Vol. 31, U.S.A. Status of Bacterial Toxins and Their ... · BONVENTRE, LINCOLN, ANDLAMANNA procedureto use intact bacilli rather than culture filtrates for the purification oftype

BACTERIOLOGICAL REviEws, June 1967, p. 95-109 Vol. 31, No. 2Copyright © 1967 American Society for Microbiology Printed in U.S.A.

Status of Bacterial Toxins and Their Nomenclature:Need for Discipline and Clarity of Expression

PETER F. BONVENTRE, RALPH E. LINCOLN, AND CARL LAMANNADepartment of Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45219;U.S. Army Biological Laboratories, Fort Detrick, Maryland 21701; and Life Sciences Division,

Office of the Chief of Research and Development, Department of the Army,Washington, D.C. 20310

INTRODUCTION........................................................ 95NOMENCLATURE OF Toxis.96

Different Bases of Nomenclature ...................................... 97Nomenclature by Anatomical Location ........ ......................... 97Nomenclature by Mode or Site of Action .... .......................... 98Nomenclature by Structure of the Toxin Molecule........................ 100

General categories based on molecular structure........................ 100Theoretical consideration of three classes of toxins and means available for

their differentiation.............................................. 100Examples of "Problem" Toxins .......... ............................. 102

Diphtheria toxin................................................... 102Botulinum toxin ................................................ 102Plague murine toxin ............................................... 103Anthrax toxin .................................................... 103Staphylococcal leucocidin ........................................ 104

ToxoIDs.. 104SYMBOLIC NOTATION FOR IDENTIFICATION OFTOXINS 105

Capital Letter Notation......................................... 105Greek LetterNotation. 105Symbolic Notation for Multicomponent Toxins. 106

RECOMMENDATIONS ................................... 107LrrERATURE CrmE. ......... .......................... 107

INTRODUCrIONIn view of the exponential growth of scientific

literature, one might justifiably ask why this papershould be published, thereby adding to the over-whelming task of perusing the current literature.But it is just this growth of scientific publicationwhich prompted us to assume the task of focusingattention on the complexity and growing con-fusion surrounding the classification and nomen-clature of bacterial toxins. Since the Ehrlichianera the field has grown more or less like the pro-verbial "Topsy," and because there have notbeen any hard or fast rules governing where aparticular toxin fits in a general nomenclaturalscheme or how one should refer to it, a ratherconfused situation now exists. As increasingnumbers of individuals direct their activities to-ward the study of toxins, and as biochemicalsophistication increases our awareness of the di-versity and complexity of materials which maylegitimately be classified as toxins, the inade-quacies and inconsistencies of currently employed

terminology will become magnified. One mightargue that this situation has existed for decadesand that terms are so ingrained in our minds andthe literature that it is futile to do anything aboutit. Indeed, in a survey which we took of approxi-mately 20 active workers in the field of bacterialtoxins, this opinion was voiced by several of theworkers. The others were of the opinion thatsomething should be done to clarify the situation,but there was no agreement as to how this couldbe accomplished. Obviously, then, if those whoare most knowledgeable in the field of bacterialtoxins have no simple solution, there is none. We,however, cannot sympathize with those whoadopt the "why bother" attitude. What we haveattempted to do is to discuss toxins in historicalperspective and show that older systems do notencompass new concepts and knowledge. It isnot our purpose to lay down any hard and fastrules which the scientific community is asked toadopt. On the contrary, we hope to show theneed and advantages of clarity of thought and

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BONVENTRE, LINCOLN, AND LAMANNA

expression, and to begin the dialogue which willhopefully end as a universally agreed uponnomenclature of toxins. The "why bother" atti-tude may be tolerated at the moment, but a morerational approach is mandatory for the nextgeneration of toxinologists.The specific objectives of this communication

are threefold: (i) to describe the old and currentlyemployed nomenclature of bacterial toxins and tosuggest some ideas to make the terminologyclearer and more uniform; (ii) to stimulatethought and awareness of the problem in thescientific community with the hope that this willlead to analysis of a constructive nature andultimately to the adoption of a consistent ter-minology; (iii) to suggest where older nomencla-ture has been and remains useful when employedin the proper context.The material covered is restricted primarily to

bacterial toxins, but there is no reason on theo-retical grounds why the concepts and suggestionsmade do not apply equally to all toxins of plantand animal origin.

NOMENCLATURE OF TOXINS

Toxins are usually referred to in two senses. Incommon parlance, a toxin refers to any poisonousmaterial derived from living organisms. In thescientific literature, notably of bacteriology, theterm toxin has been a label for a specific class ofpoisons. Poisons are any substances, either or-ganic or inorganic in nature, which when ingested,inhaled, adsorbed through the skin, or injectedparenterally produce damage to tissues or dis-ruption of normal physiological functions. Onthe other hand, as a specific class of poisons,toxins are distinct from the simple chemical poi-sons by their cellular origin, high molecularweight, and antigenicity. Since scientific nomen-clature should contribute to clear thinking by in-sistence on precise definitions of terms, the termtoxin should be restricted to a particular definedclass of poisons of animal or plant origin. Theexistence of poisonous substances as a class ofproteins demands a word to categorize these pro-teins. The fact that substances traditionallycalled toxins have proven to be proteins is thejustification for restricting the term toxin topoisonous proteins. This does not imply thatliving organisms do not produce other kinds ofpoisons which are not proteins. It simply meansthat these other kinds of poisons should not becalled toxins. Toxins should not be confused withvenoms. The term venom is derived from theLatin venenum, meaning a drink of the GoddessVenus (a love potion!), and should be restrictedto animal poisons inflicted either by a sting orbite. Venoms are usually mixtures of distinct

chemically unrelated entities, and they can in-clude one or more toxins among their constitu-ents (50).The property of antigenicity is a subtle one. It

would be inappropriate to call an antigen or ahapten a toxin if it causes lesions by participationin a harmful antigen-antibody reaction and ofitself is not toxic (32). This restriction on the useof the term toxin would eliminate calling thetraditional bacterial endotoxins a class of truetoxins, if in the future the scientific communityshould become convinced that these complexlipopolysaccharides of bacterial origin causedamage exclusively by participation in antigen-antibody reactions. In that case, the invention ofa new term such as "endobacterial poisons" couldbe employed legitimately to describe the endo-toxins, so that they would not be included amongthe true toxins. At the present time, however, theendotoxins should not be disqualified, since thereis sufficient evidence that they are directly harmfuland do not depend upon antigen-antibody reac-tions for all of their toxic properties (27). New-born piglets, free from immunoglobulins, havebeen found to be extremely sensitive to the lethaleffect of endotoxin. The development of toleranceto endotoxin (68), on the other hand, suggeststhat some of the toxic reactions evoked by endo-toxins are antigen-antibody mediated. Therefore,it is likely that the complex effects elicited byendotoxins are due to both their inherent toxicityand antigenicity.

Implied, if not always stated, in the definitionof a toxin is the property of specificity in modeand sites of harmful activities. Experience sug-gests that the great majority and possibly all ofthe toxins are not general cellular poisons, be-cause, in those cases where information is avail-able, the primary anatomical (morphological) orfunctional (biochemical) lesions have been foundto be restricted to a limited spectrum of relatedtissues or organ systems; e.g., neurotoxin, cardio-toxin, etc. As will be discussed, these descriptiveterms, though useful, hardly suffice in the charac-terization of all bacterial toxins.

Generally speaking, bacterial toxins possess norecognized function in the metabolism or struc-ture of the organisms producing them (excludingendotoxins which represent a portion of the bac-terial soma). The hypothesis that diphtheria toxinwas part of the cytochrome system of Corynebac-terium diphtheriae (43) has never been substan-tiated. It seems highly improbable, however, thatall of these complex molecules would be synthe-sized as waste products and nonsense molecules.Rather than being a general characteristic ofbacterial toxins, the absence of known functionfor these materials may be an indictment of our

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BACTERIAL TOXINS AND THEIR NOMENCLATURE

scientific ignorance. Some toxins in venoms serveto immobilize and prevent the escape of potantialfood, and in some reptilian venoms several toxinsact to immobilize prey prior to and during inges-tion (11).Any kind of disturbance of normal function or

structural change may be employed for the detec-tion of a toxin. Oakley (39), who feels that theterm toxin has outlived its usefulness and mightbe replaced by "soluble bacterial antigen," hasreviewed various methods employed to detecttoxins, most of them of an immunological orserological nature. Although immunologicalmethods have the desirable elements of specificityand sensitivity, they require that specific anti-serum of high titer be available. This will becomeincreasingly a more difficult logistic problem forthe average laboratory as new and more complextoxins are characterized. Immunological assaysalso have the disadvantage that a toxin may re-tain its immunological specificity while havinglost all or part of its biological activities. Thelethal toxins are usually detected by the death ofsuitable laboratory animals. Methods of varyingdegrees of sophistication have been devised toquantitate lethal toxins, but they all depend on astatistical treatment of one kind or another. Theyall assume a typical "normal" host populationwhich may or may not always be true (35). Sev-eral excellent reviews on this subject are available(7, 61). For reasons of economy and convenience,many investigators prefer in vitro assays for de-tection and quantification of toxins. Unfortu-nately, in many cases, the animal assay is the onlyreliable method available; e.g., botulinum andanthrax toxins. In recent years, tissue culturemethods have been explored as a substitute forobservation of whole animals. Although reason-able success has been achieved with diphtheriatoxin (18), it has not been useful for many otherbacterial toxins because of the absence of anyvisible cytopathic effect specifically induced bythe toxins. The enthusiasm for tissue culture as-says is understandable, but it should be temperedby a recognization of two possible pitfalls. Tissuecultures can be extraordinarily sensitive to im-purities present in toxin preparations. Therefore,unless a highly purified toxin or antiserum withantibody directed against only the true toxic com-ponent is available, extreme caution must beexercised in attributing observed cytologicalchanges to the toxin itself. Secondly, a cytopathiceffect on a tissue culture may not be meaningfulwhen extrapolated to an intact animal. Sincetissue cultures tend to dedifferentiate, in vitro cellsare often not identical metabolically with the invivo cells from which they were originally derived.At the very minimum, tissue cultures are removed

from nervous system and endocrine stimuli andrestraints. Therefore, it should be kept in mindthat prominent effects of toxins on tissue culturecells can be of minor or no significance for thewhole organism. An area of research which mightimprove the utility of tissue culture assays is theformulation of environmental conditions requiredto prevent dedifferentiation in vitro.

Different Bases of NomenclatureHistorically, the nomenclature of toxins has

developed along three major paths. This oc-curred in response to three distinct points of em-phasis which investigators placed on their studies:(i) the intracellular or extracellular nature of thetoxin, i.e., whether it is associated with a struc-tural component of the bacterium or found pri-marily in the extracellular menstruum; (ii) con-cern with the structural or biochemical lesionscaused by the toxin; and (iii) attempts to under-stand the relationship between chemical structureand toxicity. Nomenclatures based on each ofthese different foci of interest are not mutually ex-clusive, and each of them can be individually anduniquely useful in communication of conceptsand data. Which system an investigator or authoremploys should be related to the major theme ofhis effort or discussion. The nomenclature basedon the relation of chemical structure to toxicityhas been least developed and requires thoughtfulconsideration, since this is a field in which we canexpect the most rapid development of knowledge.The following commentary attempts to sum-marize present usage and, wherever deemed neces-sary, suggests new terms or ideas which mightserve to fill recognized gaps in the currently em-ployed nomenclature.

Nomenclature by Anatomical LocationThe classical nomenclature of toxins based on

their intracellular or extracellular nature spawnedthe concept of exotoxins as opposed to endotox-ins. Although this is useful as an operationalclassification, it is by no means completely accu-rate. The exotoxins were considered to be meta-bolic products excreted into the growth men-struum during active growth of the bacterium oralternatively as a result of autolysis (12). Thishas given rise to the idea that little or no toxin ofthis kind can be obtained from intact cells. It hasbeen found, however, that tetanus toxin (54) andbotulinum toxin (8) can be extracted from intactcells in considerable quantities. In the latter case,it was observed that, if young cultures of Clostrid-ium botulinum (type A) were employed, as muchas nine times more toxin could be obtained fromthe cells than from the extracellular menstruum.Indeed, in recent years it has become routine

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procedure to use intact bacilli rather than culturefiltrates for the purification of type E botulinumtoxin (58, 59).The second class of toxins according to the

classical scheme is the endotoxin. These complexmaterials are considered to be derived from an in-trinsic part of the cellular structure, and, althoughthey are currently defined as lipopolysaccharides,the exact nature of the toxic moiety is still uncer-tain. At the moment, the nature and properties ofendotoxins are being investigated by many re-searchers, and, rather than becoming involved inpolemics of the day, it may be judicious to post-pone consideration of where the endotoxinsshould be placed in a general scheme until moreinformation concerning their chemistry and bio-logical properties becomes available. For thetime being, however, a useful differentiation be-tween exotoxins and endotoxins can be madeprimarily on their chemical and biological prop-erties rather than on their association with bac-terial structures. The classical exotoxins whichhave been purified are pure unconjugated pro-teins, heat-sensitive, highly antigenic, and spe-cific regarding their biological activities and thebacterial species producing them. Purified endo-toxins apparently do not contain protein in thetoxic complex (57), are heat-stable, are antigenicbut do not appear to stimulate classical neu-tralizing antibodies (5), and possess biologicalactivities which are identical regardless of thebacterial species from which they are derived. Itcan be speculated that the antigenic nature of theendotoxins depends on their association with pro-teins in the in vivo situation.Although none has been described to date,

another class of toxins based on anatomical loca-tion is conceivable. These would be materialswhich are synthesized on the cell membrane andwhich can be found only in the extracellularenvironment, or which form a portion of themembrane. The former is analogous with specificexoenzymes, such as the penicillinase of Bacilluscereus (44). Although no bacterial toxin has beenshown to fall into this category, the lethal toxinproduced by B. cereus may prove to be an exam-ple. This toxin is found only extracellularly, andlethality is not expressed by whole cells or ex-tracts derived from them (Bonventre, unpublisheddata). Toxins which might belong to the mem-brane-bound category have also not been de-scribed with any certainty. The plague murinetoxin, however, has been demonstrated both in-tracellularly and closely associated with the cellmembrane (37). It has not been possible to de-termine as yet whether the intracellular proteinand the membrane-bound material are identical,

or whether the demonstration of two distinctmolecules is an artifact of purification. A self-explanatory term which could be applied to anymembrane-bound toxins to differentiate themfrom the exo- and endotoxins is "ectotoxin."

Nomenclature by Mode or Site of ActionA second system of toxin nomenclature in use

is one based on tissue or organ affinity. Thisclassification describes a toxin in terms of eitherits apparent site or mode of action in a sensitivehost. In practice, because of our limitedknowledge of the biochemical mode of action,most toxins are referred to in terms of the tissuesaffected. For example, the toxins of C. botulinumand C. tetani and several others are described asneurotoxins. The accuracy of this terminology isbased on sound clinical and pharmacologicalevidence (78). A more specific description of thebotulinum and tetanus neurotoxins based on thistype of nomenclature must await the elucidationof the exact biochemical lesions they induce in thenervous tissues. Although there is good evidencethat tetanus toxin acts primarily at the level of thecentral nervous system after it is bound by spe-cific sphingolipids of the brain (71), and thatbotulinum toxin acts primarily on peripheralnerve elements (13), it probably would not bejustifiable to include this information in the de-scriptive terminology. If the toxins, for example,were shown conclusively to affect the metabolismof acetylcholine or other nerve cell componentsin a specific fashion, then it would be logical tobroaden their description from the generic term,neurotoxin, to include the mode of action.One example of a true bacterial toxin being

shown to possess enzymatic activity is the leci-thinase (phospholipase) of Clostridium perfrin-gens. The toxin is an enzyme which cleavesphospholipid substrates to yield phosphoryl-choline and diglycerides and, according to cur-rently accepted terminology, is a phospholipaseC. Although this is a perfectly acceptable descrip-tion, because we feel strongly that a toxin shouldbe described in biochemical or enzymatic termswhenever possible, it is not known whether theenzymatic activity of the clostridial lecithinasecontributes significantly to the pathogenesis ofgas gangrene infections. Proof of enzymaticaction would reduce the problem to a proper useof enzyme nomenclature. To ensure recognitionof the relationship of the enzymatic activity totoxicity, a compound name representing a mar-riage of enzymatic and toxin nomenclature couldbe employed. The phospholipase C produced byB. cereus is neither hemolytic nor lethal, althoughthe organism produces a lethal toxin and ahemolysin as well (24). Therefore, it may be that

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description of the clostridial a toxin (alas, anotherclassification!) as a lecithinase is accurate butalso misleading in that its enzymatic activitymay have nothing to do with its in vivo modeof action. In such a case, the term lecithinasedescribes a property but not a toxin as toxin.The hemolytic toxins include many examples of

descriptive terms which do not clarify their truenature. As already pointed out, the phospholipaseC of C. perfringens is most often designated by theGreek letter a. The term a toxin of course hasprecedence, and it is understandable that it wouldbe accepted and used at a time when none of itsbiochemical properties was known. There areseveral reasons why we feel that such a name isno longer useful. First, in the light of our cur-rent knowledge, there are several descriptiveterms which are preferable. Second, one of thestaphylococcal hemolysins is also referred toas a toxin, and this at times may be a source ofconfusion if, as often happens, the Greek letteris not coupled with the generic or specific nameof the organism which synthesizes the toxin.Since the hemolytic nature of the staphylococcaland C. perfringens a toxins is coincidental withtheir lethal property, there is no question as tothe correctness of calling them true toxins. Manyof the other hemolysins produced by microor-ganisms, however, probably are not true toxins,since their activity seems to be restricted tohemolysis of erythrocytes of one or more animalspecies and probably has little or no significancein vivo (34).

Diphtheria toxin is an example where no otherdescriptive term has been applied. Although thetoxic protein has been characterized extensivelyboth chemically and immunologically (31, 42,47, 72), its mode and sites of action have beenextremely difficult to pinpoint. A recent studyin the sensitive guinea pig has provided strongevidence that the action of the toxin in vivo maybe restricted to inhibition of protein synthesisin cardiac tissue only (10). This biochemicallesion correlates well with clinical findings inwhich fatal cases of diphtheria have been as-cribed to cardiac failure (2). If and when thisevidence is accepted by the scientific communityat large, it will be possible to include diphtheriatoxin in the nomenclature based on mode andsite of action. Since a phrase describing its ca-pacity to inhibit cardiac protein synthesis wouldbe cumbersome, it might be referred to simply asdiphtheria cardiotoxin.The classification of toxins associated with food

poisoning within the framework of mode or siteof action is difficult. The toxin associated withstaphylococcal food poisoning has been calledenterotoxin, a term implying direct action on the

alimentary tract. To be able to differentiate clearlybetween directly and indirectly acting toxins, werecommend restricting the term enterotoxin tosubstances acting directly on the alimentarytract. Focusing on this disctinction presentsanother challenge to the pathophysiologist inthe fundamental understanding of site and modeof action. Enterotoxins are proteins synthesizedby specific strains of Staphylococcus aureus inseveral immunologically distinct forms (6, 14).When a toxin is ingested or formed within thegastrointestinal tract by infectious organisms,its harmful effects can be due to direct action onthe intestinal tissues. In this case, the toxin isproperly spoken of as an enterotoxin, since bydirect contact it specifically affects the normalbehavior of cells which comprise the intestinaltract. Observations of gastrointestinal disturb-ances, however, are not of themselves conclusiveevidence of a direct intestinal site of toxic activity.The alimentary tract is notorious for its sensi-tivity to nervous-system stimuli. When gastro-intestinal signs are prominent but it is not certainthat the primary site of action is the alimentarytract, it might be logical to employ the term"nutriotoxin" rather than enterotoxin. Thiswould specify the source of the toxin withoutnomenclatural commitment to site of action.Three experimental approaches can be con-

sidered to determine whether a toxin acts directlyon the gastrointestinal tract. If toxic activity isdirect and limited to the intestinal tissue, paren-teral injection might be expected to be withouteffect or of reduced effect in producing gastro-intestinal disturbances. Since the absorption ofproteins from the intestine is often by way of thelymphatics (23, 33), another approach would beto cannulate the thoracic duct or cisterna chyliof animals exposed to the toxin by the oralroute. Theoretically, such a procedure shouldprevent toxin from entering the general circula-tion. Therefore, if the primary site of toxin actionis remote from the intestinal tract, the intestinalsymptoms should either not be seen or the illnessof the cannulated animals should be of a mildernature. A third experimental possibility would beto observe the effects of the toxin on isolatedgut segments or primary tissue cultures derivedfrom intestinal tissues (60).The nomenclature of toxins based on site and

mode of action is potentially a very useful one.However, from the examples given of currentusage, it should be clear that the classificationmay be misleading when it is used indiscrimi-nately. We would recommend that toxins bedescribed in this fashion whenever possible, butonly when there is good experimental evidenceto support it.

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Nomenclature by Structure of the Toxin MoleculeA nomenclature based on the chemical struc-

ture and related physicochemical properties oftoxin molecules is not well developed at thepresent time. In view of the intense effort beingdevoted to the biochemical characterization oftoxins, it would appear that such a system willbecome needed and most useful in the future.The following discussion considers examples oftoxins, both real and theoretical, which can bedescribed on the basis of the types of proteinmolecular structures related to their specificbiological activities.

General categories based on molecular structure.On the basis of present knowledge, three gen-eral types of toxins can be distinguished. We havechosen to call them simple toxins, complex toxins,and toxic mixtures. A fourth kind of possibletoxin is a conjugated protein: a protein to whichis attached a biologically active nonproteinprosthetic group. Classical endotoxin wouldhave to be considered a conjugated toxin iflipopolysaccharide were proven to be associatedwith a protein, and when in the conjugated stateto possess specific toxic activity absent in theprotein-free state.Two kinds of simple toxins can be described.

The first is a toxin which when purified consistsof one molecular species. In addition, it is alwayssynthesized in the fully biologically active form.The toxin molecule may polymerize to formdimers or other poisonous aggregates. Tetanustoxin would legitimately fit into this category,since it is a homogeneous protein, is synthesizedas a completely active molecule, and dimeriza-tion has been described (55). The second type ofsimple toxin is one which also is unimolecularbut which may exist in an inactive or partiallyactive precursor form. The biological inactivitymay be the result of polymerization or tertiarystructure, either of which may mask toxophoricchemical groups. Examples of toxins which canbe put into this category are the type E botulinumtoxin and the e toxin of C. perfringens type D.The maximal biological activities of these toxinscan be obtained by a brief exposure to a proteo-lytic enzyme, such as trypsin (15, 69). That asimilar process may occur endogenously duringgrowth has been suspected for type A botulinumcultures (9). The mechanism by which activa-tion occurs is not certain, but it may be either afragmentation of the molecule into toxic smallerunits (21) or merely a change in configuration ofthe polypeptide chain. In either case, the endresult would be an exposure of chemical groupsresponsible for toxicity. Though neither termhas achieved universal acceptance, the partially

active and nonactive states of simple toxins havebeen called protoxins or prototoxins. It may bedesirable to limit these terms to the completelyinactive state and to use another as yet unin-vented term for the partially active stage con-vertible to full toxicity. The universality of thephenomena of activation for toxins remainsunexplored.Complex toxins and toxic mixtures are similar

in that more than one molecular species is neces-sary for toxicity to be expressed. A complextoxin is one which consists of two or more com-ponents which must bond in the chemical senseto form a biologically active entity. The individualcomponents by themselves do not demonstratethe toxicity of the complex, and in a true com-plex the bond is easily broken to yield unalteredoriginal components. Toxic mixtures, on theother hand, also require more than one com-ponent to form an active toxin, but the com-ponents retain their molecular identity and do notundergo a chemical union. The toxins in bothof these categories can be considered multi-component toxins. Although several cases ofmulticomponent toxins are known, it is not yetpossible to say with certainty whether they arecomplex toxins or toxic mixtures. As with thesimple toxins, the possibility exists that the indi-vidual components of toxic mixtures and com-plex toxins might exist in a precursor protoxicstate. In addition, it is conceivable that one ormore components of a multicomponent toxinmight be nonprotein in nature. At least onecomponent must be a protein for the substanceto be classified as a true toxin.

Theoretical consideration of three classes oftoxins and means available for their differentia-tion. A schematic representation and possiblenomenclature of the multicomponent toxins andsimple toxins are shown in Fig. 1. This overlysimplified diagram illustrates the unique mannerin which the three toxin types would interactwith a sensitive host.With the simple toxin, there is a direct effect of

the toxic material on sensitive host cells ortissues. The host responds in a predictable man-ner and the symptoms or signs of the specifictoxemia are manifested.The complex toxins consist of two or more

components which must have the opportunityto form a chemical entity before biologicalactivity of the toxin is expressed. The complexconceivably could be formed in vitro before com-ing into contact with the host, or in vivo duringthe course of an infection or intoxication. In ahypothetical experimental in vivo situation, thesequence in which the components are introducedinto the host would probably have no effect on

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BACIERIAL TOXINS AND THEIR NOMENCLATURE

SIMPLE TOXINS

Th-iProtoxin

or (IDimer

or ( )etc.+ E* >

Polymer

COMPLEX TOXINS

PpIa.syn/oi,n Poisy SyProparios~vntox~in Pariosyntoxin Syntoxin

TOXIC MIXTURES

+ +0+

Es U-'i'. .

0

Proporiomixtoxins Pariomixloxins

+*'>Mixfoxins

FIG. 1. Schematic illustration and suggested nomenclature for the types oftoxins that may exist. The susceptiblehost is represented by *, an altered but not fully poisoned host by X, and a fully poisoned host by O. The inactiveor partially active toxin precursor is represented by broken circles, and the toxin component or fully active toxin by0. A simple toxin may require activation from a protoxic state before it exerts toxicity on a susceptible host. Also,a simple toxin may be biologically active in both a nonpolymerized and a polymerized state. The components ofacomplex toxin (syntoxin) or a toxic mixture (mixtoxin) might be called pariotoxins. Pario derives from the Latin,to give birth to. Any components ofa complex toxin or toxic mixture existing in an inactive state can be referred toeither as proloxin, as is the casefor the simple toxin, or more specifically as propariotoxins.

toxic activity, providing that the complex isformed.The toxic mixture presents a somewhat more

complicated situation since two alternativemechanisms are possible. In the first case, theindividual components of the mixture might actindependently in the host, with each componentexerting a necessary function before full toxicityis expressed. A component by itself would notelicit the symptoms elicited by the completetoxic mixture, even though it still retains theunique activity associated with that particularcomponent. If one component is inactivated insome fashion, biological activity of the mixtureis lost. A second mechanism by which a toxicmixture might express its biological activity invivo is dependent upon the sequence in whichthe individual components come into contactwith sensitive host tissues. The first componentmight act so that the result is an altered hostnot obviously showing signs of poisoning. Thesecond component then is able to act on thealtered host, in which case the effects of the multi-

component toxin are expressed as characteristictoxic signs. In the normal host, the introductionof the second component alone would not betoxic since the first had not prepared the host.The introduction of the first component after thesecond might or might not result in overt toxicactivity. Observable toxicity would depend inthis case on the length of time elapsed betweenthe exposure of the host to the individual com-ponents. The same rationale would hold if thereverse situation occurred (i.e., introduction ofthe first component followed by the second com-ponent). Since this is the sequence in which thetoxic mixture must act, however, overt toxicitywould more likely be expressed in this case thanthe other.The terms syntoxin and mixtoxin can be con-

sidered as synonyms for complex toxin andtoxic mixture, respectively. They have the ad-vantages of being simple and etymologicallyprovocative terms. Conceptually, it is not diffi-cult to visualize the difference between a syntoxinand a mixtoxin, but a problem does exist in dis-

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tinguishing them in practice. The criteria forjudging whether a particular toxin is a complexor mixture can be physiological, serological, orbiophysical. When injected separately, thoughconcurrently, components constituting a complextoxin should cause a slower or less intense toxicreaction than when injected after prior mixingin vitro. This result is expected, since separateinjection of components would result in dilutionand decreased opportunity for complex forma-tion. On the other hand, the response to a toxicmixture would be predicted to be independentof whether or not the individual componentswere mixed together prior to their injection,provided that they were injected simultaneously.This physiological test would be limited to caseswhere individual components of a toxin havebeen separated. This requirement does not existfor serological and biophysical tests. When theOudin (41) and Ouchterlony (40) techniquesare used, a syntoxin should show a specific pre-cipitin line for the complex in addition to anylines for individual components of the complex.A toxic mixture would show a number of specificprecipitin lines corresponding to the number ofcomponent precipitinogens in the mixture. Bio-physical measurements, such as sedimentation,diffusion, and gel filtration, can also be applied.Unlike a mixture, a complex with each of thesetechniques would be expected to reveal a larger-sized component (the complex itself) than re-corded for any individual component in thecomplex. In addition, a complex would haveeither an additive or reduced electrophoreticmobility relative to individual components, sincecharged groups could be expected to be exposed,neutralized, or masked by the formation of thecomplex.

Theoretically, the characterization of simpletoxins would not appear to be as difficult asthat of the multicomponent toxins. Yet, theymay present considerable difficulties. Since theunimolecular toxins are proteins, the problem isone of protein purification as well as the criteriaemployed as indices for determining purity orhomogeneity (3). As in the case of the multi-component toxins, physicochemical and im-munological methods are applicable. These in-clude electrophoretic mobility, sedimentationrates, diffusion coefficients, viscosity, aminoacid analysis, agar-gel diffusion, immunoelec-trophoresis, and neutralization of biologicalactivity with specific antisera. As these methodsand others become more readily available andare put to use for this purpose, it should bepossible in the not too distant future to purifyand characterize all of the toxins.

Examples of "Problem" Toxins

At the present time, there are only two bac-terial toxins which on the basis of sound experi-mental evidence can be classified as multi-component toxins: the anthrax toxin and staph-ylococcal leucocidin. However, there are severalothers which possess some properties whichdeserve comment within this context.

Diphtheria toxin. On the basis of biophysicaland biochemical criteria, diphtheria toxin is ahomogeneous protein which warrants its classifi-cation as a simple toxin. Immunological evidence,on the other hand, suggests that even the highlypurified, crystalline preparations do not satisfythe requirements for a homogeneous preparation.The extensive studies of Pope and Stevens haveshown that solutions of crystalline diphtheriatoxin yield several distinct precipitin lines bygel diffusion when reacted with specific antitoxin(45). In addition, they have shown that thecomponents differ in stability to phosphate saltsand to the action of pepsin and trypsin (46, 48,49). In spite of the apparent heterogeneity, ithas not been possible to associate toxicity withindividual fractions, nor has it been shown thatthe fractions must act in tandem for toxicity tobe expressed. Poulik and Poulik (51) showedthat diphtheria toxin could be separated intoseveral components by means of electrophoresis,but that all of the protein fractions retainedtoxicity to a greater or lesser degree. Therefore,although there is some evidence that diphtheriatoxin is not a single protein species and thereforemay not be a simple toxin, its known propertiesare such that at this time it cannot be consideredto be a complex toxin or toxic mixture. Theimmunological heterogeneity may merely reflecta group of closely related but not identical pro-tein molecules which all possess the same chem-ical groupings responsible for the unique biolog-ical activity of diphtheria toxin. In such a case,the diphtheria toxin could be considered to be anumber of closely related simple toxins.

Botulinum toxin. The botulinum toxins alsodemonstrate several peculiar characteristics whichmake it difficult to assign them to the ranks ofthe simple unimolecular toxins. The early workdone concerning the purification and charac-teristics of botulinum toxins type A (1, 29) andB (30) revealed that they were pure proteinsof typical amino acid composition. Originally,it was estimated that the molecular weight oftype A approached 1 million (52), and that oftype B, approximately 60,000 (30). It has beenobserved by several investigators, however,that type A toxin can be separated into fractions

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either by ultracentrifugation (73, 74), columnchromatography (56), or high-voltage electro-phoresis (28), and that toxicity is distributed(not necessarily equally) throughout the frac-tions. The molecular weights of toxic componentshave been re-evaluated recently (20, 22), and themuch lower estimates of between 9,000 and 16,000have been assigned.

In the light of these reports, several interestingquestions can be asked. Are the low and highmolecular weight toxic units of a particulartype identical in amino acid composition and,therefore, likely to be immunologically identicalas well? Is it conceivable that a botulinum toxintype is composed of nonidentical polypeptideunits each of which has the specific toxic moiety(s)incorporated within its three-dimensional struc-ture? Must each such unit be considered a simpletoxin in its own right? Depending on environ-mental conditions, these units may or may notaggregate and thus account for the fact that dif-ferent methods of purification are reported toyield toxins of different molecular weights. Thesequestions may be raised, but it should be pointedout that no experimental evidence suggests thebotulinum toxins to be either complex toxins ortoxic mixtures.

Plague murine toxin. The plague murine toxinalso possesses several properties which makecategorization within the chemical system ofnomenclature difficult.At the present time, it is not possible to say

whether it is composed of two distinct unimolec-ular simple toxins or whether it is a single molec-ular species associated with both the cell mem-brane and cytoplasm. Pasteurella pestis bacillihave been extracted, and two antigenicallydistinct toxic proteins have been isolated by discelectrophoresis (38). Each of the purified toxinsis highly toxic for mice, and there is no evidencethat the mixture of both proteins is a necessityfor toxicity to be expressed. Toxin A (molecularweight, 240,000) appears to be associated withthe cell membrane, whereas toxin B (molecularweight, 120,000) is associated with the cyto-plasmic fraction. Kadis et al. (25) have specu.lated that, if the demonstration of the twotoxins is not an artifact of purification, it may bepossible that the smaller molecular weight toxinB found in the cytoplasm is a precursor of thetoxin A which is incorporated enzymatically intothe cell membrane after dimerization. Ajl (per-sonal communication) is of the opinion that thetwo proteins are distinct toxins which act to-gether to elicit the toxic signs observed in mice.What is not certain at present is whether the

toxic response engendered by the individualcomponents is identical to the response elicitedby both proteins injected simultaneously. If thetoxic response is found to be the same in bothinstances, then the plague murine toxin is mostlikely composed of two distinct molecular formsof the same toxin (e.g., a monomer and dimer).On the other hand, if the individual proteinsdo not elicit the same physiological responsesin the sensitive host, then it is likely that theplague toxin consists of two distinct unimoleculartoxins which act independently of each other invivo.

Anthrax toxin. The anthrax toxin is undoubt-edly a multicomponent toxin. As such, it is anexample of a situation in which nomenclaturalproblems can be discussed in a specific manner.The situation can best be appreciated by describ-ing the historical development of the anthraxtoxin's nomenclature.The initial demonstration of an in vivo toxin

produced by Bacillus anthracis was accomplishedby the fine efforts of the English group at Porton(26, 62). Their observations also establishedthat the toxin was the major contributing factorleading to anthrax death in guinea pigs.

Subsequent investigations established that thetoxin was composed of three components, whichthe English group (66) named factors 1, 11, andIII. American scientists, on the other hand, as-signed the descriptive terms of edema factor (EF),protective antigen (PA), and lethal factor (LF),respectively, to the three components (4). TheEnglish usage has historical precedence, but thisin and of itself is not sufficient cause to continuesuch a nomenclature if a better one can be found.Although historical precedence is a useful con-cept, it should not be an inviolate rule when newknowledge makes a change desirable and useful.Each component of anthrax toxin by itself is

nontoxic, but is immunogenic. In the terminologyused by Americans, components EF plus PAproduce an edema when injected intracutaneouslyinto the skin of the guinea pig; PA plus LF causedeath of several animal species, with the Fisher344 rat being the most uniformly susceptiblehost. The three components together produceall of these effects, which parallel the symptomsof the infectious disease in many ways. Althoughthere are certain discrepancies in the publishedliterature regarding the antigenicity and pro-tective ability of each of the components, thereseems little doubt that the Sterne strain vaccineresults in the production of antibodies for eachof the three components (65).We recommend that the three components be

called an edema component, a protective-antigen

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component, and lethal component. The edemaand protective-antigen components have possiblybeen separated or converted into a second orderof molecular species. The edema componentwas shown to be converted spontaneously duringpurification to a form designated as fraction Y(64). It was shown by serological means thatmore than one component might be present in"Factor I" (edema component). Unfortunately,the interpretation of these findings is difficultsince the sample was contaminated with guineapig serum. Working with the protective-antigencomponent, Strange and Thorne (67) observedmultiple lines of precipitation on Ouchterlonyplates and suggested that these were due toproducts of enzymatic degradation. Wright andLuksas (77) showed that protective-antigencomponent, after purification, contained threecomponents that were closely related serologicallybut differed in electrophoretic mobility. Fish(19), who purified and separated the three com-ponents, presented evidence that the toxin existsas a molecular aggregation of variable compo-sition. Toxin produced in vivo demonstratedan increase in the number of protective-antigencomponent bands after the serum was storedat 4 C, whereas all the purified componentsdeveloped multiple lines of precipitation onOuchterlony plates. The identity of the sub-components described by these investigators isnot clear, but at this time the components appearto be related molecular species and might beidentified by Arabic numbers as edema com-ponent 1 and edema component 2. For com-pleteness, it should be noted that several authorshave described a biologically inactive toxin orinactive components (36, 65, 67).Although it can be stated unequivocally that

the anthrax toxin is multicomponent in nature,the manner in which the components interact toelicit toxicity in the sensitive host is not suffi-ciently understood so that a choice betweencomplex toxin or toxic mixture can be made. Onthe basis of the fact that death can be broughtabout in the absence of the edema component,present evidence suggests that the complete toxinis a mixture rather than a complex. On the otherhand, the possibility that the protective-antigencomponent and the lethal component must com-plex before full toxicity is expressed cannot beignored. If this proves to be the case, then,what has been called the anthrax toxin might beboth a toxic mixture and a complex toxin.

Staphylococcal leucocidin. Staphylococcal leu-cocidin (Panton-Valentin leucocidin) is the otherexample of a known multicomponent toxin.Called the "true" leucocidin to distinguish itfrom the a hemolysin and A hemolysin of

Staphylococcus aureus, it is nonhemolytic innature but produces extensive morphologicalchanges of both rabbit and human leukocytes(72). The studies of Woodin have shown that theleucocidin causes an extrusion of lysosomes aftertheir fusion at or on the cell membrane (76). Thetoxin consists of two components which havebeen separated by fractionation on Dowex andAmberlite columns. The two fractions are im-munologically distinct and have been designatedas the F (fast) and S (slow) components accord-ing to their respective rates of elution from thecolumns. Since the molecular weights of thecomponents are quite similar (75), the differencesin their rates of elution can probably be attributedto differences in the net charge of the two pro-teins. Although it is known that either or bothof the leucocidin components are adsorbed outof solution onto cell surfaces (75), and that bothmust be present for poisoning to occur, the man-ner in which they ineract to exert toxicity isunknown. Therefore, it is not possible to saywhether the staphylococcal leucocidin is a com-plex toxin or a toxic mixture. It would appear onthe basis of the availability of purified compo-nents that investigation directed toward makingsuch a distinction is now feasible. Such an effortmight provide guidelines which could be usedin determining how other multicomponent toxinsexert their toxicity in vivo.

TOXOIDS

Ehrlich originated the term toxoid to identifydiphtheria toxin preparations which upon aginglost their biological potency without concomitantloss of antigenicity (16). In practice, the termhas been extended to any toxin retaining anti-genicity in the face of loss of toxicity by anymechanism whatsover. Commercial toxoids pre-pared by formaldehyde treatment have been themost successful and widely employed for immuni-zation. Therefore, the suggestion has sometimesbeen made that toxoid refer only to formaldehyde-treated toxins. Indeed, several of the expertsqueried in our survey felt that this should be thecase. Others, however, saw no necessity for form-aldehyde treatment as a requisite for inclusionof a material in the toxoid league, providing thatit satisfied the criteria of biological inactivityand antigenicity. We tend to agree with the latterviewpoint. Ramon (53), the discoverer of thesuperior toxoiding properties of formaldehyde,employed the term anatoxin for such prepara-tions. It would be in keeping with historicalprecedent and systematic usage of language toretain the word toxoid for the entire class ofantigenic, nontoxic preparations of toxin, andanatoxins for the special class of formaldehyde-

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treated preparations. There appears to be nocompelling need, however, to make a termino-logical distinction between naturally occurringand experimentally induced toxoids.We believe that it is incorrect to consider a

component of a complex toxin as a toxoid whenthere has been no change in the fundamentalmolecule either in configuration or number ofatoms. Toxoiding could be proven for the com-ponents of a complex toxin if it were shown thatthey were inactive when mixed with the othercomponent or components necessary for lethalityor other expected biological activity, and yetremained immunologically active.SYMBOLIC NOTATION FOR IDENTIFICATION OF

ToxNsAlthough symbolic notation of toxin nomen-

clature has not been discussed as such, severalexamples have been given in other contexts (e.g.,a toxin, type A, factor 1, etc). It is clear that sucha terminology has inherent weaknesses but canbe useful if used intelligently and in a consistentmanner. Inconsistencies do occur in the symbolicnotations used today, and they are likely to be-come more pronounced as more toxins arecharacterized. We would like to point out severalinstances in which symbolic notation is usedinconsistently and finally make some recom-mendations which might help in the clarificationand standardization of toxin nomenclature.

Capital Letter NotationIt has become common practice to identify

the botulinum toxins by capital letters. The classi-fication of C. botulinum types A, B, C, D, E,and F is based on the immunological specificityof toxic protein produced by the individualstrains. Although the proteins are distinct interms ofamino acid composition and antigenicity,they evoke essentially the same neurologicaldisease in sensitive animal hosts. Although thereis no objection to the use of the letters A throughF to identify the toxins of botulism, it is anarbitrary classification. Once a system of classifi-cation is used, however, it should no longer re-main arbitrary but must be guided by specificrules. These rules should be neither too rigidnor too flexible so that they can accommodatevarious situations while remaining meaningful.No one has ever suggested that capital lettersbe used only to identify immunologically distincttoxins having the same biological activity, as isthe case for the botulinum toxins. In retrospect,however, this appears to be a reasonable sug-gestion. Indeed, the rule, without having everbeen stated, was applied in the case of thestaphylococcal enterotoxins, and, therefore, no

inconsistency in nomenclature resulted. As previ-ously noted, enterotoxins A, B, and C have beenidentified as immunologically distinct proteins(6, 14) and presumably have the same mode ofaction in vivo. Therefore, the use of capitalletters to identify the staphylococcal enterotoxinsis valid and presents no difficulties, even if inthe future other immunological species are dis-covered. However, in the absence of rules, in-consistencies can and will arise. The two toxicproteins extracted from Pasteurella pestis havebeen designated as toxin A and toxin B (25).In view of the uncertain status of the relation-ships among the plague toxins, the use of capitalletters may prove to be inconsistent with theiruse as applied to the botulinum toxins andstaphylococcal enterotoxins. As more and moretoxins are discovered and characterized, there isno doubt that further inconsistencies will ariseif guidelines concerning the use of capital lettersare not applied consistently by present andfuture investigators.

Greek Letter NotationThe origin of the Greek letter system for the

identification of bacterial toxins is obscure buthas gained considerable popularity. As the systemhas developed, Greek letters have been assignedto different toxins produced by either one bac-terial species or a strain within a species. Thissystem of nomenclature is used for the staphylo-coccal toxins and for the toxins of the gas-gangrene group of clostridia. If this classificationis to retain any validity, then it should be re-stricted to the case where a number of differenttoxins are produced by the same organism. Itmay very well be that the murine plague toxins,if they are found to be separate and distinct,should be placed within this category (i.e., aand # toxins) rather than being designated astoxin A and toxin B.One overt weakness of the Greek letter system

is that it has been applied indiscriminately forall of the bacterial products without taking intoconsideration whether they are true toxins. Thisis misleading, since many of the a to At productsof the C. perfringens group may be completelyirrelevant to pathogenicity, or at most onlyauxiliary virulence mechanisms (Miles, personalcommunication). The same situation may existfor the y and A hemolysins of S. aureus. In ouropinion, materials of this kind should notbe classified as toxins. It should be pointed out,however, that some investigators feel just asstrongly that they be considered toxins for wantof a better niche in which to put them (70).We do not intend to reopen the old aggressinversus toxin polemic, since no constructive pur-

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pose would be served. Suffice it to say that theexistence of the controversy proves that therehas been genuine doubt about including thosematerials which have been labeled aggressins astrue toxins. We feel that only those materialswhich exert a direct toxic effect upon sensitivehost cells and tissues should be classified astoxins. Materials such as hyaluronidase, deoxy-ribonuclease, and several others should be dif-ferentiated in some manner and should not bereferred to as true toxins unless evidence can beadduced for their role in generating signs ofillness. Until a better term is found, these sub-stances might be classified as auxiliary virulencefactors, a phrase employed originally by A. A.Miles. Within this context, Miles (34) referredto the enzyme-substrate fallacy in a discussionconcerning the relevance of these substances topathogenicity. He pointed out quite correctlythat the mere presence of the appropriate sub-strate in host tissues for enzymatic products ofpathogenic bacteria does not necessarily implythat the enzymes contribute to the pathogenesisof the infections. We would like to broaden thisconcept to the antigen-antibody fallacy as well.Although the bacterial aggressins for the mostpart are antigenic and elicit specific antibodiesin mammalian hosts, these substances do notnecessarily contribute significantly, or at all,to the diseases with which they are associated.

Symbolic Notation for Multicomponent ToxinsIt is likely that the multicomponent toxins

will eventually present the most difficult nomen-clatural problems of all. At this point, however,the anthrax toxin is the only one which is ap-propriate as a model for discussion and recom-mendation. The English investigators, who wereinstrumental in originating and perpetuating inthe literature the terms factors 1, II, and III of thethree components of anthrax toxin, still feelstrongly that this is the simplest and best nomen-clature and that there is no reason for changingit (Smith, Keppie, Belton, Strange, and Stanley,personal communication). We have already statedthat their terminology has historical precedence,but that this should not constitute a barrier if amore suitable nomenclature were available. Inthe case of the multicomponent toxins, descrip-tive names based on sound experimental evidenceare preferable to symbolic notation. Readers ofthe anthrax literature, especially those who arenot experts in the field, would be much betterinformed if the terms edema component, pro-tective-antigen component, and lethal componentwere used rather than factors I, II, and III. Weprefer to use the term component rather than

factor even in the descriptive terminology. Factoris defined as one of the facts, circumstances, orinfluences which leads to a result. Implied in thisdefinition is the element of dependence on otherfactors for the result to be manifested. Compo-nent, on the other hand, may be defined as adistinct element or constitutent of a larger, com-plex entity. In this case, each component mayact independently to bring about the final result.Admittedly, the distinction is a fine one butnevertheless real. At best, a descriptive termi-nology should create a mental image which isaccurate, and only by the proper choice of wordsis this possible.

Experimental evidence will not always be avail-able to allow a satisfactory descriptive termi-nology. In this case, we should be prepared toadopt a uniform set of symbols to identify themulticomponent toxins. It is obvious that no onesystem will appeal to all scientists, and we are notsuggesting that the following possibilities arethe best ones. To reiterate, our primary objec-tive is to stimulate thought along these lines andand even to provoke some healthy controversy(preferably of the nonvindictive variety).Two possibilities might be considered. With

the anthrax toxin again as an example, the multi-component toxin might be referred to as anthraxtoxin component 1, component 2, component 3.The Arabic numerals are suggested because oftheir universal use and recognition by scientiststhroughout the world. Another advantage whichcannot be ignored is their suitability for program-ming in computer systems. It may be argued thatthe suggested nomenclature is too cumbersome,but it could be simplified by choosing an appro-priate abbreviation for the component terms.Another system which was suggested by A. A.

Miles (personal communication) might also beconsidered. A system similar to the one usedfor serum complement could be adopted. If thesymbol MT' were accepted as a symbol for amulticomponent toxin, the individual compo-nents could be designated as MT'1, MT'2, etc.The description of the activities and even thesequence of action in vivo could be expressedif this information were available. This systemwould also facilitate the addition of anothernumber for a newly discovered component of thetoxin or, alternatively, the deletion of a numberedcomponent in the symbolic notation if it wasproved to be irrelevant to toxin activity in vivo.The anthrax toxin according to current knowledgewould be identified as anthrax toxin -MT'1,MT'2, MT'3. By use of the same system, thestaphylococcal leucocidin would be formulatedas leucocidin -MT'1, MT'2.

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RECOMMENDAIONS

In conclusion, we would like to suggest thatour colleagues in microbiology recognize theparallelism which exists in the inherent difficultiesof toxin nomenclature with the problems faced bythe biochemists and enzymologists. It should berealized that inevitably confusion arises frompoorly conceived and ill-defined systems ofnomenclature. Thus, something should be doneto clarify and systematize the nomenclature oftoxins while the confusion is still relativelyminor. Recently, the International Union ofBiochemistry (17) published its recommendationson the nomenclature of 875 enzymes and, byvirtue of its authority and recognition, provideda workable scheme for the overall nomenclatureof enzymes. In spite of the completion of thismonumental effort, general acceptance will notbe immediate, and the confusion caused by theearlier names for enzymes will make itself feltfor many years to come. As our knowledge ofthe biochemistry of toxins increases, the toxinnomenclature will by necessity be forced tochange. The ultimate nomenclature which atpresent is only a distant goal is one based onknowledge of amino acid sequence of both theentire molecule and the active sites within themolecule. For the time being, however, a logicalstep that can be taken is for a scientific society,such as the American Society for Microbiology,to initiate a study (i) from within the Society'smembership, (ii) in cooperation with interna-tional microbiology organizations, or (iii) incooperation with organizations outside of themicrobiological specialty (e.g., American Chem-ical Society, American Association for theAdvancement of Science, Federation of AmericanSocieties for Experimental Biology), with thepurpose of examining the growing problem oftoxin nomenclature and making concrete recom-mendations for a rational solution. It is a projectworthy of the concern of the American Societyfor Microbiology, and one which thoughtfulanalysis will show it has an obligation to initiate.

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Vol. 31, U.S.A. Status of Bacterial Toxins and Their ... · BONVENTRE, LINCOLN, ANDLAMANNA procedureto use intact bacilli rather than culture filtrates for the purification oftype