Vol. Priiited Biochemical Challenge Microbial Pathogenicity · might be experiments in vitro...

BACTERIOLOGICAL REVIEWS, Sept., 1968, p. 164-184 Vol. 32, No. 3Copyright c 1968 American Society for Microbiology Priiited in U.S.A.

Biochemical Challenge of Microbial PathogenicityH. SMITH

Depcartmenit of Microbiology, The Uuiiversity, of Birminiglhamii, Bir-iniglhami1, Eniglantd

INTRODUCTION.. .....

STUDIES OF BACTERIAL PATHOGENICITY ..... ....

Difficulty of'Inivestigati,ig Biochenmicail Mechanlisms of PathogenlicitY.Studies of Bacterial Behavior In Vivo....

ASPECTS OF BACTERIAL PATHOGENICITY REQUIRING ATTENTION-Intitiationl of InlfectioniGrowth and Multiplication In Vivo.Aggressive Activity...........

Inhihitors of blood atnd tissie hbactericidins............Inihibitors of' the actioni of plipagocvtes............................

(i) Iihibitors of conltac t. .....

(ii) l liihi tor-s of intgestionl .

(iii) Inihibitors of initralcellutlair hactericidins: prom2otioni of intracellular growvth.(iv) Repercussionu of ietertogeiueity of pliagocytic finIuctiOni Oni microbial requiire-ments for caggressitis. .

Toxic Activity.......................Toxi,is of overridling importalnce in diseaseToxi,Is which aire siglnificanlt bhlt niot the o;uly factors respoursible.for dlisecase.Toxilns produced ini vitro hblt of ioiknown importance ill disea.se.Toxic effe(cts in vivo ofhacteria which aippear to produice lo rolevait to-viii ifi vitro

(i) Toxilns revealedbl vsticlyinig hbacteria in more iiactuiral enviro.iciltsWi(ii) Rolev of hypersensitiviity ili toxic maniJe*stations of disesn.A.

Relcatioli o' Protective Anitigens to Virule,ice Factors...Biochenicall Bases of Host anid Tissuce Specificity,....

Role ofucireaise ili kidney, localizations of Coryniehacteriuon rena(ile and(i Proteuis mirabilis.Role of ervthritol in the tissute specificity of the brlucellae

UNANSWERED QUESTIONS RELATING TO THE PATHOGENICITN' OF VIRUSES, FUNGI,PROTOZOA, AND MALIGNANT CELLS.Viriuses.Fui.igi. ..

Protozoa.. .... .. ...................... ................

Ca,icer Cells . .... ..... ..

WHY STUDY BIOCHEMICAL MECHANISMS OF MICROBIAL PATHOGENICIT'I.........LITERATURE CITED .. ... .. .. .....

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170171171171171171172173174174175175

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INTRODUCTION

During this century, man's success in control-ling infectious disease has been dramatic. Al-though diseases such as cholera, trachoma, ma-laria, and influenza are still major problems insome areas, many infectious diseases can now becontrolled-by vaccination, by drugs, and, mosteffectively, by strict public health measures. Thissuccess in controlling infectious disease has beenparalleled and promoted by the equally successfulrecognition and description of the causativemicroorganisms-bacteria, protozoa, fungi, orviruses. In contrast, the biochemical mechanismswhereby these microorganisms produce diseaseare still obscure. What is known is confinedalmost entirely to bacterial diseases, and evenhere information is scanty. Although the toxinsresponsible for the classical toxemias (tetanus,

diphtheria, gas gangrene, and botulism) and theendotoxins of gram-negative bacteria have beenstudied in detail (49. 93), and the chemistry ofbacterial substances which inhibit the action ofphagocytes is known (17, 33), many of the mech-anisms of bacterial pathogenicity are still notclear. Thus, the bacterial products responsiblefor many disease syndromes remain obscure, andlittle if anything is known about the biochemicalbases for communicability, for survival andgrowth of certain bacteria within phagocytes, forhost and tissue specificities, and for long-termmicrobial survival in chronic diseases. Thesephenomena are not confined to bacterial diseases;they occur in even less understood infectionscaused by other microbes.

In this review, it is not intended to describe themost recent investigations of well-known bacterial

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toxins and substances which prevent ingestion byphagocytes, nor to catalogue further differencesexhibited in vitro between virulent and avirulentstrains of the same pathogenic species. There isrecent literature on these subjects (17, 33, 49, 93).The intention here is to point out gaps in ourpresent knowledge of the biochemistry of micro-bial pathogenicity and to suggest possible meth-ods for filling them. Particular attention will bedirected to areas where there are now some in-dications of the direction future research shouldtake. The review is restricted to microorganismspathogenic for animals. Because bacteria havereceived more attention in virulence studies thanother microorganisms, they are dealt with moreextensively than the other pathogenic microbes.However, if only to emphasize the dearth ofknowledge in this field, the mechanisms of patho-genicity of viruses, fungi, protozoa, and cancercells are discussed briefly in the context of con-cepts applied to bacterial pathogenicity. The nearsynonymous terms pathogenic and virulent willbe used as suggested by Miles (55), i.e., the formerin respect to species and the latter in respect todegrees of pathogenicity of strains within species.

STUDIES OF BACTERIAL PATHOGENICrrYPathogenic bacteria are peculiarities. The

great majority of bacteria are harmless and oftenbeneficial. Obviously, pathogenic bacteria have achemical armory which enables them to invade ahost and produce disease. The problem is toidentify the weapons in this armory, their relativeimportance, their chemical nature, and their modeof action on the host. This task is relatively simplewhen pathogenicity is determined by a singlebacterial product easily produced in vitro, as indiphtheria and tetanus. In the majority of infec-tious diseases, however, pathogenicity cannot berelated to a single microbial product and its bio-chemical bases are difficult to identify for thereasons given below.

Difficulty of Investigating BiochemicalMechanisms of Pathogenicity

The main factor contributing to difficulty inthis field is that virulence-the disease-producingcapacity of a population of microbes-is detect-able only in vivo and is markedly influenced bychanges in growth conditions due to selection oftypes and to phenotypic change (84).Most pathogenic species contain attenuated

strains which are often indistinguishable fromvirulent strains in the available tests in vitro. Thus,virulence is determined by small genetic differ-ences which may be fully expressed only underthe conditions of the test for virulence; i.e., duringgrowth in vivo. The decisive nutritional condi-

tions, those of host tissues under microbialattack, are not physiological but pathologicaland continually changing (110, 116); at present,they are not reproducible in vitro. Changes inmetabolism would therefore be expected whenbacteria from infected animals are cultured invitro (116), and such changes have been demon-strated (see below). In turn, these changes inmetabolism could affect virulence. Bacterialvirulence is usually reduced by subculture invitro, because bacteria lose the capacity to formone or more of the full complement of virulenceattributes manifested in infected animals (110,116). Also, apparent virulence factors might beproduced in vitro which are not formed, andtherefore not relevant, in vivo (116). Thus,bacteria grown in vitro can be incomplete or mis-leading with regard to the possession of virulenceattributes; this, coupled with the fact that bac-terial behavior in vivo is not easily examined (seebelow), forms the essence of the difficulties en-countered in studies of pathogenicity.

Studies of Bacterial Behavior In VivoObviously, virulence factors can be produced in

laboratory cultures if the requisite nutritionalconditions are known. This has already beenaccomplished in studies of classical bacterialtoxins and some antiphagocytic substances. How-ever, for problems of pathogenicity which haveso far defied solution by conventional procedureswith in vitro cultures, the above discussion sug-gests the study of bacterial behavior in vivo. Thereare no vitalistic leanings behind this suggestion,merely a realistic assessment of an approach thatmight reveal aspects of pathogenicity which latercould be reproduced in vitro by appropriatechanges in cultural conditions.

Information on bacterial behavior in vivo canbe gained in several ways. First, bacteria and theirproducts can be separated directly from the dis-eased host for biological examination and forchemical and serological study in vitro. Second,the behavior of organisms growing in vivo andtheir repercussion on the host can be examined,either in the whole animal or in restricted tissues.The largest gaps in our knowledge of pathogenic-ity occur here; detailed experimental pathologyand precise biochemical determinations are noteasily accomplished during infection, and only ina few cases have such studies supplemented theclinical pictures. Yet this information is vital, ifmechanisms of pathogenicity are to be understoodand if relevant biological tests for potential viru-lence factors are to be designed. Not the leastamong the difficulties is the lack of suitable lab-oratory animals in which human infections, e.g.,bacillary dysentery and typhoid or meningo-

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coccal and gonococcal infections, can be trulysimulated. Third, light can be shed on particularphases of microbial behavior in vivo by makingobservations in tissue, or preferably organ, cul-ture. Finally, tests in vitro can be made morerelevant to behavior in vivo. These methods forgaining knowledge of bacterial behavior in vivohave been described elsewhere (116); severalexamples are included in the sections below.A classical method of studying bacterial viru-

lence is to compare the properties of virulent andavirulent strains. Properly used, this method ofbacteriology is a powerful tool which could beapplied to studies of pathogenicity of othermicrobes (see below). The techniques of microbialgenetics have increased the scope of the method,and studies in vitro on enzymes, metabolic char-acteristics, and antigens of different bacterialstrains (17) have indicated many virulence mark-ers; i.e., factors associated with virulence. How-ever, relatively few of these factors have beenshown to be virulence determinants; i.e., producedduring infection and having biological activitiesdirectly connected with virulence, such as thepower to inhibit or destroy phagocytes (110).Studies of virulence can benefit, therefore, fromcomparisons of virulent and avirulent strains andfrom examinations of the influence of the prod-ucts of a virulent strain on the behavior of anavirulent strain, provided tests are carried out invivo or in simulant conditions in vitro. In thesestudies, it must be remembered, however, that iffull virulence is due to the possession of a numberof factors an avirulent strain may possess all butone of these factors (110).

ASPECTS OF BACTERIAL PATHOGENICITYREQuIRING ATNTIoNInitiation of Infection

Most infections occur through the mucousmembranes. Hence, to initiate infection, patho-genic bacteria must first survive on the mucoussurfaces in competition with commensals andthen penetrate into the tissues. Together withother factors (33), a differential ability of bac-terial species to accomplish these early stages ofinfection might explain why some diseases (e.g.,brucellosis) are more communicable than others(e.g., anthrax). Furthermore, interactions be-tween different pathogenic species on the mucoussurfaces might explain some of the marked effectsone infection can have on another in mixed infec-tions (116).The biochemical mechanisms whereby bacteria

survive on and penetrate mucous membranes areunknown. Two facts should be stressed in relationto survival: microbial populations on mucosal

surfaces are mixed, and only small numbers ofthe pathogenic component may be present at thestart of infection. Preoccupation with pure cul-tures can make microbiologists forget that naturalinfection involves bacterial behavior in mixedculture about which little is known even in vitro.Furthermore, metabolic studies on survival andgrowth of small inocula are rare even in vitro(116). In conventional studies with large inocula,the results reflect the activities of the bulk of thepopulation, whereas in vivo only initially atypicalorganisms in the inoculum may succeed (116). Arecent study (46) of the growth of Pasteurellatularensis from small inocula showed heterogene-ity of nutritional requirements among the popu-lation, only a small proportion of which wereable to grow initially. A useful beginning to theunderstanding of early processes of infectionmight be experiments in vitro on mixed culturesof relevant organisms using small inocula of thepathogenic components. In this respect, recentwork (25, 80) on survival and growth of mixedbacterial populations in continuous culture,which demonstrated striking effects of one organ-ism on another, is encouraging. With regard tomechanisms of penetration of mucosal surfaces,electron microscopy of the early stages of infec-tion, comparable to studies of leukocyte migrationduring inflammation (79), would be a usefulprelude to examining the biochemical processesinvolved.

Growth and Multiplication In Vivo

Once within tissues, virulent bacteria must beable to grow and multiply in order to producetheir disease syndrome, either by increasing in alocal lesion or by spreading throughout the hostvia the lymphatics and blood. To grow andmultiply, two qualities are needed by bacteria: (i)an inherent biochemical ability to grow under thenutritional conditions provided by host tissuesand (ii) an ability (see below) to combat defensemechanisms that would otherwise kill or removethem. The effects of these two qualities in vivoare not easy to separate, and consequently it isdifficult to assess their relative importance, eitherin the increase of a single infecting population orin the differential behavior of virulent and at-tenuated strains. Avirulence can arise from in-ability of bacteria to grow and divide in the en-vironment in vivo. Thus, nutritionally deficientmutants of pathogenic species were avirulentunless injected with their required nutrients (17,93). However, for most bacteria, the tissues andbody fluids probably contain sufficient nutrientsto support some growth. Hence, few naturallyoccurring strains (93) will be avirulent solely

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because of simple inability to grow in the host.Nutritional considerations will, however, affectrates of growth in vivo (and the production ofvirulence factors) and hence will influence degreesof virulence. Furthermore, differences in nutri-tional conditions in different hosts and in differenttissues within the same host may be responsiblefor host and tissue preferences of pathogenicbacteria (see below).The degree to which pathogenic bacteria multi-

ply in a host is in most cases unknown. At anyinstant, the viable bacteria can be counted, butthey only represent the result of bacterial divisionand bacterial destruction or removal. Recently(83, 85, 130), division rates in vivo have beenmeasured by using pathogenic bacteria, geneti-cally labeled with recognizable characteristicsretained by a known proportion of the progeny ateach division. Remarkable results were obtained;the doubling time of Salmonella typhimurium inmice was 8 to 10 hr compared with 0.5 hr incultures in vitro.Woods and Foster (116) have discussed the

almost complete lack of knowledge of bacterialmetabolism in vivo and the reasons why it prob-ably differs from metabolism in vitro. The me-tabolism of Bacillus anthracis in vivo is differentfrom that in vitro (110), and, although observa-tions in vitro on the metabolism of bacteria iso-lated directly from infected animals will not detectall aspects of metabolism in vivo (116), suchobservations on tubercle bacilli (15, 69, 107),plague bacilli (40), staphylococci (12), and strep-tococci (44) indicated marked differences inmetabolism between these organisms and thosegrown in vitro. These observations should beextended, because knowledge of microbial me-tabolism in vivo might be important not only instudies of virulence but for the design of new anti-bacterial drugs. The ideas and techniques used instudying host-dependent bacteria (47) might beapplied to other bacteria when the latter are grow-ing in vivo.

Aggressive ActivityIn addition to a metabolic ability to grow in

host tissues, virulent bacteria must produce sub-stances which act positively against the host; theyfall into either or both of two classes. First, thereare factors, not necessarily toxic, which promotemicrobial growth in vivo by inhibiting host de-fense mechanisms. These substances, called hereby the old term aggressins (137) because itdescribes so well their biological role, form thesubjects of this section. (The term is used as anoperational definition without any implication ofrelatedness between different aggressins either ofchemical nature or mode of action.) The second

class of substances, those which cause the diseasesyndrome and possibly death of the host, will bediscussed later.

Aggressins act first during the decisive, primarylodgment period (87) of infection, when the fewinvading bacteria are most vulnerable to the pro-tective reactions of the host. At this early stage,aggressins must inhibit not only those nonspecificbactericidal mechanisms already existing in thetissues (86) but also those agencies, especiallyphagocytic cells, which are mobilized by inflam-matory processes soon after the tissues are irri-tated (123). If some bacteria survive the primarylodgment and grow, spread of infection is opposedby the fixed phagocytes of the reticuloendothelialsystem; here again, to make headway bacterianeed aggressins possibly different from thoseoperating during the early lodgment phase. Tobreak through the protection of immunized ani-mals, bacteria must be either numerous or wellendowed with aggressins, since the host defensemechanisms are of increased efficiency and aresupplemented by antibodies capable of directneutralization of microbial products (26). Theclinical outcome depends on the interplay of thesereactions of bacteria and host, and varies fromcomplete subjugation of the host to completedestruction of the bacteria, including near stale-mate in chronic infections. The following descrip-tions of various bacterial aggressins are prefacedby brief descriptions of the host defense mecha-nisms they inhibit.

Inhibitors ofblood and tissue bactericidins. Bodyfluids and tissues contain bactericidal factors (17,26, 97, 109) which include: #3-lysins, heat-stablesubstances acting against gram-positive organ-isms; complement, acting against gram-negativeorganisms sensitized either by antibody or pos-sibly by nonspecific substances; lysozyme, actingdirectly against some gram-positive organismsand enhancing the action of complement andantibody on gram-negative organisms; and basicpolypeptides. Despite evidence that some bac-tericidins are artifacts which leak or are ex-creted from cells during manipulations in vitro(51, 112), there is little doubt that blood andtissue bactericidins play roles in host defense (17),being especially important during the first 3 hrafter infection (86). Since, clearly, there are sev-eral different types of bactericidins, virulent bac-teria must produce different types of aggressins toinhibit them.

Although resistance to bactericidins has beenassociated with virulence in strains of manyspecies [e.g., Enterobacteriaceae (17), Staphylo-coccus aureus (30), Leptospira spp. (59), B.anthracis (64), and Brucella abortus (34)], onlyrarely have the microbial products responsible

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been identified. These products, prepared fromvirulent strains, inhibited the destruction ofavirulent strains by bactericidins, and some wereimmunizing antigens. Thus, the "anti-anthra-cidal" activity ofB. anthracis was due to capsularpolyglutamic acid and to the complex immuno-genic toxin (64). Also, the resistance of B. abortusto the action of bovine serum was due to an im-munogenic ceU wall component which containedprotein, carbohydrate, formyl residues, and lipid[35 to 42% (34)]. The identification in otherpathogenic species (e.g., S. aureus and Leptospiraspp.) of aggressins active against host bactericid-ins might lead to the design of better protectivemeasures against the corresponding diseases. Thechemical mechanisms of aggressin-bactericidinreactions are unknown and cannot be investigateduntil more knowledge of the chemistry of thereactants has accumulated.

Inhibitors of the action of phagocytes. Once amicrobe has penetrated the mucous membranesor skin, the phagocytic activity of the wanderingand fixed cells of the reticuloendothelial systemforms the main protective mechanism of the body,a mechanism which acts nonspecifically but whichis greatly enhanced by immunization (26). Phago-cytes vary in origin, morphology, constituents,and bactericidal function. There are two maintypes, each of which has two subdivisions: poly-morphonuclear (neutrophils and eosinophils) andmononuclear (blood monocytes and tissue macro-phages) phagocytes. Polymorphonuclear phago-cytes are end cells with a short life derived fromstem cells different from the stem cells of long-lived mononuclear phagocytes. Inflammatoryexudates contain cells of all types; the polymor-phonuclear cells predominate initially but laterdie, leaving the mononuclear phagocytes ascend-ant. Macrophages form the fixed phagocyticsystem in the lymph nodes, spleen, and liver.

Phagocytosis of bacteria involves three stages:contact, ingestion, and intracellular killing anddigestion (33). Virulent bacteria may produceaggressins which inhibit any of these stages.

(i) Inhibitors of contact. Contact with bacteria iseffected by random hits, by trapping on unevensurfaces in confined tissue spaces, by filtrationsystems in lymph nodes, spleen, and liver, and bychemotaxis (26). Bacterial products could hardlyinterfere with the mechanical processes, but theymight inhibit nonspecific and specific chemotaxis(26, 33). The writer is unaware of any clear ex-ample of the latter, but certain fractions fromtubercle bacilli inhibit leukocyte migration (33).

(ii) Inhibitors of ingestion. Ingestion of bacteriainvolves engulfment within a phagocytic vacuole(the phagosome), the wall of which is derived byinversion of the phagocyte membrane. Specific

and nonspecific opsonins enhance this process ofingestion (26, 33).Once inside phagocytes, many bacteria (e.g.,

Streptococcus pneumoniae, S. pyogenes, B. an-thracis, Salmonella typhi, and Pasteurelia pestis)are rapidly killed and digested. Resistance toingestion, which allows bacteria to avoid intra-cellular bactericidins, is therefore essential forthe survival of virulent strains of these species.Aggressins that inhibit ingestion have been recog-nized, but the chemical basis for their activity isunknown. They fall into two main types.

First, there are surface and capsular productswhich do not harm phagocytes. Examples are thecapsular polysaccharides of Streptococcus pneu-moniae (33), the cell wall M protein and capsularhyaluronic acid of S. pyogenes (17), the capsularpoly-D-glutamic acid of B. anthracis (64), the 0somatic antigens of some gram-negative organ-isms (74, 98), the Vi antigen (poly-N-acetyl-D-galactosaminuronic acid) of S. typhi (24, 33), andthe protein carbohydrate envelope substance of P.pestis (33). Further investigation of the detailedchemistry of these aggressins may reveal commonstructural features, but at present no such featuresare apparent that might determine activity. Al-though acidic components occur in most of theabove compounds, including the various pneumo-coccal polysaccharides (33), they are absent fromsome active compounds (e.g., type IV and XIIpneumococcal polysaccharides) and hence do notappear to determine aggressive activity. The sug-gestion that the antiphagocytic activity of some 0somatic antigens might be due to a lipophiliccharacter determined by deoxy- and acetyl groupsin the polysaccharide side chains of the highersalmonella chemotypes (74, 98) awaits proof.Thus, there is no obvious connection betweenstructure and aggressive activity, and at the sametime the modes of action of aggressins are notclear. Interference with ingestion may be purelymechanical, but other mechanisms can be in-volved, such as inhibition of adsorption of serumopsonin (64) or possibly an inability of the hostto recognize the bacterial surface as foreign. Al-though several aggressins are capsular in origin,a common impression, possibly arising from theclassical work on the pneumococcal polysacchar-ides, that all capsulated bacteria resist phagocyticingestion and are virulent, is not true. The chemi-cal nature of the surface material determinesvirulence, not the presence of a capsule per se.

Excreted bacterial products which have a toxicaction on phagocytes form the second type ofaggressins interfering with ingestion. The leuco-cidins of the staphylococci (33) and the anthraxtoxic complex (64) are examples; the former areproduced by staphylococci within phagocytes,

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thus incapacitating the latter for further ingestion.The chemical basis of the effects of these aggres-sins is unknown.

(iii) Inhibitors of intracellular bactericidins:promotion of intracellular growth. Knowledge ofthe intracellular bactericidal mechanisms ofphagocytes is fragmentary but increasing. Neutro-phils contain lysozyme and basic proteins called"phagocytin" (33) or "leukin" (108), which arebactericidal for gram-positive and gram-negativeorganisms and are associated with granules whichdischarge into the phagocytic vacuoles containingbacteria. In addition, hydrogen peroxide maycontribute to bacterial killing in polymorpho-nuclear cells (94). In contrast to neutrophils, littleis known about the bactericidal mechanisms ofeither wandering or fixed mononuclear cells;peritoneal macrophages contain no phagocytinor lysozyme, and alveolar macrophages containlysozyme but no phagocytin (33). Thus, the bac-tericidal capacities of phagocytes differ, and theycould vary with the species of microorganismsbeing ingested.

Virulent strains of some bacteria (tuberclebacilli, gonococci, meningococci, and brucellae)resist the phagocytic bactericidins which destroyother microorganisms, and grow intracellularly.This phenomenon, which is probably the mostimportant aspect of the pathogenicity of thesebacteria, occurs both in infected animals and incell maintenance culture in vitro (112, 116).Obviously, virulent strains possess aggressinswhich interfere with bactericidal mechanisms ofthe phagocytes. However, the nature of these ag-gressins and the mechanisms of intracellular sur-vival and growth are unknown, even for the tu-bercle bacillus whose biology and chemistry havebeen the subject of a half century of work. Thisis not surprising in view of the limited knowledgeon bactericidal mechanisms of phagocytes them-selves. With more information accumulatingabout phagocytic defense mechanisms, investiga-tions should begin on the microbial aggressinsthat counteract them. As an example of whatmight be attempted, a recent study of the chemi-cal basis for the intracellular survival and growthof B. abortus in bovine phagocytes will be sum-marized.

Cell maintenance cultures were used and,J incontrast to previous work with monocytes oflaboratory animals employing a high bacterium-phagocyte ratio (116, 125), attempts were madeto simulate natural infection: the mixed phago-cyte population of bovine "buffy coat" (i.e., poly-morphonuclear cells, monocytes, and lympho-cytes) was infected with a small number of B.abortus cells. Then fresh bovine serum with aminimal quantity (2 ,ug/ml) of streptomycin was

used to kill extracellular organisms (112). In thephagocytes of this system, virulent strains of B.abortus survived and grew, whereas avirulentstrains were progressively destroyed (112).A factor which might have favored virulent

strains was their ability to use nutritional condi-tions within phagocytic cells more effectively thanavirulent strains (17). However, ultrafiltrates ofextracts of bovine phagocytes did not stimulategrowth of virulent strains of B. abortus any morethan that of avirulent strains (20). Thus, a simplenutritional explanation for the different behaviorof the strains seemed unlikely, and attention wasthen concentrated on their differential abilities toinhibit destructive mechanisms of phagocytes.The higher catalase content of virulent strains

of B. abortus compared with avirulent strains (17)might have afforded intracellular protection,since bactericidal hydrogen peroxide is present inpolymorphonuclear cells (94). A correlation be-tween the intracellular behavior (and virulence) ofstrains and sensitivity to hydrogen peroxide wasestablished, and survivors from the almost com-plete intracellular destruction of an avirulentstrain were more resistant to hydrogen peroxidethan the parent strain (37). However, these en-couraging results were not supported by similarexperiments with B. melitensis and B. suis; here,there was no correlation between sensitivity tohydrogen peroxide and intracellular behavior andvirulence (37). In addition, the intracellular be-havior of virulent and avirulent strains of B.abortus in bovine cells was not influenced by in-creasing the intracellular content of bovine cata-lase, either by allowing phagocytes to ingestcarbon particles on which catalase had beenadsorbed, or by allowing them to ingest B. abortusfrom a solution containing bovine catalase (37).In the first method, catalase might have remainedsolely in the phagocytic vacuoles surrounding thecarbon particles, but in the second methodcatalase would probably have been incorporatedin the same phagocytic vacuole as the bacteria.The high catalase content of virulent strains of B.abortus did not appear to play a significant partin intracellular survival and growth.The following experiments suggest that the

ability of virulent B. abortus to survive and multi-ply intracellularly is due to a cell wall substancewhich interferes with the intracellular bactericidalmechanisms of phagocytes. Virulent B. abortus,obtained either from cultures in guinea pig mono-cytes or from infected bovine placental tissue, hadan increased ability to survive intracellularly com-pared with the same strain grown in laboratorymedia (112, 125). In addition, the cell wall mate-rial of the organisms from infected bovine pla-centa inhibited intracellular destruction of an

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avirulent strain of B. abortus, and this effect wasneutralized by an antiserum against live virulentB. abortus. This inhibitory activity was not shownby cell wall material obtained from either thevirulent or an avirulent strain grown in vitro(112). The material which prevented intracellulardestruction of B. abortus differed from the im-munogenic cell wall material which interferedwith the bactericidin of bovine serum (see above).The latter material, which was produced byvirulent and to some extent by avirulent bac-teria in vitro, interfered with an extracellularbactericidal activity of bovine buffy-coat cellsbut not with the intracellular activity (34, 77,112).Once the celi wall material contributing to

intracellular survival had been recognized by ob-servations on B. abortus grown in vivo, organismshaving this material were produced in vitro bysupplementing laboratory cultures with bovinefetal fluids (38). Thus, organisms resistant tointracellular bactericidins can be prepared inquantity and extracted to determine the natureof the material contributing to intracellular sur-vival. It might prove structurally similar to thematerial which interferes with the extracellularbactericidins but different in the detailed chem-istry of its side chains [cf. the different 0 somaticantigens of the Enterobacteriaceae (74, 98)].

In addition to frankly intracellular bacteria likebrucellae, other bacteria such as straphylococciappear to survive within phagocytes, at least forshort periods. Hence, virulent staphylococci pre-sumably have the ability to inhibit the bacte-ricidins of polymorphonuclear cells, either intra-cellularly when surviving within the phagocytes,or possibly extracellularly if the bactericidins areliberated from the dead (leucocidin killed) cellsof pus. Unlike bactericidins acting on B. abortus,which could not be demonstrated in phagocyteextracts but only in the cell cultures describedabove, bactericidins acting on staphylococci arepresent in extracts of polymorphonuclear cells.Recently, Adlam, Pearce, and Smith (1) showedthat Staphylococcus aureus grown in rabbits isnot only more virulent for rabbits but is also moreresistant to the bactericidins of rabbit polymor-phonuclear cells (and to rabbit serum bacte-ricidins, which may or may not be the same as thecellular bactericidins) than the same strain grownin vitro. This observation may lead to the identi-fication of staphylococcal aggressins responsiblefor these antibactericidal effects [cf. studies of B.abortus and other bacteria grown in vivo (110,116)].

(iv) Repercussion of heterogeneity oJ phagocyticfunction on microbial requirements for aggressins.There is increasing evidence that phagocytic

capacity and function vary between differenttypes of phagocytes and within the same type.Thus, mononuclear phagocytes are less effectivethan polymorphonuclear cells for kifling somebacteria. P. pestis survived and grew withinmouse and guinea pig monocytes, yet was killedwhen ingested by polymorphonuclear cells (21).Staphylococci were killed more effectively by rab-bit polymorphonuclear cells than by macrophages(76). Avirulent strains of B. abortus were killed,and the growth of virulent strains was inhibitedmore by polymorphonuclear phagocytes than bymonocytes in bovine blood (H. Smith, P. W.Harris-Smith, and R. B. FitzGeorge, unpublisheddata). Populations of the same cell type showheterogeneity with respect to bactericidal capacity;members of populations of rabbit and mousemacrophages differed in their capacities to killstaphylococci and S. typhimuiriuim, respectively(76, 101). Also, in the syndrome termed chronicgranulomatous disease of childhood (61), poly-morphonuclear cells appeared to lose theircapacity to kill certain bacteria (e.g., S. atureus,Escherichici coli, and Klebsiella spp.) but not others(e.g., streptococci).The reasons for heterogeneity of phagocytic

function are becoming clearer as the nature of theintracellular bactericidins is being investigated.The lack, in macrophages, of some of the bacte-ricidins present in neutrophils has been mentionedpreviously. Recently (138, 139), the lysosomalcationic proteins of rabbit polymorphonuclearcells were separated by electrophoresis into frac-tions with different arginine-lysine ratios anddifferent bactericidal activities for streptococci,Proteus spp., and E. coli. Similarly, "leukin' (108)from rabbit polymorphonuclear cells was hetero-geneous and appeared to contain different bacte-ricidins for gram-positive and gram-negative bac-teria. The presence or absence of these differenttypes of bactericidal proteins in various phago-cytes could explain heterogeneity of function asregards intracellular killing. However, phagocytesmight also differ in their capacity to ingest differ-ent bacteria, and heterogeneity of function mightbe evident at this early stage of phagocytosis.

Heterogeneity of phagocytic function has twomain repercussions on studies of microbial ag-gressins. First, bacteria may have to produce dif-ferent aggressins to combat the activities of differ-ent kinds of phagocytes. Thus, a strain of P. pestisresistant to phagocytosis by wandering phago-cytes was ingested and killed by cells of thereticuloendothelial system (33). A similar sus-ceptibility to the reticuloendothelial system mightalso explain the avirulence of type III A66pneumococcus for rabbits, despite the posses-sion of capsular polysaccharide, which, like that

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of virulent strains (e.g., type SV III), protected itfrom phagocytosis by wandering phagocytes (33).Second, bacteria lacking sufficient aggressins tocombat powerful destructive mechanism, eitherin serum or in other phagocytes, might be pro-tected by ingestion into phagocytes that are lessbactericidal. This situation might occur to theadvantage of invading bacteria during the earlystages of infection when they have not yet ac-quired, under the conditions of growth in vivo,their full complement of aggressins. Indeed, aftera period within less destructive monocytes, P.pestis became resistant to subsequent ingestion bypolymorphonuclear cells (21) and B. abortusbecame more resistant to the bactericidal actionof bovine serum (125). A similar protection withinless bactericidal phagocytes might also play a rolein long-term survival of bacteria in chronic andcarrier states and in drug-resistant infections (52).

Toxic ActivityThe toxic activities of bacteria can be divided

roughly into four categories.Toxins of overriding importance in disease.

Clostridium tetani, C. botulinum, and Corynebac-terium diphtheriae produce in vitro powerful, well-characterized exotoxins that are responsible forthe disease syndromes in vivo. Immunization withtoxoid protects against disease. The biochemicalactivities of these toxins at the cellular level (17,33, 49, 93) will not be discussed here. The major-ity of pathogenic bacteria do not produce toxinsof this type.

Toxins which are significant but not the onlyfactors responsible for disease. These toxins areproduced in vivo and are responsible for somepathological effects of infection. However, theyare not the sole determinants of disease: often asmuch toxin is produced by avirulent as by viru-lent strains; sometimes injection of toxin does notreproduce all the pathological effects of disease;and usually immunization with toxoid does notconfer solid protection against infection. Sincethese toxins are involved in disease, biochemicalinvestigations of the toxins themselves and theiractivities at the cellular level should be continued.The a-toxin of staphylococci and the erythrogenictoxin of streptococci (33) are examples, but themost important representatives are the endotoxinsin cell walls of gram-negative bacteria. Theirchemistry has been elucidated (74); however, theemphasis has been on serological activity ratherthan on toxicity. Their toxic manifestations-pyrexia, diarrhea, prostration, and death inshock (137)-are similar, no matter from whichspecies they are prepared (33). In some infections,e.g., typhoid fever, endotoxin seems to be re-sponsible for fever, leucopenia, and possibly

death (33, 137). However, avirulent strains ofmany gram-negative species contain much endo-toxin; mice bred resistant to endotoxin are sus-ceptible to infection (50); the endotoxin of nor-mal alimentary tract E. coli has no apparentnoxious effect; and, despite their similar endo-toxins, different gram-negative organisms producedifferent disease syndromes (33, 110). Clearly,factors other than endotoxins are important ingram-negative bacterial infections. These factorsmay be aggressins (e.g., the K antigen of E. coliand the Vi antigen of S. typhi), differing nutri-tional requirements of various species, and toxinsdifferent from endotoxin (110). The productionof such toxins by Vibrio cholerae, E. coli, andPseudomonas aeruginosa is described later.

Toxins produced in vitro but of unknown impor-tance in disease. Many substances producing toxiceffects related or unrelated to disease syndromeshave been isolated from laboratory cultures. Someof these products may be laboratory artifactshaving no relevance to disease in vivo [cf. lyco-marasmin in plant disease (116)1. Even if formedduring infection, the question remains whetherthey play significant roles. Hence, the relevanceof these substances to disease should be deter-mined before their biochemical study in depth isattempted. Examples are the many "toxic" prod-ucts of staphylococci (33) and streptococci (33),the "neurotoxin" of Shigella shiga (33, 49), andthe "murine" toxin of P. pestis (93, 110, 116).

Toxic effects in vivo of bacteria which appear toproduce no relevant toxin in vitro. Apart from bac-teria, very few pathogenic microbes have beenshown to form toxins responsible for the patho-logical effects of infection, and, despite theirintensive study in vitro, some pathogenic bacteriastill fall into this category. Examples are Strepto-coccus pneumoniae (33), Shigella spp. (17), andMycobacterium tuberculosis (33); until recently,V. cholerae, enteropathogenic strains of E. coli,and P. aeruginosa would have been included.The primary problem here is to demonstrate

the microbial factors causing the disease syn-dromes so that biochemical investigations ofthese factors can proceed. There are two explana-tions for the apparent lack of toxins. First, toxinsexist but have yet to be demonstrated. Second, thehost is harmed by means other than the directaction of a toxin. For example, in diseases wherelarge bacterial populations accumulate before thehost succumbs, the growing bacteria might depletesome tissues of essential nutrients. This explana-tion does not appeal to the writer because of thedemonstrated versatility of replacement mecha-nisms of mammalian hosts; it is also improbableif the host still dies when the large terminal popu-lation of organisms is removed by treatment with

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antibiotic [cf. studies on anthrax (65)]. A morelikely explanation for host damage in the absenceof toxin is the evocation of hypersensitivity reac-tions by nontoxic bacterial products.

(i) Toxins revealed by studying bacteria in morenatural environments. It has been suggested pre-viously that, for dealing with unsolved problemsof pathogenicity, the behavior of bacteria in vivoshould be examined for virulence attributes whichmight then be reproduced in vitro for closer in-vestigation. The preliminary examination shouldbe made in experimental animals or in biologicaltests in which aspects of the natural disease aresimulated as far as possible. In the past decade,hitherto unknown toxins have been demonstratedby this approach.The cause of death in anthrax was clarified by

studies (110, 115, 116) in guinea pigs, followed byappropriate experiments in vitro. Until this work,neither the general nature of the lethal effect ofB. anthracis on the host nor the products responsi-ble were known; no lethal toxin had been foundin laboratory cultures. The main findings were (i)that the massive terminal bacteremia was not theprimary cause of death, because removal of organ-isms by streptomycin did not prevent death, whichwas therefore probably caused by a toxin; (ii)that the fatal syndrome was oligemic secondaryshock; (iii) that the plasma of infected guinea pigscontained a specifically neutralizable, edema-producing, lethal toxin; (iv) that the immunogenictoxin originally recognized in vivo was reproducedin vitro; (v) that the toxin comprised at leastthree nontoxic components, two of which wereproteins and one a chelating agent containingprotein, carbohydrate, phosphorus, and a groupabsorbing at 260 nm; and (vi) that the compo-nents interacted synergically in toxicity and im-munogenicity tests and formed the basis of effec-tive vaccination against anthrax. This work hasnow been confirmed (91). The anthrax toxic com-plex has been found in many infected animals,including rhesus monkeys, and is now generallyaccepted as responsible for death from anthrax(91). At the biochemical level, its mode of actionis unknown, but in animals it produced fluid loss,leading to secondary shock, and in some speciesto pulmonary complications (115).

P. pestis and its products obtained from in-fected guinea pigs were investigated (17, 116) toresolve the following anomaly regarding the causeof death in plague. Live virulent P. pestis killedboth guinea pigs and mice, but a product, "mu-rine" toxin, obtained from cultures in vitro killedonly mice. An extracellular toxin comparable tothat in anthrax could not be demonstrated ininfected guinea pigs, but an extract of P. pestisfrom guinea pigs killed guinea pigs as well as

mice. Later, the guinea pig toxin was found inP. pestis grown in vitro and fractionated into twosynergistically acting components, both proteinsand unconnected with either the "murine" toxinor lipopolysaccharide (124). The question as towhether the guinea pig toxin or the "murine"toxin or both are involved in death of man fromplague is unresolved.

Recently, an enterotoxin of V. cholerae hasbeen demonstrated; it appears to be responsiblefor the dramatic loss of fluid from the intestinewhich occurs in cholera and which results in de-hydration and death in shock. Previous workershad attempted, without much success, to impli-cate in cholera the endotoxin, a mucinase, andother materials from V. cholerae. The lack ofprogress was due to the difficulty of obtainingeither an experimental animal or a biological testin which the effects of cholera could be simulated;for example, although mice were killed by intra-peritoneal injection of living V. cholerae and itsendotoxin, in neither case were the signs ofcholera evident. Recent advances have resultedfrom the use of more natural biological systemsand the examination in them of the behavior ofliving V. cholerae before study of its productsformed in vitro.

In ligated intestinal loops of rabbits, De andhis associates (31, 32) produced the gross fluidloss and mucosal damaging effects of cholera,first by young living cultures of V. cholerae andthen by filtrates from such cultures. The extracel-lular or easily liberated enterotoxin was heat-labile, neutralized by an antiserum to culturefiltrates, and not related to mucinase, hemolysin,or endotoxin. Recently (62), this rabbit gut tech-nique was used for titration of cholera entero-toxin.

Datta, Finkelstein, and their colleagues (35)produced some cholera signs in starved 8- to12-day-old suckling rabbits by introducing youngliving V. cholerae into their washed stomachs witha catheter. The cholera effects were then pro-duced by filtrates from young, well-aerated cul-tures of V. cholerae in simple media. Like theproduct described by De, the enterotoxin washeat-labile, specifically neutralized by antisera toculture filtrates, and not connected with endo-toxin. At first, the enterotoxin was thought toconsist of two components, procholeragens Aand B, but later (36) procholeragen A was demon-strated to be the important factor.

Craig (27, 28), recognizing that a possiblecholera enterotoxin should increase capillarypermeability, used for his work skin tests foredema in rabbits and guinea pigs. Furthermore,he examined the extracellular products of V.cholerae growing in the natural host, i.e., those

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in filtrates of rice water stools from humancholera patients. These filtrates produced skinedema, in contrast to filtrates from the stools ofpatients with diarrhea from causes other thancholera. Filtrates from young aerated cultures ofV. cholerae also produced the skin edema, againin contrast to similar filtrates from cultures ofShigella spp., E. coli, and noncholera vibrios.Serum of humans who had survived choleraneutralized the edema-producing activity of fil-trates from human stools and of the cultures ofV. cholerae. The enterotoxin was heat-labile,unaffected by trypsin, and distinct from endo-toxin. Like log-phase cultures of V. cholerae (102),it produced in mongrel dogs a dramatic fluid losscharacteristic of cholera (J. P. Craig, personalcommunication).The enterotoxin of V. cholerae can now be

investigated biochemically. The same active com-pound(s) is probably present in all the crude prep-arations. The first step must be to purify the en-terotoxin to remove extraneous compounds whichmight interfere with subsequent investigations ofits chemistry and mode of action. The entero-toxin appears to enter the blood stream (136), soits effect could be systemic as well as local. It willbe interesting to see whether Formalin-treatedenterotoxin (28) will immunize against cholera.The success of the ligated rabbit gut technique

in revealing the enterotoxin of V. cholerae ap-pears to have been repeated with regard to thediscovery of the toxin of enteropathogenic strainsof E. coli. Until recently, no toxin was knownwhich could explain the production of diarrheaand scours in young humans and domestic ani-mals by certain strains of E. coli (116). However,Taylor and her colleagues (127, 128) showed thatliving E. coli serotypes, isolated from babies withdiarrhea, distended ligated rabbit gut prepara-tions, whereas E. coli from healthy children andother sources had no effect; they also noted thata similar gut-distending action was produced bychloroform-killed suspensions of the active sero-types but not by similarly killed suspension ofthe other serotypes. The enterotoxic activity ofthe chloroform-killed suspensions was extremelylabile and not connected with endotoxin activity.Using E. coli in domestic animals, Williams-

Smith and Halls (119) showed that dilation ofligated gut preparations from pigs, lambs, andcalves reflected the enteropathogenicity of E. colistrains for the appropriate animal species. How-ever, although there was some cross-reactivity, agut preparation from one animal species, e.g.,from pigs, might not detect a strain pathogenicfor another species, e.g., calves. By use of appro-priate ligated gut preparations, enterotoxin wasdemonstrated in filtrates from soft-agar cultures

of enteropathogenic strains of E. coli but not incorresponding filtrates from nonenteropathogenicstrains (120). The enterotoxins were extracellular,heat-labile, and nonlethal for mice. They wereunrelated to hemolysins and endotoxin; indeed,three preparations of the latter failed to dilate anappropriate ligated gut preparation. Thus, as isthe case with V. cholerae, the enterotoxin of E.coli can now be investigated biochemically.

Studies of Shigella spp. in ligated rabbit gutpreparations have not proceeded as far as thoseon V. cholerae and E. coli, but they appear to betaking a similar course. Signs of dysentery wereproduced in gut preparations 12 hr after the intro-duction of live Shigella spp. (5). Only freshlyisolated strains had these effects, and subculturein vitro soon destroyed this activity. A toxindifferent from endotoxin is the most likely causeof the enteropathogenic action. However, such atoxin has yet to be demonstrated, and hypersen-sitivity to endotoxin might also play a role indysentery (see below).

P. aeruginosa can infect burns with fatal con-sequences. In vitro, it forms endotoxin, a leci-thinase, a protease, and a hemolysin, but none ofthese compounds appears to explain the effects ofinfection. Recently (71), the toxic activity of ex-tracts from lesions produced in rabbit skin bywidespread injections of P. aeruginosa (i.e., theproducts of growth in vivo) was compared withthat of filtrates from vigorously shaken cultures ofP. aeruginosa in rabbit serum and in broth. Sero-logically, the toxin produced in vivo appeared tobe identical to that produced in vitro; both prep-arations killed mice in shock, lowered the bloodpressure of rabbits, and appeared distinct fromany of the previously described products of P.aeruginosa. The toxin now awaits biochemicalinvestigation.

(ii) Role of hypersensitivity in toxic manifesta-tions of disease. The classical work with M.tuberculosis showed that evocation of hypersensi-tivity reactions by pathogenic bacteria and theirproducts can have unpleasant and even fatal con-sequences for the host (33, 137). Furthermore,skin tests clearly indicate that hypersensitive statesoccur in numerous bacterial diseases, e.g., tuber-culosis, staphylococcal infections, streptococcalinfections, pneumococcal infections, brucellosistularemia, glanders, leprosy, Johne's disease, andsalmonellosis (33, 137). Usually the reactions areof the delayed type, indicating that cellular mech-anisms are involved, although Arthus-type reac-tions can also occur, e.g., against bacterial poly-saccharides (33). Thus, in some diseases, nontoxicbacterial products can produce some of the toxicmanifestations by evoking hypersensitivity reac-tions. These reactions are perhaps more likely in

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chronic rather than in acute disease, and espe-cially where the parasites are intracellular; aller-gized cells (26) could be provoked from time totime by small amounts of microbial productsliberated from the few surviving intracellular or-ganisms. However, just as production of a toxinin vitro does not mean automatically that it isrelevant in vivo, mere demonstration of a state ofhypersensitivity by a skin test, is no proof of theimplication of hypersensitivity reactions in themain pathological effects of the disease. Moreextensive investigations are needed; the mainsystemic and local effects of the disease must besimulated by hypersensitivity reactions evoked ina sensitized host by products of the appropriatemicrobe.

Evidence for the implication of hypersensitivityreactions in the pathology of the disease is noteasily obtained and is often equivocal. It is evenharder to identify the particular bacterial productsinvolved. Almost certainly, for any one species[e.g., B. abortus (114)], these products will bemore numerous than those showing overt toxicity.Descriptions (33, 137) of the extensive work ontuberculosis and rheumatic fever emphasize thedifficulties ofobtaining precise biochemical knowl-edge in this field. Now, there seems little doubtthat the pathology of tuberculosis is largely dueto hypersensitivity to products, particularly thewaxes, of M. tuberculosis. Also, the cardiac andother lesions of rheumatic fever appear to be ex-plained by hypersensitivity to streptococcal anti-gens or to host tissue products altered by reactionwith streptococcal products, or to both. The roleof hypersensitivity is less clear in bacterial diseasesother than tuberculosis and rheumatic fever, forexample in pneumococcal pneumonia (48). Itseems to be involved in some nephritic syndromesand in chronic brucellosis (33, 137). However, inacute brucellosis of susceptible pregnant animals,abortion is due to the direct action of endotoxinliberated from brucellae concentrated in certainfetal tissues (133). Nevertheless, in some diseasescaused by other gram-negative organisms, endo-toxin might act more by provoking hypersensitiv-ity reactions than by direct toxic action (33). Thismight occur in dysentery, since the intestinal tractcan participate in hypersensitivity reactions, andsome infections of primates and other animalswith Shigella spp. produce the signs of dysenteryonly after a second challenge (17).The possibility of auto-allergy entering into the

pathology of chronic infectious disease should bementioned. The biochemical activities of a para-site may change host products sufficiently forthem to evoke a tissue-damaging host response.This seems particularly possible for viruses, andsome bacteria may operate in this manner, e.g.,

the role of streptococci in rheumatism (33, 137).The difficulties of proving a significant role forauto-allergy in the pathology of an infectiousdisease are similar to those outlined above forhypersensitivity reactions. And, of course, thequestion arises as to whether a host productaltered by microbial activity is then a host or amicrobial product and thus whether auto-allergyin the strictest sense is really involved. To myknowledge, the nature and mode of production ofany compound involved in "auto-allergy" duringinfectious disease has not been elucidated. Withthe increasing importance in medicine of chronicmicrobial disease, the biochemical mechanismsunderlying the possible involvement of hyper-sensitivity and "auto-allergy" in pathology shouldbe investigated.

Relation of Protective Antigens andVirulence Factors

Many bacterial products are antigens, and oftenthey form the bases of diagnostic tests. However.only a few of these antigens are protective (im-munogenic), i.e., actively immunize against dis-ease. Of necessity, these immunogenic antigensare factors involved in virulence, either toxins(e.g., that of C. tetani) or aggressins (e.g., thepolysaccharides of S. pneumoniae). However,some antigenic virulence factors are not protec-tive (e.g., the a toxin of staphylococci); possiblythese factors do not operate significantly in thoseearly stages of infection which must be inhibitedif vaccination is to be effective. Some virulencefactors are not even antigenic [e.g., capsularhyaluronic acid of S. pyogenes and polyglutamicacid of B. anthracis (33)]; this might account forthe lack of immunity which sometimes occurseven after overt diseases, as in staphylococcalinfections. If such nonantigenic virulence factorscould be identified and rendered antigenic bycoupling to proteins, effective vaccination mightfollow.

Biochemical Bases of Host and TissueSpecificity

Two aspects of microbial pathogenicity whichembrace the behavior of all types of microbes inboth animals and plants, and about which weknow practically nothing in biochemical terms,are host and tissue specificities. In the bacterialfield, it is not known why dysentery is confined toprimates and Johne's disease occurs only in cattleand related species, nor is it known why, in man,S. pneumoniae grows so well in the lung to pro-duce pneumonia. Some discussion of host andtissue specificity is included here because recentinvestigations of the tissue specificities of patho-

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genic bacteria suggest a pattern for future bio-chemical investigations in this field. However,since the subject has already been reviewed (116),only the main aspects are considered.

Differences in susceptibility to infection occur-ring between different species of the same host(and among different tissues in the same host)often exceed those between immunized and unim-munized animals of the same host species. Hence,if the chemical bases of resistance of a certainspecies (or tissue) to infection could be identified,it might be possible to confer on an otherwise sus-ceptible species a higher level of resistance thanthat produced by immunization.The two most likely explanations for differ-

ences in susceptibility to infection of differenthosts and tissues are differential distributions ofbactericidal mechanisms and differential distribu-tions of nutrients for which the metabolism of theparasite is specially adapted.The extracellular and cellular bactericidal

mechanisms of animal hosts are many and varied,and there is increasing evidence of heterogeneityof phagocytic function (see above). Undoubtedly,variations of bactericidal mechanisms in differentspecies and tissues (33, 92, 116, 137, 139) couldaccount for differences in specificities of infection;e.g., resistance of animal species to anthrax mightdepend on their level of tissue "anthracidal sub-stance" (13). However, despite much effort, at-tempts to lay the responsibility for specificity ofinfection unequivocally on such variations ofdefined bactericidal mechanisms have so far failed(54, 116).More success has been achieved in recent in-

vestigations of the influence of nutrition on speci-ficities of infection. Clearly, different nutritionalconditions in different hosts and tissues coulddetermine different levels of bacterial invasionand growth. Such an explanation for host speci-ficity has not yet been demonstrated, but it ap-pears to apply in the following examples of tissuespecificity.

Role of urease in kidney localizations of Coryne-bacterium renale and Proteus mirabilis. C. renalein cattle and P. mirabilis in man persist longerand cause more severe pyelonephritis than theother bacteria (e.g., E. coli) which cause kidneyinfections (63, 116). Nonspecific factors, such asinhibition of phagocytic response by high salt andurea concentrations (23, 88, 99) and inactivationof complement by ammonia (11) probably con-tribute to the general susceptibility of kidneytissue to infection. However, a more specificmechanism, namely, the ability of urease tometabolize urea, also appears to influence thepersistence and severity of infections with C.renale and P. mirabilis. Thus, C. renale became

localized in the kidneys of mice, whereas otherdiphtheroids did not, utilized urea when growingin bovine urine and urea-enriched peptone-water,and, in contrast to C. equi and E. coli, yielded ex-tracts which formed ammonia from urea (72).P. mirabilis also contains a urease (16) and pro-duces in rats, as in man, more severe renaldamage than is produced by E. coli or enterococci(16, 103). Furthermore, in tissue cultures of kid-ney epithelium growth of intracellular P. mirabilisbut not that of E. coli, was stimulated by urea,and the optimal concentration (0.2%) of urea wasthe same as that found in kidney homogenate(16). Hence, ability to utilize urea for growthcontributes to the localization of C. renale andP. mirabilis. However, other factors must also beinvolved in renal localizations, because organ-isms lacking a urease, e.g., E. coli, also localize inthe kidney.

Role of erythritol in the tissue specificity of thebrucellae. In many animals (humans, rats, guineapigs, and rabbits), brucellosis is relatively mildand chronic; the causative organisms do not growprolifically and have no marked affinity for par-ticular tissues. However, in pregnant cows, sheep,goats, and sows, an enormous growth of brucellaein the placentae, the fetal fluids, and the chorionsleads to the characteristic climax of the disease,abortion (113). Recent investigations (4, 66, 95,111, 118, 133, 134) have provided the followingdata which explain this tissue localization in thesusceptible animal species. A survey of extractsof fetal and maternal bovine tissues showed thata material which stimulated the growth of B.abortus was concentrated in the fetal fluids, thefetal placenta, and the chorion; this growth-stimulating material was isolated and identified aserythritol. Analyses of fetal and maternal bovinetissue extracts showed that erythritol was concen-trated in those tissues heavily infected in brucel-losis. It was observed that erythritol enhancedB. abortus infections in newborn calves and stim-ulated the growth of B. melitensis and B. suis invitro and in vivo. The placentae of cattle, goats,sheep, and sows were found to contain erythritol,but it was not found in those of humans, rats,guinea pigs, and rabbits. Male genitalia of sus-ceptible species contained erythritol, thus corre-lating its presence with localization of infectionin the male. During growth, B. abortus had agreat affinity for erythritol; in a complex mediumcontaining glucose at a concentration 1,000 timesthat of erythritol, B. abortus used 1.5 times its ownweight of erythritol as a general energy source.Analogues of erythritol inhibited the growth ofB. abortus both in vitro and in vivo.

Hence, the presence of erythritol, a growthstimulant for brucellae, in susceptible tissues of

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susceptible species explains tissue specificity inbrucellosis. Ability to grow rapidly with erythritolexplains localization in an already infected host,but it does not account for primary invasion ofthe host, an aspect of virulence. The primaryinvasion is controlled by ability to grow intracel-lularly, and this process does not involve erythri-tol (134). The growth of both virulent and at-tenuated strains of all three species of brucellaewas stimulated by erythritol (29, 60, 67, 82, 134).Only for B. abortus was the growth of virulentstrains stimulated more than that of attenuatedstrains (134), although growth stimulation of thelatter occurred with high concentrations of eryth-ritol (67). A most striking and satisfying findingwas that growth of the S19 vaccine strain whichhas been used safely in the field was inhibited byerythritol (60, 67).The work on kidney infections and on brucel-

losis encompasses practically all of our preciseknowledge of the chemical bases for tissue andhost specificity, and suggests a pattern for futureresearch. Indeed, preliminary studies on V. fetus(the cause of vibrionic abortion in domestic ani-mals) indicate that growth of the organism isgreater in extracts of fetal placenta than in ex-

tracts of maternal tissues or conventional media,thus suggesting that localization of vibrio is deter-mined by a preferred nutrient, as in brucellosis(D. B. Lowrie and J. H. Pearce, personal communi-cation). These studies on tissue and host specific-ity also show that knowledge of the bases of thesespecificities might indicate approaches to chem-otherapy.

UNANSWERED QUESTIONs RELATING TO THE

PATHOGENICITY OF VIRUSES, FUNGI,PROTOZOA, AND MALIGNANT CELLS

Although many aspects of bacterial patho-genicity are still unexplained in biochemicalterms, far more is known about bacteria thanabout other pathogenic microbes. There are manydifferences between these other pathogens andbacteria, e.g., the necessity for viruses to grow inliving cells, the polymorphism of pathogenicfungi, the complex life cycles and the antigenicplasticity of pathogenic protozoa, and the simi-larity to the host of cancer cells [considered hereas analogues of pathogenic microbes (39, 104)];however, like pathogenic bacteria, all of thesemicrobes invade hosts and produce disease. Inspite of their differences from bacteria, furtherstudies of the other pathogenic microbes mightbe helped by briefly discussing their mechanismsof pathogencity in the context of what has al-ready been said for bacteria. The following ques-tions might be considered. Can differences invirulence be detected and measured in vivo? Are

reasonably stable virulent and avirulent strainsavailable so that virulence markers and deter-minants can be recognized (i) by comparing thebehavior of the strains in vitro and in vivo and (ii)by observing the effects of products of the virulentstrain on the behavior of the avirulent strain?How far can avirulence be due to an inherentinability to grow in the host tissues (as distinctfrom an inability to combat host defense mech-anisms)? What host defense mechanisms actagainst the microbe and what aggressins inhibitthem? Are the pathological effects of disease dueto production of toxins (acting intracellularly orsystemically, or both), depletion of nutrients,mechanical blockage of vital tissues, or evocationof hypersensitivity or auto-allergic reactions? Canhost and tissue specificities be explained either bydifferential distribution of microbial inhibitors ordifferential suitability of tissues for microbialgrowth? The remaining pages record an attemptto answer some of these questions from the avail-able relevant investigations of microbes other thanbacteria. The reader is reminded that, as for bac-teria, studies of viruses, fungi, and protozoagrown in vitro (for viruses in tissue culture) canbe misleading with regard to mechanisms of path-ogenicity, since such organisms can be consider-ably different from the fully virulent organismsgrown in vivo (116).

VirusesThe difficulty of identifying factors responsible

for virus virulence and the present lack of knowl-edge are apparent from reviews (6, 18, 45, 89,116, 121, 131). The first essential quantitativecomparison of the virulence of different strains(or different viruses) is difficult. The effects inanimals (LD5o, lesion size, or mean death time forthe "same" dose) must be related to amounts ofvirus particles indicated by plaque formation oregg infection. These tests may detect only a smallproportion of the total virus particles present andtherefore may not be a measure of the numbers ofparticles (which may vary for different strains)capable of multiplying in the experimental ani-mals. Hence, only strains for which the availabletests have indicated the greatest possible dif-ference in virulence should be used for compara-tive studies. Although such comparisons are rare(3, 6, 45, 89, 131), they have yielded much of theinformation summarized below. The writer isunaware of any major study of the effect of theconstituents or products of a virulent strain on thebehavior of avirulent or attenuated strains.A virulent virus must be able to grow in host

cells and to spread from one cell to another; anychange in these abilities will almost certainly re-sult in changes in virulence, and this may be re-

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flected in the size of plaques formed in cell cul-ture (116). The marked effects of temperature onthe virulence of many viruses (6, 116, 122) prob-ably depend on different abilities of viruses togrow and survive at the various temperatures.Inability to grow at low pH (116), e.g., in thecells of an inflammatory exudate, may explainsome examples of avirulence of viruses. Differ-ential ability to respond to small molecular con-stituents in host tissues may also determine differ-ences in virulence, in view of the growth-stimula-tory effect of arginine on herpes simplex virus(10).As for bacterial infections, there are few de-

tailed examinations of early stages of virus infec-tions (45, 89); hence, the mechanisms of virusaggressive activity are unknown. Host defenseappears to include inhibitors in serum (122), inter-feron (8), antibodies (26), and macrophages (6,45, 89). The role of polymorphonuclear leuko-cytes appears less important than in bacterialinfections but needs further investigation (45,89). Virulent viruses appear to possess in theirvirions or to induce in cells aggressins whichinhibit defense mechanisms. Thus, virulent strainsof influenza virus resist serum inhibitors morethan avirulent strains (122), virulent strains ofsome viruses induce less interferon than avirulentstrains (8, 116), virulent strains of ectromeliainfect mouse macrophages more readily thanavirulent strains (45, 89), and influenza virus andmumps virus reduce the phagocytic activity ofguinea pig and mouse leukocytes against bacteria(45, 81, 105). Nevertheless, a viral constituent orproduct which might reasonably be termed a virusaggressin has yet to be identified.There seems little doubt that the cytopathic

effects of some viruses (poliovirus, influenza virus,Newcastle disease virus, mumps virus, poxviruses,and mengovirus) on tissue culture cells are notdue solely to intracellular growth but to the for-mation of cytotoxic compounds (3, 42, 96, 106,121, 131). Similar cytopathic effects, which eithertake place in localized areas of animal hosts (e.g.,poliovirus in the cells of the anterior horn) or aremore widespread (e.g. poxviruses), are probablyresponsible for many of the pathological effectsof virus diseases (121). Hence, factors responsiblefor cytotoxic effects are probably virulence fac-tors and, in certain cases [e.g., Newcastle diseasevirus (6, 131) and mengovirus (3)], virulent strainshave been shown to have greater cytopathic effectsthan avirulent strains. The larger plaques pro-duced in cell cultures by the virulent strains weredue to their higher cytotoxicity and not to a fastergrowth rate, the burst population of virulentstrains being lower than that of avirulent strains.Apart from the neurominidases of influenza and

Newcastle disease viruses (106, 131) and the cyto-toxin of adenovirus (42, 43), viral cytotoxins havenot been characterized.

In addition to a greater cytotoxicity, virulentstrains of some viruses appear to have a greaterability than avirulent strains to grow in tissueswhere their cytotoxicity can have maximal effect.Thus, virulent strains of poliovirus and New-castle disease virus have a greater ability thanavirulent strains to grow in spinal cord tissue andbrain tissue, respectively (6, 121). The biochemi-cal bases for these effects are unknown.

Viruses can produce their pathological effectson the host by mechanisms other than directcytotoxicity. Cumulative damage could resultfrom cells bursting after acting merely as hostsfor virus multiplication. Furthermore, both hyper-sensitivity and auto-allergy occur in virus dis-eases, e.g., in small pox, mumps, measles, andviral encephalitides (137). Whether the evocationof hypersensitivity or auto-allergic reactions isresponsible for the main pathological effects ofsome virus diseases remains to be proved. Decid-ing between such mechanisms and direct virustoxicity is not made easier by the present lack ofknowledge of the latter. However, it appears thathypersensitivity or auto-allergic reactions may beinvolved in the pathology of some rashes (90), oflymphocytic choriomeningitis (53), and of viralencephalitides (132).Host and tissue specificities occur in virus in-

fections, but the biochemical bases for thesespecificities are unknown (116, 121). A differ-ential distribution of antiviral mechanisms couldexplain some of these specificities; this might bethe reason why the susceptibilities of differentstrains of mice to mouse hepatitis virus and WestNile virus were paralleled by the susceptibility toinfection of appropriate macrophages (45, 89).Differential suitability of cells for growth of thevirus might also explain some specificities. Thismight be a question of the presence or absence ofreceptors which allow viruses to penetrate intocells. On the other hand, there may be equalpenetration but the cells of susceptible hosts ortissues may have a biosynthetic apparatus forvirus growth that is more favorable than that ofother cells. Differential influences of simple fac-tors like temperature (116), pH (116), or lowmolecular nutrients (10) might also have someinfluence. To investigate the biochemical bases forhost and tissue specificities, whole animals ororgan cultures must be used, because, in contrastto normal, nondifferentiated, tissue culture cells,organ cultures appear to retain their parentspecificities for viral infection (7, 129). Futurestudies on the biochemical bases of virus specific-ities should be encouraged by reports of the

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transfer of mouse hepatitis virus susceptibility tootherwise insusceptible mouse (C3H) macro-phages by extracts of susceptible mouse (P.R.I.)macrophages (45).

Fungi

Although quantitative methods are availablefor comparing the virulence of strains of patho-genic fungi, and virulent and avirulent strains ofvarious species exist for comparison, the factorsresponsible for fungal pathogenicity in animalsare largely unknown (33, 55, 68, 100). This issurprising because the dimorphic fungi seem par-ticularly suitable for studies of pathogenicity.The yeast forms predominate in vivo and appearto be more pathogenic and immunogenic than themycelial/arthrospore forms which, although theyoccur, are less prevalent in vivo (55, 68, 100, 116).The two forms are antigenically and chemicallydifferent (68, 70, 100), and studies of pathoge-nicity could benefit from comparing them inappropriate biological tests, and from observa-tions of the influence of the products of one onthe behavior of the other. Chemical extraction ofany relevant product could follow. Such studieswould be facilitated by methods that are nowavailable for producing some yeast forms invitro (116), although some reservations have beenexpressed regarding their in vivo nature (100).Neither the host defense mechanisms againstpathogenic fungi nor the fungal aggressins thatcombat them are clear. The noninvasive, mycelialdermatophytes probably lack powerful aggres-sins, and this may be related to the lack of theyeast form (55, 100). Powerful antifungalmaterials in body fluids have not been reported,and hence possible aggressins that act againstthem are a matter of conjecture. Interference withphagocytic ingestion appears important in thevirulence of some fungi, e.g., Crvptococcuts neo-formans, in which the capsulalr polysaccharide isthe aggressin possessed by virulent strains (19).Mere size may prevent ingestion (68) since somefungi are larger than phagocytes; hence, rapidityof growth in host tissues may be a factor in patho-genicity. Some fungi, e.g., Histophisinia capsulatumand Coccidioides inmmitis, appear to survive andgrow within phagocytes (68); hence, aggressinscomparable to those described previously forB. abortus and which combat intracellular killingmay be produced. Such intracellular aggressinscould be connected with various ill-defined pro-teins, carbohydrates, and protein/carbohydratecomplexes isolated from fungi that appear toproduce immunity to the appropriate disease (68).The toxic mechanisms of pathogenic fungi are

obscure. Clearly, toxins such as aflatoxin (whichin the fungal field is comparable to botulinum

toxin in the bacterial field: i.e., a toxin, signi-ficant in disease but produced outside thehost) could be responsible for the effects of infec-tion, but the writer is unaware of such a toxin(other than aflatoxin) being unequivocallydemonstrated and purified. Peptidases may beinvolved in the toxic action of dermatophytes(22). Hypersensitivity undoubtedly occurs inmany fungal diseases and probably explains to alarge degree the pathology of some fungal skindiseases; however, the degree to which hypersen-sitivity is implicated in the main pathology ofdeep mycoses is a matter of conjecture (55, 68).The compounds responsible for these hypersen-sitivities are many (even for one fungus) and areill-defined chemically, although some progresshas been made with products of dermatophytes(9).Host and tissue specificities occur in fungal

diseases (33, 55), and the explanations for thesespecificities may be similar to those discussedabove for bacterial diseases. For example, thelack of sebaceous glands which excrete mycostaticfatty acids may allow the growth of Trichophytonspp. in certain areas, e.g., between the toes (55).Also, the growth of Aspergillus fumigatus, thecause of mycotic abortion, in the placenta ofdomestic animals could be determined by nutri-tional factors in a manner similar to the placentallocalization of B. abortus.

Protozoa

Although the pathology of protozoal infectionshas been studied, the microbial factors responsiblefor the pathological effects on the host havereceived little attention (2, 41, 55, 56, 57, 75, 78,116). Since protozoa can be counted easily andsuitable experimental animals exist, quantitativecomparisons of virulence present no insuperabledifficulties. The major difficulty in identifyingvirulence factors appears to lie in the versatilityof the protozoa. The different phases of theirlife cycles, their different morphologicalforms, and their antigenic plasticity all influencepathogenicity (26, 41, 55, 75, 116). Keeping onestrain in one form for repeated experiments on aparticulair problem in pathogenicity appears tobe a major operation, and valid comparisonsbetween corresponding forms of different strainsare even more difficult, if not impossible. Perhapsthis is an area in studies of microbial patho-genicity where comparison between strains ofdifferent virulence may not be a good approach.Observations of a single strain and the changesin its biochemical behavior that affect pathoge-nicity may be more rewarding.

Microbial nutrition plays a role in protozoalpathogenicity; e.g., p-aminobenzoic acid has

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been shown to influence malarial infections (55).A nutritional influence could also determine somecases ofhost and tissue specificities, e.g., the invas-ion of red blood cells by the malarial parasite(55). In the host defense mechanisms againstprotozoal attack, humoral factors, particularlylysis by antibody and complement, appear to beimportant in some infections [e.g., trypanoso-miasis (26, 55)] and phagocytes are important inothers [e.g., malaria and leishmaniasis (2, 56)].Thus, virulent protozoa must produce aggressinswhich interfere with extracellular lysis, ingestionby phagocytes, or intracellular killing [e.g., forLeishmania spp. (2)]. No protozoal aggressinseems to have been identified, although enta-moebae appear to produce factors which killleukocytes (58). Some protozoa avoid extra-cellular lysis by antibody and complement bychanging their surface antigens periodically(26).The toxic effects of protozoa on their hosts have

been investigated (55), with special attentiongiven to Plasmodium spp., which cause red celldestruction, anaemia, and shock involving circu-latory and renal failure (41, 56, 78). However, noprotozoal toxin has yet been recognized as beingunequivocally responsible for the main patho-logical effects of a disease. A malarial toxinappears likely but has yet to be demonstrated(41); the lytic effects of Entamoeba spp. on tissuesare known, but the microbial enzymes responsiblefor them have not been isolated (55); and, al-though toxic products of trypanosomes (55, 75)and toxoplasmas (57) have been investigated,their importance in disease is still not clear. Un-doubtedly, hypersensitivity and auto-allergicphenomena occur in protozoal infections (2, 41,55), and may be responsible for important patho-logical effects in some cases, e.g., in the destruc-tion of normal red blood cells by phagocytosisafter opsonization by antibodies to antigenschanged by the red blood cell parasitization (41).

Cancer CellsThe existence of common features between the

behavior of malignant cells and pathogenicmicrobes has been noted (39, 104). Malignancy,i.e., the ability of cancer cells to invade and kill ahost, like microbial virulence, appears to varyand to depend on genetic and environmentalfactors. Thus, cancer "strains" of differingmalignancy exist, and malignancy can beincreased by animal passage and decreased byculture in vitro (39, 104, 117). Additional similari-ties (117) between cancer and infectious diseaseinclude the importance of nutrition [e.g., theeffect of asparagine on leukemias (14)] and hostdefense mechanisms in tumor growth, the tend-

ency of cancer cells to grow better in some tissuesthan others, and the production of activeimmunity by malignant cells. There is even apossibility that cancer cells produce toxic factors(126). The analogy must not be taken too far, ascancer cells are more similar to host cells than areinvading microbes, and the differences betweenanimal cells which determine malignancy areprobably even more subtle than those which deter-mine virulence in microorganisms. Nevertheless,the analogy suggests an approach to studies ofmalignancy by the microbial methods outlinedin this review.

Sublines of differing malignancy were preparedfrom two tumors, a carcinogen-induced rat tumorand a spontaneous lymphoma in AKR mice, byserial passage in isogenic animals. Tumor progres-sion occurred, and, as for microbial virulence,cells from late passage generations were moremalignant than cells from early passage genera-tions (117). The two sublines of the ascitic rattumor showed differences in intraperitonealgrowth and blood and visceral invasion. In par-ticular, when compared with the less malignantsubline, the more malignant subline showed agreater initial decrease in free tumor cells in theperitoneal cavity (possibly indicating a greatercapacity to invade) and subsequently a greaterincrease. Furthermore, the rats having the moremalignant tumor, which died before those receiv-ing the same dose of the less malignant tumor,contained far less tumor than the latter rats; thus,the toxic activity of the more malignant cells wasgreater than that of the less malignant cells(135). Similar results were obtained with the twostrains of the lymphoma in AKR mice (73). Thecontinued investigation of these sublines will be asmall contribution from microbiologists to thevast effort of others in the cancer field.

WHY STUDY BIOCHEMICAL MECHANISMS OFMICROBIAL PATHOGENICITY?

This review ends with an answer to the questionof why biochemical mechanisms of microbialpathogenicity should be studied when so manyinfectious diseases can be controlled satisfactorilywithout the information such studies yield. Thereasons are twofold. First, there is the intellectualsatisfaction in studying the interactions of themicrobe and host at the biochemical level by useof the rapidly developing techniques of biochem-istry, microbiology, and experimental pathologywhich offer increasing chances of success in suchstudies. This review has indicated some areasrequiring attention. Second, there is a practicalreason for such studies. Infectious diseases aredangerous and, despite public health measures,outbreaks still occur. An effective chemotherapy

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of virus diseases is lacking, and drug resistance ofbacteria, protozoa, and fungi is increasing. Manyvaccines remain unsatisfactory, either because ofincomplete immunogenicity or because of thehazard of injecting live microorganisms. Thus,new measures for attacking microbial disease are

needed, and such measures may arise from an

increasing knowledge of the biochemical mecha-nisms whereby these microbes invade and damagethe tissues of a host. The biochemical challenge ofmicrobial pathogenicity should be accepted.

ACKNOWLEDGMENTS

In preparing this review, I benefited from many dis-cussions with J. H. Pearce, J. Stephen, A. E. Williams,0. Basarab, K. R. Wood, and other colleagues inthe Department of Microbiology, University of Bir-mingham.

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