and other active-site residues of DT and other ADP-ribosyltransferases have been extensively probed by directed mutagenesis (Blanke et al., 1994; Wilson et al., 1990). Besides taking the understanding of toxin action to a new level of detail, the discovery of Glu-148 had the bene®t of allowing an enzymically attenuated form of the holotoxin to be eloned and expressed in E. coli (Barbieri and Collier, 1987). Cloning of wild-type DT in E. coli had been prohibited by NIH, except under the most stringent containment conditions, but cloning of DT containing a 3 bp substitution at this site (E148S) was permitted under less-stringent conditions. DT-E148S exhibited a reduction in ADP-ribosylation activity of 2±3 orders of magnitude. This substantially eliminated risk of expression in an heterologous bacterium, but the protein was nonetheless still active enough to inhibit of protein synthesis in cultured cells, thereby facilitating studies of its structure and activity. The ability to express DT-E148S in E. coli opened a path to detailed mapping functional sites throughout the molecule by directed or random mutagenesis. 2.5. The crystallographic structure of DT In 1992, the three-dimensional structure of DT was revealed for the ®rst time. Collier and coworkers obtained X-ray grade crystals of DT a decade earlier (Collier et al., 1982), and Choe, Eisenberg and coworkers solved the structure (Choe et al., 1992) (Fig. 3). The molecule was seen to have three discrete folding domainsÐC, T, and RÐcorresponding to the three major functions of the toxin: catalysis,

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Fig. 3. Crystallographic structure of diphtheria toxin. Active-site cleft of C-domain contains the endogenous dinucleotide ApUp.

translocation, and receptor-binding, respectively. The C domain therefore corresponded to fragment A, and the T and R domains to fragment B. The domains are arranged in the shape of a Y, with the T domain forming the lower segment and the T and R domains the upper, angled segments of the Y. The active-site cleft of the C domain was seen to be blocked from access to its bulky substrate, EF-2, by the R domain, explaining why the A fragment must separate from the B fragment for its ADP-ribosylation activity to be expressed. The C domain was seen to be of the a 1 b type. In the active site was an endogenous dinucleotide, ApUp, that had been found in certain preparations of DT (Barbieri et al., 1981). ApUp binds with high af®nity and apparently acts as an NAD analog, but does not appear to be relevant to toxin action. The T domain was seen to be an a helical bundle, and the R domain a ¯attened b -barrel. The trypsin-sensitive loop between the C and T domains was not observed in the structure, due presumably to its ¯exibility. The T domain was seen to correspond almost exactly to B45, the hydrophobic N-terminal domain of the B chain

of CRM45, which had been shown earlier to be capable of forming pores in membranes under acidic conditions. The ®rst crystallographic structure of DT solved was of a nontoxic, metastable dimeric form. Carroll, Barbieri, and Collier showed that this form is a laboratory artifact generated when the toxin is frozen in buffers, notably sodium phosphate, that undergo a dramatic reduction in pH upon freezing (Carroll et al., 1986). (This raises a warning ¯ag in choosing buffers for storage of biological substances in frozen form.) The dimer slowly dissociated to native monomers at neutral pH and could be rapidly dissociated with dimethyl sulfoxide. The dimer was unable to bind receptors, and Bennett, Choe, and Eisenberg later showed that the R domains of each monomer within the dimer were associated with the partner monomer in an interesting manner (Bennett et al., 1994b). That is, these domains had been swapped between the two monomers and were functionally impaired. This seminal discovery led Eisenberg and coworkers to identify a wide variety of instances in the crystallographic literature of domain swapping as a means of oligomerization

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(Bennett et al., 1994a; Schlunegger et al., 1997). The structure of monomeric DT was solved later and showed that the structures of the individual domains were virtually identical to their counterparts in the dimer (Bennett and Eisenberg, 1994). Bell and Eisenberg solved the structure of dimeric DT in complex with NAD (Bell and Eisenberg, 1996; Bell et al., 1997). This was the ®rst crystal structure of an ADPribosyltransferase in complex with NAD and suggested features that this class of enzymes might use in NAD binding and catalysis. NAD within the complex was seen to be in an extended form with the Glu-148 carboxylate group in proximity to the scissile, N-glycosidic bond of the dinucleotide. This conformation is consistent with the known requirement of Glu-148 for ADP-ribosylation. 2.6. The T domain and translocation The crystallographic structure of DT provided an invaluable framework for understanding the various functions of the molecule in detail. An immediate bene®t was the ability to localize the ®rst mutation in DT that speci®cally blocked the translocation function. This mutation, E349K, was identi®ed by O'Keefe et al., following expression of DT-E148S in E. coli (O'Keefe et al., 1992; O'Keefe and Collier, 1989). The mutant toxin contained a leader sequence, which promoted its secretion to the periplasmic space, and it was found that the presence of DT in this location made the bacterial cells extremely sensitive to acidic pH. Lowering the pH to 5 caused rapid killing of the cells as the toxin inserted into the inner membrane, permeabilizing it and disrupting membrane potential. This system provided a means to select for mutant forms of DT that were de®cient in membrane insertion. The E349K mutation was seen in the crystallographic structure to lie in an inter-helix loop at the tip of a long hydrophobic helical hairpin (helices TH8 and TH9) buried in the T domain. Because a positively charged lysine residue at this location would be expected impede membrane insertion, this ®nding supported the suggestion from the structure alone that this hairpin might represent a membrane-insertion motif, which could function in pore formation and translocation. Results of a variety of studies have supported this model, including ones by London and coworkers using single-Cys mutants of T domain labeled with ¯uorophores (Kachel et al., 1998; Ren et al., 1999), experiments by Oh, Hubbell and coworkers with spin-labeled T domain (Oh et al., 1996, 1999b, 2000; Zhan et al., 1995), and hydrophobic photolabeling experiments of Lala (D'Silva and Lala, 2000). The translocation process and its dependence on pore formation have been investigated on cells and in various model membranes. Olsnes and coworkers have made major advances in understanding these processes by measuring arti®cial translocation of labeled toxin across the plasma membrane induced by low pH medium (Olsnes et al., 1990; Stenmark et al., 1991, 1992). Introducing

arti®cial disul®de bonds into the fragment A moiety has been found to block translocation, suggesting that the C domain must unfold at least partially to cross the membrane (Falnes et al., 1994; Falnes and Olsnes, 1995). Consistent with this model, fragment A is known to refold readily into an active form following denaturation by high temperature or extremes of pH. In addition, from careful analysis of proteolytic products obtained with toxins containing arti®cial disul®des at different locations in the C domain it has been prediced that translocation of this domains occurs from C- to N-terminus (Falnes and Olsnes, 1995); that is, the Cterminus crosses the membrane ®rst and the N-terminus last. This model has recently received support from studies of channel conductance in planar phospolipid bilayers by Senzel, Finkelstein, Collier, and coworkers (Oh et al., 1999a; Senzel et al., 1998, 2000). The results show that, when isolated T domain containing an N-terminal His6 af®nity tag inserts into a bilayer from the cis chamber and forms a pore, the His6 tag is spontaneously translocated to the opposite (trans) side of the membrane. This approach has also yielded other evidence indicating that a large Nterminal segment of T domain is translocated to the trans compartment under these conditions. Further, when extended to whole DT, the approach showed that the entire C-domain (fragment A) was translocated under these conditions. Thus the translocation process may be effectively reproduced in a model membrane containing no other proteins. London and coworkers have reported data consistent with this conclusion from experiments in liposomes (Jiang et al., 1991). Hence the T domain apparently contains the entire molecular machinery for mediating transfer of the C domain of DT across membranes. Tetanus and botulinum neurotoxins also contain central a -helical domains that appear to serve the same basic function, but the folds are entirely different from that of T domain (Lacy et al., 1998). 2.7. The DT receptor The identities of two components of the DT receptor complex were also reported in 1992. Culminating a long effort to identify the receptor, Eidels and coworkers succeeded by expression cloning in identifying heparinbinding EGF-like growth factor precursor, or HB-EGF, as a DT receptor (Naglich et al., 1992). Mekada reported that a 27 kDa membrane protein that copuri®ed with the DT receptor was CD9 (Mitamura et al., 1992). The interaction between HB-EGF and CD9, and their relative roles in toxin action, have been the subject of continuing investigation, and it has recently been reported that CD9 increases the af®nity of DT for HB-EGF (Cha et al., 2000). Choe and coworkers have obtained a crystallographic structure of DT bound to HB-EGF, which details the interaction of this moiety with the R domain of the toxin (Louie et al., 1997). Chimeric toxins in which the R domain has been replaced with another cell-binding moiety are highly toxic, indicating that the role of the HB-EGF:CD9 complex may

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be mainly to bind DT to the cell and mediate its endocytosis and traf®cking (Foss et al., 1998; Francis et al., 2000). 2.8. Activation of receptor-bound DT Recently there has been insight into another longstanding question, namely the identity of the protease, or proteases, that activate DT and other toxins in vivo. In early structure± function studies Collier, Gill and their coworkers activated DT in vitro with trypsin. Recent results of Leppla and coworkers (Gordon et al., 1995) revealed that the furin family of cell-associated proteases are capable of activating toxins. These proteases, whose normal function is the processing of cellular proteins, including certain hormone receptors, recognize a sequence that contains basic residues, and therefore is also cleaved by trypsin. The other step in toxin activation, reduction of interchain disul®de bonds, has been reported by Papini, Montecucco and coworkers to be the rate-limiting step in DT action (Papini et al., 1993). Recent data from experiments in planar bilayers indicate that the translocation is not blocked by the disul®de bridge connecting the C and T domains (Oh et al., 1999a). This lends credence to the hypothesis that reduction of the disul®de in vivo occurs on contact with cytosolic reducing agents, such as glutathione. Whether or not a disul®de bond reductase accelerates this reaction in vivo remains unknown. 2.9. Challenges In view of all that has been learned about how DT acts, what gaps remain in our knowledge? Although many remain, let me mention only two. Perhaps the most challenging is an atomic level understanding of the process of translocation. While we have an increasingly sophisticated understanding of the process, we still cannot answer the fundamental question of how the T domain mediates translocation of the C domain across a bilayer. For this we need to know more about the structure of the T domain in the membrane. What is the structure of the pore? Is it monomeric or oligomeric? What conformations do the TH8±TH9 helical hairpin and the remainder of the domain adopt in the membrane? What is the sequence of events involved in insertion of the T domain? What energetic parameters drive insertion and translocation? Answers to such questions bear not only on our underestanding of toxin action, but also on the more general questions of how proteins interact with membranes. Another longstanding question pertains to the physiological function of diphthamide. The fact that this posttranslational modi®cation has been retained throughout the eukaryotes implies a signi®cant function, but current evidence indicates that the function is subtle. Mutational analysis shows that the residue is not required for cell viability, and blocking its synthesis does not have a major effect on cell growth.

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Beyond these questions, it can be said that our notions of the sequence of events leading from toxin action on various tissues and organs in the body to death of the host remain vague and are likely to remain so, given the dif®culty of investigations at this level. Further, our understanding of the selective advantage of toxin production to C. diphtheriae and phage carrying the tox gene are likely to remain speculative for similar reasons.

3. Pseudomonas aeruginosa Exotoxin A To date it appears that, whatever the pathway by which DT evolved, no truly close relatives of this molecule appear to have survived (although they may exist among the vast array of bacteria that occupy obscure and inaccessible ecological niches in nature). The closest known relative is exotoxin A (ETA) produced by Pseudomonas aeruginosa. P. aeruginosa contrasts with C. diphtheriae in many ways. Whereas the diphtheria organism is a pathogen exclusively of man, P. aeruginosa is an opportunistic pathogen that is ubiquitous in nature. Whereas C. diphtheriae relies primarily on a single major virulence factor (DT), P. aeruginosa releases a broad array of biologically active substances into its environment (toxins, proteases, phospholipase, exopolysaccharide). In addition P. aeruginosa has acquired the ability to transfer enzymically active factors (e.g. exoenzyme S) directly into the mammalian cell via the relatively recently discovered Type III transport systems. Clearly, P. aeruginosa has adopted a life style that enables it to occupy a diverse array of environmental niches and requires a more extensive armamentarium. ETA was discovered in the 1960s through the studies of Liu and coworkers, and found to be the most potent toxin produced by P. aeruginosa (50% lethal dose in mice ,200 ng, injected intraperitoneally) (Liu, 1966). The discovery of Iglewski and Kabat that ETA uses the same biochemical mechanism of action as DTÐADP-ribosylation of EF-2Ðraised obvious questions about the structural, functional, and evolutionary relationships between the two molecules (Iglewski and Kabat, 1975; Iglewski et al., 1977). Besides having ADP-ribosyltransferase activity in common with DT, ETA is also synthesized as a single polypeptide chain with three folding domains. The catalytic domains of the two toxins have similar folds (Allured et al., 1986; Collier and McKay, 1982), an active-site Glu residue (Glu-553 in ETA) that is photolabeled with NAD under the in¯uence of UV light (Carroll and Collier, 1987), and other active-site residues in common (Douglas and Collier, 1987; Lukac and Collier, 1988a,b). Also, the native proteins are proenzymes that are activated by proteolysis and reduction of an interchain disul®de bridge (Chiron et al., 1994; Gu et al., 1996; McKee and FitzGerald, 1999; Ogata et al., 1990). Nevertheless, other properties show that DT and ETA are

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only distantly related. The ADP-ribosyltransferase activity of ETA, unlike that of fragment A from DT, is heat-labile and neutralized by antibody to ETA, but not by antibody to fragment A (Iglewski and Kabat, 1975). Sequence similarity between the two toxins is weak and limited to the catalytic domain (Carroll and Collier, 1988). The order of the domains is reversed; in contrast to DT, the catalytic domain of ETA is C-terminal and the receptor-binding domain Nterminal (Allured et al., 1986). The middle domain (domain II) of ETA is a -helical, like the T domain of DT, but the number, arrangement, and properties of the helices of domain II differ. The proteolytic activation site, which is located in a loop between the C and T domains of DT, is located within domain II of ETA, in the loop connecting helices A and B. These structural differences are re¯ected in functional differences. ETA binds to a different receptor, low-density lipoprotein receptor-related protein, or LRP (FitzGerald et al., 1995; Kounnas et al., 1992), and has a different pro®le of species and cell sensitivity. Further, domain II and Tdomain may serve different functions. While domain II has been generally assumed to function in translocation, by virtue of its location and helicity (in comparison with T domain), it does not appear form discrete pores in membranes, and data relevant to its function are limited. It could be that the primary function of domain II is simply to present the protease cleavage site. This difference in properties of the central domain re¯ects the fact that ETA appears to have evolved an entirely different translocation mechanism from that of DT. The Cterminus of the toxin contains a KDEL-like endoplasmic retention sequence, which is required for toxicity (Chaudhary et al., 1990). This sequence acts to mediate retrograde transport of the toxin (or the 37-kDa enzymatic fragment) to the endoplasmic reticulum, where the enzymically active fragment may translocate to the cytosol via the ERAD (endoplasmic reticulum associated degradation) pathway, which is normally used to degrade proteins that misfold in the ER (Hazes and Read, 1997). This pathway has been proposed to be used by a number of other toxins, including ricin, cholera toxin and the E. coli heat-labile toxins, to translocate their enzymatic subunits to the cytosolic compartment. From available data one can only say that it is likely, from the similarity in folds of the catalytic domains of DT and ETA, that these domains evolved divergently from a common ancestral domain. There is no evidence that either of the other two domains of these toxins share a common lineage. It was suggested by Uchida et al. that, because DT enzymically modi®es a eukaryotic protein, the gene encoding it (or at least its catalytic subunit) may have been captured from a eukaryote at some time in the past (Uchida et al., 1971). While there is no clear evidence to support this, it is a possibility that deserves to be investigated as the complete genomes of organisms emerge.

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