BACTERIOLOGICAL REVIEWS, Sept., 1968, p. 164-184 Vol. 32, No. 3 Copyright c 1968 American Society for Microbiology Priiited in U.S.A. Biochemical Challenge of Microbial Pathogenicity H. SMITH Depcartmenit of Microbiology, The Uuiiversity, of Birminiglhamii, Bir-iniglhami1, Eniglantd

INTRODUCTION...... 164 STUDIES OF BACTERIAL PATHOGENICITY ...... 165 Difficulty of'Inivestigati,ig Biochenmicail Mechanlisms of PathogenlicitY. 165 Studies of Bacterial Behavior In Vivo.... 165 ASPECTS OF BACTERIAL PATHOGENICITY REQUIRING ATTENTION- 166 Intitiationl of Inlfectioni 166 Growth and Multiplication In Vivo. 166 Aggressive Activity...... 167 Inhihitors of blood atnd tissie hbactericidins...... 167 Inihibitors of' the actioni of plipagocvtes...... 168 (i) Iihibitors of conltact...... 168 (ii) l liihi tor-s of intgestionl . 168 (iii) Inihibitors of initralcellutlair hactericidins: prom2otioni of intracellular growvth. 169 (iv) Repercussionu of ietertogeiueity of pliagocytic finIuctiOni Oni microbial requiire- ments for caggressitis. . 170 Toxic Activity...... 171 Toxi,is of overridling importalnce in disease 171 Toxi,Is which aire siglnificanlt bhlt niot the o;uly factors respoursible.for dlisecase. 171 Toxilns produced ini vitro hblt of ioiknown importance ill disea.se. 171 Toxic effe(cts in vivo ofhacteria which aippear to produice lo rolevait to-viii ifi vitro 171 (i) Toxilns revealedbl vsticlyinig hbacteria in more iiactuiral enviro.iciltsWi 172 (ii) Rolev of hypersensitiviity ili toxic maniJe*stations of disesn.A. 173 Relcatioli o' Protective Anitigens to Virule,ice Factors... 174 Biochenicall Bases of Host anid Tissuce Specificity,.... 174 Role ofucireaise ili kidney, localizations ofCoryniehacteriuon rena(ile and(i Proteuis mirabilis. 175 Role of ervthritol in the tissute specificity of the brlucellae 175 UNANSWERED QUESTIONS RELATING TO THE PATHOGENICITN' OF VIRUSES, FUNGI, PROTOZOA, AND MALIGNANT CELLS. 176 Viriuses. 176 Fui.igi. .. 178 Protozoa...... 178 Ca,icer Cells ...... 179 WHY STUDY BIOCHEMICAL MECHANISMS OF MICROBIAL PATHOGENICIT'I...... 179 LITERATURE CITED ...... 180

INTRODUCTION diphtheria, gas gangrene, and botulism) and the During this century, man's success in control- endotoxins of gram-negative bacteria have been ling infectious disease has been dramatic. Al- studied in detail (49. 93), and the chemistry of though diseases such as cholera, trachoma, ma- bacterial substances which inhibit the action of laria, and influenza are still major problems in phagocytes is known (17, 33), many of the mech- some areas, many infectious diseases can now be anisms of bacterial pathogenicity are still not controlled-by vaccination, by drugs, and, most clear. Thus, the bacterial products responsible effectively, by strict public health measures. This for many disease syndromes remain obscure, and success in controlling infectious disease has been little if anything is known about the biochemical paralleled and promoted by the equally successful bases for communicability, for survival and recognition and description of the causative growth of certain bacteria within phagocytes, for microorganisms-bacteria, protozoa, fungi, or host and tissue specificities, and for long-term viruses. In contrast, the biochemical mechanisms microbial survival in chronic diseases. These whereby these microorganisms produce disease phenomena are not confined to bacterial diseases; are still obscure. What is known is confined they occur in even less understood infections almost entirely to bacterial diseases, and even caused by other microbes. here information is scanty. Although the toxins In this review, it is not intended to describe the responsible for the classical toxemias (tetanus, most recent investigations of well-known bacterial 164 VOL. 32, 1968 MICROBIAL PATHOGENICITY 165 toxins and substances which prevent ingestion by tions, those of host tissues under microbial phagocytes, nor to catalogue further differences attack, are not physiological but pathological exhibited in vitro between virulent and avirulent and continually changing (110, 116); at present, strains of the same pathogenic species. There is they are not reproducible in vitro. Changes in recent literature on these subjects (17, 33, 49, 93). metabolism would therefore be expected when The intention here is to point out gaps in our bacteria from infected animals are cultured in present knowledge of the biochemistry of micro- vitro (116), and such changes have been demon- bial pathogenicity and to suggest possible meth- strated (see below). In turn, these changes in ods for filling them. Particular attention will be metabolism could affect virulence. Bacterial directed to areas where there are now some in- virulence is usually reduced by subculture in dications of the direction future research should vitro, because bacteria lose the capacity to form take. The review is restricted to microorganisms one or more of the full complement of virulence pathogenic for animals. Because bacteria have attributes manifested in infected animals (110, received more attention in virulence studies than 116). Also, apparent virulence factors might be other microorganisms, they are dealt with more produced in vitro which are not formed, and extensively than the other pathogenic microbes. therefore not relevant, in vivo (116). Thus, However, if only to emphasize the dearth of bacteria grown in vitro can be incomplete or mis- knowledge in this field, the mechanisms of patho- leading with regard to the possession of virulence genicity of viruses, fungi, protozoa, and cancer attributes; this, coupled with the fact that bac- cells are discussed briefly in the context of con- terial behavior in vivo is not easily examined (see cepts applied to bacterial pathogenicity. The near below), forms the essence of the difficulties en- synonymous terms pathogenic and virulent will countered in studies of pathogenicity. be used as suggested by Miles (55), i.e., the former in respect to species and the latter in respect to Studies of Bacterial Behavior In Vivo degrees of pathogenicity of strains within species. Obviously, virulence factors can be produced in laboratory cultures if the requisite nutritional STUDIES OF BACTERIAL PATHOGENICrrY conditions are known. This has already been Pathogenic bacteria are peculiarities. The accomplished in studies of classical bacterial great majority of bacteria are harmless and often toxins and some antiphagocytic substances. How- beneficial. Obviously, pathogenic bacteria have a ever, for problems of pathogenicity which have chemical armory which enables them to invade a so far defied solution by conventional procedures host and produce disease. The problem is to with in vitro cultures, the above discussion sug- identify the weapons in this armory, their relative gests the study ofbacterial behavior in vivo. There importance, their chemical nature, and their mode are no vitalistic leanings behind this suggestion, of action on the host. This task is relatively simple merely a realistic assessment of an approach that when pathogenicity is determined by a single might reveal aspects of pathogenicity which later bacterial product easily produced in vitro, as in could be reproduced in vitro by appropriate diphtheria and tetanus. In the majority of infec- changes in cultural conditions. tious diseases, however, pathogenicity cannot be Information on bacterial behavior in vivo can related to a single microbial product and its bio- be gained in several ways. First, bacteria and their chemical bases are difficult to identify for the products can be separated directly from the dis- reasons given below. eased host for biological examination and for Difficulty of Investigating Biochemical chemical and serological study in vitro. Second, Mechanisms the behavior of organisms growing in vivo and of Pathogenicity their repercussion on the host can be examined, The main factor contributing to difficulty in either in the whole animal or in restricted tissues. this field is that virulence-the disease-producing The largest gaps in our knowledge of pathogenic- capacity of a population of microbes-is detect- ity occur here; detailed experimental pathology able only in vivo and is markedly influenced by and precise biochemical determinations are not changes in growth conditions due to selection of easily accomplished during infection, and only in types and to phenotypic change (84). a few cases have such studies supplemented the Most pathogenic species contain attenuated clinical pictures. Yet this information is vital, if strains which are often indistinguishable from mechanisms of pathogenicity are to be understood virulent strains in the available tests in vitro. Thus, and if relevant biological tests for potential viru- virulence is determined by small genetic differ- lence factors are to be designed. Not the least ences which may be fully expressed only under among the difficulties is the lack of suitable lab- the conditions ofthe test for virulence; i.e., during oratory animals in which human infections, e.g., growth in vivo. The decisive nutritional condi- bacillary dysentery and typhoid or meningo- 166 SMITH BACTERIOL. REV. coccal and gonococcal infections, can be truly surfaces are mixed, and only small numbers of simulated. Third, light can be shed on particular the pathogenic component may be present at the phases of microbial behavior in vivo by making start of infection. Preoccupation with pure cul- observations in tissue, or preferably organ, cul- tures can make microbiologists forget that natural ture. Finally, tests in vitro can be made more infection involves bacterial behavior in mixed relevant to behavior in vivo. These methods for culture about which little is known even in vitro. gaining knowledge of bacterial behavior in vivo Furthermore, metabolic studies on survival and have been described elsewhere (116); several growth of small inocula are rare even in vitro examples are included in the sections below. (116). In conventional studies with large inocula, A classical method of studying bacterial viru- the results reflect the activities of the bulk of the lence is to compare the properties of virulent and population, whereas in vivo only initially atypical avirulent strains. Properly used, this method of organisms in the inoculum may succeed (116). A bacteriology is a powerful tool which could be recent study (46) of the growth of Pasteurella applied to studies of pathogenicity of other tularensis from small inocula showed heterogene- microbes (see below). The techniques of microbial ity of nutritional requirements among the popu- genetics have increased the scope of the method, lation, only a small proportion of which were and studies in vitro on enzymes, metabolic char- able to grow initially. A useful beginning to the acteristics, and antigens of different bacterial understanding of early processes of infection strains (17) have indicated many virulence mark- might be experiments in vitro on mixed cultures ers; i.e., factors associated with virulence. How- of relevant organisms using small inocula of the ever, relatively few of these factors have been pathogenic components. In this respect, recent shown to be virulence determinants; i.e., produced work (25, 80) on survival and growth of mixed during infection and having biological activities bacterial populations in continuous culture, directly connected with virulence, such as the which demonstrated striking effects of one organ- power to inhibit or destroy phagocytes (110). ism on another, is encouraging. With regard to Studies of virulence can benefit, therefore, from mechanisms of penetration of mucosal surfaces, comparisons of virulent and avirulent strains and electron microscopy of the early stages of infec- from examinations of the influence of the prod- tion, comparable to studies ofleukocyte migration ucts of a virulent strain on the behavior of an during inflammation (79), would be a useful avirulent strain, provided tests are carried out in prelude to examining the biochemical processes vivo or in simulant conditions in vitro. In these involved. studies, it must be remembered, however, that if full virulence is due to the possession of a number Growth and Multiplication In Vivo of factors an avirulent strain may possess all but one of these factors (110). Once within tissues, virulent bacteria must be able to grow and multiply in order to produce ASPECTS OF BACTERIAL PATHOGENICITY their disease syndrome, either by increasing in a REQuIRING ATNTIoN local lesion or by spreading throughout the host via the lymphatics and blood. To grow and Initiation of Infection multiply, two qualities are needed by bacteria: (i) Most infections occur through the mucous an inherent biochemical ability to grow under the membranes. Hence, to initiate infection, patho- nutritional conditions provided by host tissues genic bacteria must first survive on the mucous and (ii) an ability (see below) to combat defense surfaces in competition with commensals and mechanisms that would otherwise kill or remove then penetrate into the tissues. Together with them. The effects of these two qualities in vivo other factors (33), a differential ability of bac- are not easy to separate, and consequently it is terial species to accomplish these early stages of difficult to assess their relative importance, either infection might explain why some diseases (e.g., in the increase of a single infecting population or brucellosis) are more communicable than others in the differential behavior of virulent and at- (e.g., anthrax). Furthermore, interactions be- tenuated strains. Avirulence can arise from in- tween different pathogenic species on the mucous ability of bacteria to grow and divide in the en- surfaces might explain some of the marked effects vironment in vivo. Thus, nutritionally deficient one infection can have on another in mixed infec- mutants of pathogenic species were avirulent tions (116). unless injected with their required nutrients (17, The biochemical mechanisms whereby bacteria 93). However, for most bacteria, the tissues and survive on and penetrate mucous membranes are body fluids probably contain sufficient nutrients unknown. Two facts should be stressed in relation to support some growth. Hence, few naturally to survival: microbial populations on mucosal occurring strains (93) will be avirulent solely VOL. 32, 1968 MICROBIAL PATHOGENICITY 167 because of simple inability to grow in the host. class of substances, those which cause the disease Nutritional considerations will, however, affect syndrome and possibly death of the host, will be rates of growth in vivo (and the production of discussed later. virulence factors) and hence will influence degrees Aggressins act first during the decisive, primary of virulence. Furthermore, differences in nutri- lodgment period (87) of infection, when the few tional conditions in different hosts and in different invading bacteria are most vulnerable to the pro- tissues within the same host may be responsible tective reactions of the host. At this early stage, for host and tissue preferences of pathogenic aggressins must inhibit not only those nonspecific bacteria (see below). bactericidal mechanisms already existing in the The degree to which pathogenic bacteria multi- tissues (86) but also those agencies, especially ply in a host is in most cases unknown. At any phagocytic cells, which are mobilized by inflam- instant, the viable bacteria can be counted, but matory processes soon after the tissues are irri- they only represent the result of bacterial division tated (123). If some bacteria survive the primary and bacterial destruction or removal. Recently lodgment and grow, spread ofinfection is opposed (83, 85, 130), division rates in vivo have been by the fixed phagocytes of the reticuloendothelial measured by using pathogenic bacteria, geneti- system; here again, to make headway bacteria cally labeled with recognizable characteristics need aggressins possibly different from those retained by a known proportion of the progeny at operating during the early lodgment phase. To each division. Remarkable results were obtained; break through the protection of immunized ani- the doubling time of Salmonella typhimurium in mals, bacteria must be either numerous or well mice was 8 to 10 hr compared with 0.5 hr in endowed with aggressins, since the host defense cultures in vitro. mechanisms are of increased efficiency and are Woods and Foster (116) have discussed the supplemented by antibodies capable of direct almost complete lack of knowledge of bacterial neutralization of microbial products (26). The metabolism in vivo and the reasons why it prob- clinical outcome depends on the interplay of these ably differs from metabolism in vitro. The me- reactions of bacteria and host, and varies from tabolism of Bacillus anthracis in vivo is different complete subjugation of the host to complete from that in vitro (110), and, although observa- destruction of the bacteria, including near stale- tions in vitro on the metabolism of bacteria iso- mate in chronic infections. The following descrip- lated directly from infected animals will not detect tions of various bacterial aggressins are prefaced all aspects of metabolism in vivo (116), such by brief descriptions of the host defense mecha- observations on tubercle bacilli (15, 69, 107), nisms they inhibit. plague bacilli (40), staphylococci (12), and strep- Inhibitors ofblood and tissue bactericidins. Body tococci (44) indicated marked differences in fluids and tissues contain bactericidal factors (17, metabolism between these organisms and those 26, 97, 109) which include: #3-lysins, heat-stable grown in vitro. These observations should be substances acting against gram-positive organ- extended, because knowledge of microbial me- isms; complement, acting against gram-negative tabolism in vivo might be important not only in organisms sensitized either by antibody or pos- studies of virulence but for the design of new anti- sibly by nonspecific substances; lysozyme, acting bacterial drugs. The ideas and techniques used in directly against some gram-positive organisms studying host-dependent bacteria (47) might be and enhancing the action of complement and applied to other bacteria when the latter are grow- antibody on gram-negative organisms; and basic ing in vivo. polypeptides. Despite evidence that some bac- tericidins are artifacts which leak or are ex- Aggressive Activity creted from cells during manipulations in vitro In addition to a metabolic ability to grow in (51, 112), there is little doubt that blood and host tissues, virulent bacteria must produce sub- tissue bactericidins play roles in host defense (17), stances which act positively against the host; they being especially important during the first 3 hr fall into either or both of two classes. First, there after infection (86). Since, clearly, there are sev- are factors, not necessarily toxic, which promote eral different types of bactericidins, virulent bac- microbial growth in vivo by inhibiting host de- teria must produce different types of aggressins to fense mechanisms. These substances, called here inhibit them. by the old term aggressins (137) because it Although resistance to bactericidins has been describes so well their biological role, form the associated with virulence in strains of many subjects of this section. (The term is used as an species [e.g., Enterobacteriaceae (17), Staphylo- operational definition without any implication of coccus aureus (30), Leptospira spp. (59), B. relatedness between different aggressins either of anthracis (64), and Brucella abortus (34)], only chemical nature or mode of action.) The second rarely have the microbial products responsible 168 SMITH BACTERIOL. REV. been identified. These products, prepared from and nonspecific opsonins enhance this process of virulent strains, inhibited the destruction of ingestion (26, 33). avirulent strains by bactericidins, and some were Once inside phagocytes, many bacteria (e.g., immunizing antigens. Thus, the "anti-anthra- Streptococcus pneumoniae, S. pyogenes, B. an- cidal" activity ofB. anthracis was due to capsular thracis, Salmonella typhi, and Pasteurelia pestis) polyglutamic acid and to the complex immuno- are rapidly killed and digested. Resistance to genic toxin (64). Also, the resistance of B. abortus ingestion, which allows bacteria to avoid intra- to the action of bovine serum was due to an im- cellular bactericidins, is therefore essential for munogenic ceU wall component which contained the survival of virulent strains of these species. protein, carbohydrate, formyl residues, and lipid Aggressins that inhibit ingestion have been recog- [35 to 42% (34)]. The identification in other nized, but the chemical basis for their activity is pathogenic species (e.g., S. aureus and Leptospira unknown. They fall into two main types. spp.) of aggressins active against host bactericid- First, there are surface and capsular products ins might lead to the design of better protective which do not harm phagocytes. Examples are the measures against the corresponding diseases. The capsular polysaccharides of Streptococcus pneu- chemical mechanisms of aggressin-bactericidin moniae (33), the cell wall M protein and capsular reactions are unknown and cannot be investigated hyaluronic acid of S. pyogenes (17), the capsular until more knowledge of the chemistry of the poly-D-glutamic acid of B. anthracis (64), the 0 reactants has accumulated. somatic antigens of some gram-negative organ- Inhibitors of the action of phagocytes. Once a isms (74, 98), the Vi antigen (poly-N-acetyl-D- microbe has penetrated the mucous membranes galactosaminuronic acid) of S. typhi (24, 33), and or skin, the phagocytic activity of the wandering the protein carbohydrate envelope substance of P. and fixed cells of the reticuloendothelial system pestis (33). Further investigation of the detailed forms the main protective mechanism ofthe body, chemistry of these aggressins may reveal common a mechanism which acts nonspecifically but which structural features, but at present no such features is greatly enhanced by immunization (26). Phago- are apparent that might determine activity. Al- cytes vary in origin, morphology, constituents, though acidic components occur in most of the and bactericidal function. There are two main above compounds, including the various pneumo- types, each of which has two subdivisions: poly- coccal polysaccharides (33), they are absent from morphonuclear (neutrophils and eosinophils) and some active compounds (e.g., type IV and XII mononuclear (blood monocytes and tissue macro- pneumococcal polysaccharides) and hence do not phages) phagocytes. Polymorphonuclear phago- appear to determine aggressive activity. The sug- cytes are end cells with a short life derived from gestion that the antiphagocytic activity of some 0 stem cells different from the stem cells of long- somatic antigens might be due to a lipophilic lived mononuclear phagocytes. Inflammatory character determined by deoxy- and acetyl groups exudates contain cells of all types; the polymor- in the polysaccharide side chains of the higher phonuclear cells predominate initially but later salmonella chemotypes (74, 98) awaits proof. die, leaving the mononuclear phagocytes ascend- Thus, there is no obvious connection between ant. Macrophages form the fixed phagocytic structure and aggressive activity, and at the same system in the lymph nodes, spleen, and liver. time the modes of action of aggressins are not Phagocytosis of bacteria involves three stages: clear. Interference with ingestion may be purely contact, ingestion, and intracellular killing and mechanical, but other mechanisms can be in- digestion (33). Virulent bacteria may produce volved, such as inhibition of adsorption of serum aggressins which inhibit any of these stages. opsonin (64) or possibly an inability of the host (i) Inhibitors ofcontact. Contact with bacteria is to recognize the bacterial surface as foreign. Al- effected by random hits, by trapping on uneven though several aggressins are capsular in origin, surfaces in confined tissue spaces, by filtration a common impression, possibly arising from the systems in lymph nodes, spleen, and liver, and by classical work on the pneumococcal polysacchar- chemotaxis (26). Bacterial products could hardly ides, that all capsulated bacteria resist phagocytic interfere with the mechanical processes, but they ingestion and are virulent, is not true. The chemi- might inhibit nonspecific and specific chemotaxis cal nature of the surface material determines (26, 33). The writer is unaware of any clear ex- virulence, not the presence of a capsule per se. ample of the latter, but certain fractions from Excreted bacterial products which have a toxic tubercle bacilli inhibit leukocyte migration (33). action on phagocytes form the second type of (ii) Inhibitors of ingestion. Ingestion of bacteria aggressins interfering with ingestion. The leuco- involves engulfment within a phagocytic vacuole cidins of the staphylococci (33) and the anthrax (the phagosome), the wall of which is derived by toxic complex (64) are examples; the former are inversion of the phagocyte membrane. Specific produced by staphylococci within phagocytes, VOL. 32, 1968 MICROBIAL PATHOGENICITY 169 thus incapacitating the latter for further ingestion. used to kill extracellular organisms (112). In the The chemical basis of the effects of these aggres- phagocytes of this system, virulent strains of B. sins is unknown. abortus survived and grew, whereas avirulent (iii) Inhibitors of intracellular bactericidins: strains were progressively destroyed (112). promotion of intracellular growth. Knowledge of A factor which might have favored virulent the intracellular bactericidal mechanisms of strains was their ability to use nutritional condi- phagocytes is fragmentary but increasing. Neutro- tions within phagocytic cells more effectively than phils contain lysozyme and basic proteins called avirulent strains (17). However, ultrafiltrates of "phagocytin" (33) or "leukin" (108), which are extracts of bovine phagocytes did not stimulate bactericidal for gram-positive and gram-negative growth of virulent strains of B. abortus any more organisms and are associated with granules which than that of avirulent strains (20). Thus, a simple discharge into the phagocytic vacuoles containing nutritional explanation for the different behavior bacteria. In addition, hydrogen peroxide may of the strains seemed unlikely, and attention was contribute to bacterial killing in polymorpho- then concentrated on their differential abilities to nuclear cells (94). In contrast to neutrophils, little inhibit destructive mechanisms of phagocytes. is known about the bactericidal mechanisms of The higher catalase content of virulent strains either wandering or fixed mononuclear cells; of B. abortus compared with avirulent strains (17) peritoneal macrophages contain no phagocytin might have afforded intracellular protection, or lysozyme, and alveolar macrophages contain since bactericidal hydrogen peroxide is present in lysozyme but no phagocytin (33). Thus, the bac- polymorphonuclear cells (94). A correlation be- tericidal capacities of phagocytes differ, and they tween the intracellular behavior (and virulence) of could vary with the species of microorganisms strains and sensitivity to hydrogen peroxide was being ingested. established, and survivors from the almost com- Virulent strains of some bacteria (tubercle plete intracellular destruction of an avirulent bacilli, gonococci, meningococci, and brucellae) strain were more resistant to hydrogen peroxide resist the phagocytic bactericidins which destroy than the parent strain (37). However, these en- other microorganisms, and grow intracellularly. couraging results were not supported by similar This phenomenon, which is probably the most experiments with B. melitensis and B. suis; here, important aspect of the pathogenicity of these there was no correlation between sensitivity to bacteria, occurs both in infected animals and in hydrogen peroxide and intracellular behavior and cell maintenance culture in vitro (112, 116). virulence (37). In addition, the intracellular be- Obviously, virulent strains possess aggressins havior of virulent and avirulent strains of B. which interfere with bactericidal mechanisms of abortus in bovine cells was not influenced by in- the phagocytes. However, the nature of these ag- creasing the intracellular content of bovine cata- gressins and the mechanisms of intracellular sur- lase, either by allowing phagocytes to ingest vival and growth are unknown, even for the tu- carbon particles on which catalase had been bercle bacillus whose biology and chemistry have adsorbed, or by allowing them to ingest B. abortus been the subject of a half century of work. This from a solution containing bovine catalase (37). is not surprising in view of the limited knowledge In the first method, catalase might have remained on bactericidal mechanisms of phagocytes them- solely in the phagocytic vacuoles surrounding the selves. With more information accumulating carbon particles, but in the second method about phagocytic defense mechanisms, investiga- catalase would probably have been incorporated tions should begin on the microbial aggressins in the same phagocytic vacuole as the bacteria. that counteract them. As an example of what The high catalase content of virulent strains of B. might be attempted, a recent study of the chemi- abortus did not appear to play a significant part cal basis for the intracellular survival and growth in intracellular survival and growth. of B. abortus in bovine phagocytes will be sum- The following experiments suggest that the marized. ability of virulent B. abortus to survive and multi- Cell maintenance cultures were used and,J in ply intracellularly is due to a cell wall substance contrast to previous work with monocytes of which interferes with the intracellular bactericidal laboratory animals employing a high bacterium- mechanisms of phagocytes. Virulent B. abortus, phagocyte ratio (116, 125), attempts were made obtained either from cultures in guinea pig mono- to simulate natural infection: the mixed phago- cytes or from infected bovine placental tissue, had cyte population of bovine "buffy coat" (i.e., poly- an increased ability to survive intracellularly com- morphonuclear cells, monocytes, and lympho- pared with the same strain grown in laboratory cytes) was infected with a small number of B. media (112, 125). In addition, the cell wall mate- abortus cells. Then fresh bovine serum with a rial of the organisms from infected bovine pla- minimal quantity (2 ,ug/ml) of streptomycin was centa inhibited intracellular destruction of an 170 SMITH BACTERIOL. REV.

avirulent strain of B. abortus, and this effect was capacity and function vary between different neutralized by an antiserum against live virulent types of phagocytes and within the same type. B. abortus. This inhibitory activity was not shown Thus, mononuclear phagocytes are less effective by cell wall material obtained from either the than polymorphonuclear cells for kifling some virulent or an avirulent strain grown in vitro bacteria. P. pestis survived and grew within (112). The material which prevented intracellular mouse and guinea pig monocytes, yet was killed destruction of B. abortus differed from the im- when ingested by polymorphonuclear cells (21). munogenic cell wall material which interfered Staphylococci were killed more effectively by rab- with the bactericidin of bovine serum (see above). bit polymorphonuclear cells than by macrophages The latter material, which was produced by (76). Avirulent strains of B. abortus were killed, virulent and to some extent by avirulent bac- and the growth of virulent strains was inhibited teria in vitro, interfered with an extracellular more by polymorphonuclear phagocytes than by bactericidal activity of bovine buffy-coat cells monocytes in bovine blood (H. Smith, P. W. but not with the intracellular activity (34, 77, Harris-Smith, and R. B. FitzGeorge, unpublished 112). data). Populations of the same cell type show Once the celi wall material contributing to heterogeneity with respect to bactericidal capacity; intracellular survival had been recognized by ob- members of populations of rabbit and mouse servations on B. abortus grown in vivo, organisms macrophages differed in their capacities to kill having this material were produced in vitro by staphylococci and S. typhimuiriuim, respectively supplementing laboratory cultures with bovine (76, 101). Also, in the syndrome termed chronic fetal fluids (38). Thus, organisms resistant to granulomatous disease of childhood (61), poly- intracellular bactericidins can be prepared in morphonuclear cells appeared to lose their quantity and extracted to determine the nature capacity to kill certain bacteria (e.g., S. atureus, of the material contributing to intracellular sur- Escherichici coli, and Klebsiella spp.) but not others vival. It might prove structurally similar to the (e.g., streptococci). material which interferes with the extracellular The reasons for heterogeneity of phagocytic bactericidins but different in the detailed chem- function are becoming clearer as the nature of the istry of its side chains [cf. the different 0 somatic intracellular bactericidins is being investigated. antigens of the Enterobacteriaceae (74, 98)]. The lack, in macrophages, of some of the bacte- In addition to frankly intracellular bacteria like ricidins present in neutrophils has been mentioned brucellae, other bacteria such as straphylococci previously. Recently (138, 139), the lysosomal appear to survive within phagocytes, at least for cationic proteins of rabbit polymorphonuclear short periods. Hence, virulent staphylococci pre- cells were separated by electrophoresis into frac- sumably have the ability to inhibit the bacte- tions with different arginine-lysine ratios and ricidins of polymorphonuclear cells, either intra- different bactericidal activities for streptococci, cellularly when surviving within the phagocytes, Proteus spp., and E. coli. Similarly, "leukin' (108) or possibly extracellularly if the bactericidins are from rabbit polymorphonuclear cells was hetero- liberated from the dead (leucocidin killed) cells geneous and appeared to contain different bacte- of pus. Unlike bactericidins acting on B. abortus, ricidins for gram-positive and gram-negative bac- which could not be demonstrated in phagocyte teria. The presence or absence of these different extracts but only in the cell cultures described types of bactericidal proteins in various phago- above, bactericidins acting on staphylococci are cytes could explain heterogeneity of function as present in extracts of polymorphonuclear cells. regards intracellular killing. However, phagocytes Recently, Adlam, Pearce, and Smith (1) showed might also differ in their capacity to ingest differ- that Staphylococcus aureus grown in rabbits is ent bacteria, and heterogeneity of function might not only more virulent for rabbits but is also more be evident at this early stage of phagocytosis. resistant to the bactericidins of rabbit polymor- Heterogeneity of phagocytic function has two phonuclear cells (and to rabbit serum bacte- main repercussions on studies of microbial ag- ricidins, which may or may not be the same as the gressins. First, bacteria may have to produce dif- cellular bactericidins) than the same strain grown ferent aggressins to combat the activities of differ- in vitro. This observation may lead to the identi- ent kinds of phagocytes. Thus, a strain of P. pestis fication of staphylococcal aggressins responsible resistant to phagocytosis by wandering phago- for these antibactericidal effects [cf. studies of B. cytes was ingested and killed by cells of the abortus and other bacteria grown in vivo (110, reticuloendothelial system (33). A similar sus- 116)]. ceptibility to the reticuloendothelial system might (iv) Repercussion of heterogeneity oJ phagocytic also explain the avirulence of type III A66 function on microbial requirements for aggressins. pneumococcus for rabbits, despite the posses- There is increasing evidence that phagocytic sion of capsular polysaccharide, which, like that VOL. 32, 1968 MICROBIAL PATHOGENICITY 171 of virulent strains (e.g., type SV III), protected it death (33, 137). However, avirulent strains of from phagocytosis by wandering phagocytes (33). many gram-negative species contain much endo- Second, bacteria lacking sufficient aggressins to toxin; mice bred resistant to endotoxin are sus- combat powerful destructive mechanism, either ceptible to infection (50); the endotoxin of nor- in serum or in other phagocytes, might be pro- mal alimentary tract E. coli has no apparent tected by ingestion into phagocytes that are less noxious effect; and, despite their similar endo- bactericidal. This situation might occur to the toxins, different gram-negative organisms produce advantage of invading bacteria during the early different disease syndromes (33, 110). Clearly, stages of infection when they have not yet ac- factors other than endotoxins are important in quired, under the conditions of growth in vivo, gram-negative bacterial infections. These factors their full complement of aggressins. Indeed, after may be aggressins (e.g., the K antigen of E. coli a period within less destructive monocytes, P. and the Vi antigen of S. typhi), differing nutri- pestis became resistant to subsequent ingestion by tional requirements of various species, and toxins polymorphonuclear cells (21) and B. abortus different from endotoxin (110). The production became more resistant to the bactericidal action of such toxins by Vibrio cholerae, E. coli, and ofbovine serum (125). A similar protection within Pseudomonas aeruginosa is described later. less bactericidal phagocytes might also play a role Toxins produced in vitro but of unknown impor- in long-term survival of bacteria in chronic and tance in disease. Many substances producing toxic carrier states and in drug-resistant infections (52). effects related or unrelated to disease syndromes have been isolated from laboratory cultures. Some Toxic Activity of these products may be laboratory artifacts The toxic activities of bacteria can be divided having no relevance to disease in vivo [cf. lyco- roughly into four categories. marasmin in plant disease (116)1. Even if formed Toxins of overriding importance in disease. during infection, the question remains whether Clostridium tetani, C. botulinum, and Corynebac- they play significant roles. Hence, the relevance terium diphtheriae produce in vitro powerful, well- of these substances to disease should be deter- characterized exotoxins that are responsible for mined before their biochemical study in depth is the disease syndromes in vivo. Immunization with attempted. Examples are the many "toxic" prod- toxoid protects against disease. The biochemical ucts of staphylococci (33) and streptococci (33), activities of these toxins at the cellular level (17, the "neurotoxin" of Shigella shiga (33, 49), and 33, 49, 93) will not be discussed here. The major- the "murine" toxin of P. pestis (93, 110, 116). ity of pathogenic bacteria do not produce toxins Toxic effects in vivo of bacteria which appear to of this type. produce no relevant toxin in vitro. Apart from bac- Toxins which are significant but not the only teria, very few pathogenic microbes have been factors responsible for disease. These toxins are shown to form toxins responsible for the patho- produced in vivo and are responsible for some logical effects of infection, and, despite their pathological effects of infection. However, they intensive study in vitro, some pathogenic bacteria are not the sole determinants of disease: often as still fall into this category. Examples are Strepto- much toxin is produced by avirulent as by viru- coccus pneumoniae (33), Shigella spp. (17), and lent strains; sometimes injection of toxin does not Mycobacterium tuberculosis (33); until recently, reproduce all the pathological effects of disease; V. cholerae, enteropathogenic strains of E. coli, and usually immunization with toxoid does not and P. aeruginosa would have been included. confer solid protection against infection. Since The primary problem here is to demonstrate these toxins are involved in disease, biochemical the microbial factors causing the disease syn- investigations of the toxins themselves and their dromes so that biochemical investigations of activities at the cellular level should be continued. these factors can proceed. There are two explana- The a-toxin of staphylococci and the erythrogenic tions for the apparent lack of toxins. First, toxins toxin of streptococci (33) are examples, but the exist but have yet to be demonstrated. Second, the most important representatives are the endotoxins host is harmed by means other than the direct in cell walls of gram-negative bacteria. Their action of a toxin. For example, in diseases where chemistry has been elucidated (74); however, the large bacterial populations accumulate before the emphasis has been on serological activity rather host succumbs, the growing bacteria might deplete than on toxicity. Their toxic manifestations- some tissues of essential nutrients. This explana- pyrexia, diarrhea, prostration, and death in tion does not appeal to the writer because of the shock (137)-are similar, no matter from which demonstrated versatility of replacement mecha- species they are prepared (33). In some infections, nisms of mammalian hosts; it is also improbable e.g., typhoid fever, endotoxin seems to be re- if the host still dies when the large terminal popu- sponsible for fever, leucopenia, and possibly lation of organisms is removed by treatment with 172 SMITH BACTERIOL. REV. antibiotic [cf. studies on anthrax (65)]. A more mice. Later, the guinea pig toxin was found in likely explanation for host damage in the absence P. pestis grown in vitro and fractionated into two of toxin is the evocation of hypersensitivity reac- synergistically acting components, both proteins tions by nontoxic bacterial products. and unconnected with either the "murine" toxin (i) Toxins revealed by studying bacteria in more or lipopolysaccharide (124). The question as to natural environments. It has been suggested pre- whether the guinea pig toxin or the "murine" viously that, for dealing with unsolved problems toxin or both are involved in death of man from of pathogenicity, the behavior of bacteria in vivo plague is unresolved. should be examined for virulence attributes which Recently, an enterotoxin of V. cholerae has might then be reproduced in vitro for closer in- been demonstrated; it appears to be responsible vestigation. The preliminary examination should for the dramatic loss of fluid from the intestine be made in experimental animals or in biological which occurs in cholera and which results in de- tests in which aspects of the natural disease are hydration and death in shock. Previous workers simulated as far as possible. In the past decade, had attempted, without much success, to impli- hitherto unknown toxins have been demonstrated cate in cholera the endotoxin, a mucinase, and by this approach. other materials from V. cholerae. The lack of The cause of death in anthrax was clarified by progress was due to the difficulty of obtaining studies (110, 115, 116) in guinea pigs, followed by either an experimental animal or a biological test appropriate experiments in vitro. Until this work, in which the effects of cholera could be simulated; neither the general nature of the lethal effect of for example, although mice were killed by intra- B. anthracis on the host nor the products responsi- peritoneal injection of living V. cholerae and its ble were known; no lethal toxin had been found endotoxin, in neither case were the signs of in laboratory cultures. The main findings were (i) cholera evident. Recent advances have resulted that the massive terminal bacteremia was not the from the use of more natural biological systems primary cause ofdeath, because removal of organ- and the examination in them of the behavior of isms by streptomycin did not prevent death, which living V. cholerae before study of its products was therefore probably caused by a toxin; (ii) formed in vitro. that the fatal syndrome was oligemic secondary In ligated intestinal loops of rabbits, De and shock; (iii) that the plasma of infected guinea pigs his associates (31, 32) produced the gross fluid contained a specifically neutralizable, edema- loss and mucosal damaging effects of cholera, producing, lethal toxin; (iv) that the immunogenic first by young living cultures of V. cholerae and toxin originally recognized in vivo was reproduced then by filtrates from such cultures. The extracel- in vitro; (v) that the toxin comprised at least lular or easily liberated enterotoxin was heat- three nontoxic components, two of which were labile, neutralized by an antiserum to culture proteins and one a chelating agent containing filtrates, and not related to mucinase, hemolysin, protein, carbohydrate, phosphorus, and a group or endotoxin. Recently (62), this rabbit gut tech- absorbing at 260 nm; and (vi) that the compo- nique was used for titration of cholera entero- nents interacted synergically in toxicity and im- toxin. munogenicity tests and formed the basis of effec- Datta, Finkelstein, and their colleagues (35) tive vaccination against anthrax. This work has produced some cholera signs in starved 8- to now been confirmed (91). The anthrax toxic com- 12-day-old suckling rabbits by introducing young plex has been found in many infected animals, living V. cholerae into their washed stomachs with including rhesus monkeys, and is now generally a catheter. The cholera effects were then pro- accepted as responsible for death from anthrax duced by filtrates from young, well-aerated cul- (91). At the biochemical level, its mode of action tures of V. cholerae in simple media. Like the is unknown, but in animals it produced fluid loss, product described by De, the enterotoxin was leading to secondary shock, and in some species heat-labile, specifically neutralized by antisera to to pulmonary complications (115). culture filtrates, and not connected with endo- P. pestis and its products obtained from in- toxin. At first, the enterotoxin was thought to fected guinea pigs were investigated (17, 116) to consist of two components, procholeragens A resolve the following anomaly regarding the cause and B, but later (36) procholeragen A was demon- of death in plague. Live virulent P. pestis killed strated to be the important factor. both guinea pigs and mice, but a product, "mu- Craig (27, 28), recognizing that a possible rine" toxin, obtained from cultures in vitro killed cholera enterotoxin should increase capillary only mice. An extracellular toxin comparable to permeability, used for his work skin tests for that in anthrax could not be demonstrated in edema in rabbits and guinea pigs. Furthermore, infected guinea pigs, but an extract of P. pestis he examined the extracellular products of V. from guinea pigs killed guinea pigs as well as cholerae growing in the natural host, i.e., those VOL. 32, 1968 MICROBIAL PATHOGENICITY 173 in filtrates of rice water stools from human of enteropathogenic strains of E. coli but not in cholera patients. These filtrates produced skin corresponding filtrates from nonenteropathogenic edema, in contrast to filtrates from the stools of strains (120). The enterotoxins were extracellular, patients with diarrhea from causes other than heat-labile, and nonlethal for mice. They were cholera. Filtrates from young aerated cultures of unrelated to hemolysins and endotoxin; indeed, V. cholerae also produced the skin edema, again three preparations of the latter failed to dilate an in contrast to similar filtrates from cultures of appropriate ligated gut preparation. Thus, as is Shigella spp., E. coli, and noncholera vibrios. the case with V. cholerae, the enterotoxin of E. Serum of humans who had survived cholera coli can now be investigated biochemically. neutralized the edema-producing activity of fil- Studies of Shigella spp. in ligated rabbit gut trates from human stools and of the cultures of preparations have not proceeded as far as those V. cholerae. The enterotoxin was heat-labile, on V. cholerae and E. coli, but they appear to be unaffected by trypsin, and distinct from endo- taking a similar course. Signs of dysentery were toxin. Like log-phase cultures of V. cholerae (102), produced in gut preparations 12 hr after the intro- it produced in mongrel dogs a dramatic fluid loss duction of live Shigella spp. (5). Only freshly characteristic of cholera (J. P. Craig, personal isolated strains had these effects, and subculture communication). in vitro soon destroyed this activity. A toxin The enterotoxin of V. cholerae can now be different from endotoxin is the most likely cause investigated biochemically. The same active com- of the enteropathogenic action. However, such a pound(s) is probably present in all the crude prep- toxin has yet to be demonstrated, and hypersen- arations. The first step must be to purify the en- sitivity to endotoxin might also play a role in terotoxin to remove extraneous compounds which dysentery (see below). might interfere with subsequent investigations of P. aeruginosa can infect burns with fatal con- its chemistry and mode of action. The entero- sequences. In vitro, it forms endotoxin, a leci- toxin appears to enter the blood stream (136), so thinase, a protease, and a hemolysin, but none of its effect could be systemic as well as local. It will these compounds appears to explain the effects of be interesting to see whether Formalin-treated infection. Recently (71), the toxic activity of ex- enterotoxin (28) will immunize against cholera. tracts from lesions produced in rabbit skin by The success of the ligated rabbit gut technique widespread injections of P. aeruginosa (i.e., the in revealing the enterotoxin of V. cholerae ap- products of growth in vivo) was compared with pears to have been repeated with regard to the that of filtrates from vigorously shaken cultures of discovery of the toxin of enteropathogenic strains P. aeruginosa in rabbit serum and in broth. Sero- of E. coli. Until recently, no toxin was known logically, the toxin produced in vivo appeared to which could explain the production of diarrhea be identical to that produced in vitro; both prep- and scours in young humans and domestic ani- arations killed mice in shock, lowered the blood mals by certain strains of E. coli (116). However, pressure of rabbits, and appeared distinct from Taylor and her colleagues (127, 128) showed that any of the previously described products of P. living E. coli serotypes, isolated from babies with aeruginosa. The toxin now awaits biochemical diarrhea, distended ligated rabbit gut prepara- investigation. tions, whereas E. coli from healthy children and (ii) Role of hypersensitivity in toxic manifesta- other sources had no effect; they also noted that tions of disease. The classical work with M. a similar gut-distending action was produced by tuberculosis showed that evocation of hypersensi- chloroform-killed suspensions of the active sero- tivity reactions by pathogenic bacteria and their types but not by similarly killed suspension of products can have unpleasant and even fatal con- the other serotypes. The enterotoxic activity of sequences for the host (33, 137). Furthermore, the chloroform-killed suspensions was extremely skin tests clearly indicate that hypersensitive states labile and not connected with endotoxin activity. occur in numerous bacterial diseases, e.g., tuber- Using E. coli in domestic animals, Williams- culosis, staphylococcal infections, streptococcal Smith and Halls (119) showed that dilation of infections, pneumococcal infections, brucellosis ligated gut preparations from pigs, lambs, and tularemia, glanders, leprosy, Johne's disease, and calves reflected the enteropathogenicity of E. coli salmonellosis (33, 137). Usually the reactions are strains for the appropriate animal species. How- of the delayed type, indicating that cellular mech- ever, although there was some cross-reactivity, a anisms are involved, although Arthus-type reac- gut preparation from one animal species, e.g., tions can also occur, e.g., against bacterial poly- from pigs, might not detect a strain pathogenic saccharides (33). Thus, in some diseases, nontoxic for another species, e.g., calves. By use of appro- bacterial products can produce some of the toxic priate ligated gut preparations, enterotoxin was manifestations by evoking hypersensitivity reac- demonstrated in filtrates from soft-agar cultures tions. These reactions are perhaps more likely in 174 SMITH BACTERIOL. REV. chronic rather than in acute disease, and espe- the role of streptococci in rheumatism (33, 137). cially where the parasites are intracellular; aller- The difficulties of proving a significant role for gized cells (26) could be provoked from time to auto-allergy in the pathology of an infectious time by small amounts of microbial products disease are similar to those outlined above for liberated from the few surviving intracellular or- hypersensitivity reactions. And, of course, the ganisms. However, just as production of a toxin question arises as to whether a host product in vitro does not mean automatically that it is altered by microbial activity is then a host or a relevant in vivo, mere demonstration of a state of microbial product and thus whether auto-allergy hypersensitivity by a skin test, is no proof of the in the strictest sense is really involved. To my implication of hypersensitivity reactions in the knowledge, the nature and mode of production of main pathological effects of the disease. More any compound involved in "auto-allergy" during extensive investigations are needed; the main infectious disease has not been elucidated. With systemic and local effects of the disease must be the increasing importance in medicine of chronic simulated by hypersensitivity reactions evoked in microbial disease, the biochemical mechanisms a sensitized host by products of the appropriate underlying the possible involvement of hyper- microbe. sensitivity and "auto-allergy" in pathology should Evidence for the implication of hypersensitivity be investigated. reactions in the pathology of the disease is not easily obtained and is often equivocal. It is even Relation of Protective Antigens and harder to identify the particular bacterial products Virulence Factors involved. Almost certainly, for any one species Many bacterial products are antigens, and often [e.g., B. abortus (114)], these products will be they form the bases of diagnostic tests. However. more numerous than those showing overt toxicity. only a few of these antigens are protective (im- Descriptions (33, 137) of the extensive work on munogenic), i.e., actively immunize against dis- tuberculosis and rheumatic fever emphasize the ease. Of necessity, these immunogenic antigens difficulties ofobtainingprecise biochemical knowl- are factors involved in virulence, either toxins edge in this field. Now, there seems little doubt (e.g., that of C. tetani) or aggressins (e.g., the that the pathology of tuberculosis is largely due polysaccharides of S. pneumoniae). However, to hypersensitivity to products, particularly the some antigenic virulence factors are not protec- waxes, of M. tuberculosis. Also, the cardiac and tive (e.g., the a toxin of staphylococci); possibly other lesions of rheumatic fever appear to be ex- these factors do not operate significantly in those plained by hypersensitivity to streptococcal anti- early stages of infection which must be inhibited gens or to host tissue products altered by reaction if vaccination is to be effective. Some virulence with streptococcal products, or to both. The role factors are not even antigenic [e.g., capsular ofhypersensitivity is less clear in bacterial diseases hyaluronic acid of S. pyogenes and polyglutamic other than tuberculosis and rheumatic fever, for acid of B. anthracis (33)]; this might account for example in pneumococcal pneumonia (48). It the lack of immunity which sometimes occurs seems to be involved in some nephritic syndromes even after overt diseases, as in staphylococcal and in chronic brucellosis (33, 137). However, in infections. If such nonantigenic virulence factors acute brucellosis of susceptible pregnant animals, could be identified and rendered antigenic by abortion is due to the direct action of endotoxin coupling to proteins, effective vaccination might liberated from brucellae concentrated in certain follow. fetal tissues (133). Nevertheless, in some diseases caused by other gram-negative organisms, endo- Biochemical Bases of Host and Tissue toxin might act more by provoking hypersensitiv- Specificity ity reactions than by direct toxic action (33). This might occur in dysentery, since the intestinal tract Two aspects of microbial pathogenicity which can participate in hypersensitivity reactions, and embrace the behavior of all types of microbes in some infections of primates and other animals both animals and plants, and about which we with Shigella spp. produce the signs of dysentery know practically nothing in biochemical terms, only after a second challenge (17). are host and tissue specificities. In the bacterial The possibility of auto-allergy entering into the field, it is not known why dysentery is confined to pathology of chronic infectious disease should be primates and Johne's disease occurs only in cattle mentioned. The biochemical activities of a para- and related species, nor is it known why, in man, site may change host products sufficiently for S. pneumoniae grows so well in the lung to pro- them to evoke a tissue-damaging host response. duce pneumonia. Some discussion of host and This seems particularly possible for viruses, and tissue specificity is included here because recent some bacteria may operate in this manner, e.g., investigations of the tissue specificities of patho- VOL. 32, 1968 MICROBIAL PATHOGENICITY 175 genic bacteria suggest a pattern for future bio- localized in the kidneys of mice, whereas other chemical investigations in this field. However, diphtheroids did not, utilized urea when growing since the subject has already been reviewed (116), in bovine urine and urea-enriched peptone-water, only the main aspects are considered. and, in contrast to C. equi and E. coli, yielded ex- Differences in susceptibility to infection occur- tracts which formed ammonia from urea (72). ring between different species of the same host P. mirabilis also contains a urease (16) and pro- (and among different tissues in the same host) duces in rats, as in man, more severe renal often exceed those between immunized and unim- damage than is produced by E. coli or enterococci munized animals of the same host species. Hence, (16, 103). Furthermore, in tissue cultures of kid- if the chemical bases of resistance of a certain ney epithelium growth of intracellular P. mirabilis species (or tissue) to infection could be identified, but not that of E. coli, was stimulated by urea, it might be possible to confer on an otherwise sus- and the optimal concentration (0.2%) of urea was ceptible species a higher level of resistance than the same as that found in kidney homogenate that produced by immunization. (16). Hence, ability to utilize urea for growth The two most likely explanations for differ- contributes to the localization of C. renale and ences in susceptibility to infection of different P. mirabilis. However, other factors must also be hosts and tissues are differential distributions of involved in renal localizations, because organ- bactericidal mechanisms and differential distribu- isms lacking a urease, e.g., E. coli, also localize in tions of nutrients for which the metabolism of the the kidney. parasite is specially adapted. Role of erythritol in the tissue specificity of the The extracellular and cellular bactericidal brucellae. In many animals (humans, rats, guinea mechanisms of animal hosts are many and varied, pigs, and rabbits), brucellosis is relatively mild and there is increasing evidence of heterogeneity and chronic; the causative organisms do not grow of phagocytic function (see above). Undoubtedly, prolifically and have no marked affinity for par- variations of bactericidal mechanisms in different ticular tissues. However, in pregnant cows, sheep, species and tissues (33, 92, 116, 137, 139) could goats, and sows, an enormous growth of brucellae account for differences in specificities of infection; in the placentae, the fetal fluids, and the chorions e.g., resistance of animal species to anthrax might leads to the characteristic climax of the disease, depend on their level of tissue "anthracidal sub- abortion (113). Recent investigations (4, 66, 95, stance" (13). However, despite much effort, at- 111, 118, 133, 134) have provided the following tempts to lay the responsibility for specificity of data which explain this tissue localization in the infection unequivocally on such variations of susceptible animal species. A survey of extracts defined bactericidal mechanisms have so far failed of fetal and maternal bovine tissues showed that (54, 116). a material which stimulated the growth of B. More success has been achieved in recent in- abortus was concentrated in the fetal fluids, the vestigations of the influence of nutrition on speci- fetal placenta, and the chorion; this growth- ficities of infection. Clearly, different nutritional stimulating material was isolated and identified as conditions in different hosts and tissues could erythritol. Analyses of fetal and maternal bovine determine different levels of bacterial invasion tissue extracts showed that erythritol was concen- and growth. Such an explanation for host speci- trated in those tissues heavily infected in brucel- ficity has not yet been demonstrated, but it ap- losis. It was observed that erythritol enhanced pears to apply in the following examples of tissue B. abortus infections in newborn calves and stim- specificity. ulated the growth of B. melitensis and B. suis in Role of urease in kidney localizations of Coryne- vitro and in vivo. The placentae of cattle, goats, bacterium renale and Proteus mirabilis. C. renale sheep, and sows were found to contain erythritol, in cattle and P. mirabilis in man persist longer but it was not found in those of humans, rats, and cause more severe pyelonephritis than the guinea pigs, and rabbits. Male genitalia of sus- other bacteria (e.g., E. coli) which cause kidney ceptible species contained erythritol, thus corre- infections (63, 116). Nonspecific factors, such as lating its presence with localization of infection inhibition ofphagocytic response by high salt and in the male. During growth, B. abortus had a urea concentrations (23, 88, 99) and inactivation great affinity for erythritol; in a complex medium of complement by ammonia (11) probably con- containing glucose at a concentration 1,000 times tribute to the general susceptibility of kidney that oferythritol, B. abortus used 1.5 times its own tissue to infection. However, a more specific weight of erythritol as a general energy source. mechanism, namely, the ability of urease to Analogues of erythritol inhibited the growth of metabolize urea, also appears to influence the B. abortus both in vitro and in vivo. persistence and severity of infections with C. Hence, the presence of erythritol, a growth renale and P. mirabilis. Thus, C. renale became stimulant for brucellae, in susceptible tissues of 176 SMITH BACTERIOL. REV. susceptible species explains tissue specificity in reasonably stable virulent and avirulent strains brucellosis. Ability to grow rapidly with erythritol available so that virulence markers and deter- explains localization in an already infected host, minants can be recognized (i) by comparing the but it does not account for primary invasion of behavior of the strains in vitro and in vivo and (ii) the host, an aspect of virulence. The primary by observing the effects of products of the virulent invasion is controlled by ability to grow intracel- strain on the behavior of the avirulent strain? lularly, and this process does not involve erythri- How far can avirulence be due to an inherent tol (134). The growth of both virulent and at- inability to grow in the host tissues (as distinct tenuated strains of all three species of brucellae from an inability to combat host defense mech- was stimulated by erythritol (29, 60, 67, 82, 134). anisms)? What host defense mechanisms act Only for B. abortus was the growth of virulent against the microbe and what aggressins inhibit strains stimulated more than that of attenuated them? Are the pathological effects of disease due strains (134), although growth stimulation of the to production of toxins (acting intracellularly or latter occurred with high concentrations of eryth- systemically, or both), depletion of nutrients, ritol (67). A most striking and satisfying finding mechanical blockage of vital tissues, or evocation was that growth of the S19 vaccine strain which of hypersensitivity or auto-allergic reactions? Can has been used safely in the field was inhibited by host and tissue specificities be explained either by erythritol (60, 67). differential distribution of microbial inhibitors or The work on kidney infections and on brucel- differential suitability of tissues for microbial losis encompasses practically all of our precise growth? The remaining pages record an attempt knowledge of the chemical bases for tissue and to answer some of these questions from the avail- host specificity, and suggests a pattern for future able relevant investigations of microbes other than research. Indeed, preliminary studies on V. fetus bacteria. The reader is reminded that, as for bac- (the cause of vibrionic abortion in domestic ani- teria, studies of viruses, fungi, and protozoa mals) indicate that growth of the organism is grown in vitro (for viruses in tissue culture) can greater in extracts of fetal placenta than in ex- be misleading with regard to mechanisms of path- tracts of maternal tissues or conventional media, ogenicity, since such organisms can be consider- thus suggesting that localization of vibrio is deter- ably different from the fully virulent organisms mined by a preferred nutrient, as in brucellosis grown in vivo (116). (D. B. Lowrie and J. H. Pearce, personal communi- cation). These studies on tissue and host specific- Viruses ity also show that knowledge of the bases of these The difficulty of identifying factors responsible specificities might indicate approaches to chem- for virus virulence and the present lack of knowl- otherapy. edge are apparent from reviews (6, 18, 45, 89, 116, 121, 131). The first essential quantitative UNANSWERED QUESTIONs RELATING TO THE comparison of the virulence of different strains PATHOGENICITY OF VIRUSES, FUNGI, (or different viruses) is difficult. The effects in PROTOZOA, AND MALIGNANT CELLS animals (LD5o, lesion size, or mean death time for Although many aspects of bacterial patho- the "same" dose) must be related to amounts of genicity are still unexplained in biochemical virus particles indicated by plaque formation or terms, far more is known about bacteria than egg infection. These tests may detect only a small about other pathogenic microbes. There are many proportion of the total virus particles present and differences between these other pathogens and therefore may not be a measure of the numbers of bacteria, e.g., the necessity for viruses to grow in particles (which may vary for different strains) living cells, the polymorphism of pathogenic capable of multiplying in the experimental ani- fungi, the complex life cycles and the antigenic mals. Hence, only strains for which the available plasticity of pathogenic protozoa, and the simi- tests have indicated the greatest possible dif- larity to the host of cancer cells [considered here ference in virulence should be used for compara- as analogues of pathogenic microbes (39, 104)]; tive studies. Although such comparisons are rare however, like pathogenic bacteria, all of these (3, 6, 45, 89, 131), they have yielded much of the microbes invade hosts and produce disease. In information summarized below. The writer is spite of their differences from bacteria, further unaware of any major study of the effect of the studies of the other pathogenic microbes might constituents or products of a virulent strain on the be helped by briefly discussing their mechanisms behavior of avirulent or attenuated strains. of pathogencity in the context of what has al- A virulent virus must be able to grow in host ready been said for bacteria. The following ques- cells and to spread from one cell to another; any tions might be considered. Can differences in change in these abilities will almost certainly re- virulence be detected and measured in vivo? Are sult in changes in virulence, and this may be re- VOL. 32, 1968 MICROBIAL PATHOGENICITY 177 flected in the size of plaques formed in cell cul- Newcastle disease viruses (106, 131) and the cyto- ture (116). The marked effects of temperature on toxin of adenovirus (42, 43), viral cytotoxins have the virulence of many viruses (6, 116, 122) prob- not been characterized. ably depend on different abilities of viruses to In addition to a greater cytotoxicity, virulent grow and survive at the various temperatures. strains of some viruses appear to have a greater Inability to grow at low pH (116), e.g., in the ability than avirulent strains to grow in tissues cells of an inflammatory exudate, may explain where their cytotoxicity can have maximal effect. some examples of avirulence of viruses. Differ- Thus, virulent strains of poliovirus and New- ential ability to respond to small molecular con- castle disease virus have a greater ability than stituents in host tissues may also determine differ- avirulent strains to grow in spinal cord tissue and ences in virulence, in view of the growth-stimula- brain tissue, respectively (6, 121). The biochemi- tory effect of arginine on herpes simplex virus cal bases for these effects are unknown. (10). Viruses can produce their pathological effects As for bacterial infections, there are few de- on the host by mechanisms other than direct tailed examinations of early stages of virus infec- cytotoxicity. Cumulative damage could result tions (45, 89); hence, the mechanisms of virus from cells bursting after acting merely as hosts aggressive activity are unknown. Host defense for virus multiplication. Furthermore, both hyper- appears to include inhibitors in serum (122), inter- sensitivity and auto-allergy occur in virus dis- feron (8), antibodies (26), and macrophages (6, eases, e.g., in small pox, mumps, measles, and 45, 89). The role of polymorphonuclear leuko- viral encephalitides (137). Whether the evocation cytes appears less important than in bacterial of hypersensitivity or auto-allergic reactions is infections but needs further investigation (45, responsible for the main pathological effects of 89). Virulent viruses appear to possess in their some virus diseases remains to be proved. Decid- virions or to induce in cells aggressins which ing between such mechanisms and direct virus inhibit defense mechanisms. Thus, virulent strains toxicity is not made easier by the present lack of of influenza virus resist serum inhibitors more knowledge of the latter. However, it appears that than avirulent strains (122), virulent strains of hypersensitivity or auto-allergic reactions may be some viruses induce less interferon than avirulent involved in the pathology of some rashes (90), of strains (8, 116), virulent strains of ectromelia lymphocytic choriomeningitis (53), and of viral infect mouse macrophages more readily than encephalitides (132). avirulent strains (45, 89), and influenza virus and Host and tissue specificities occur in virus in- mumps virus reduce the phagocytic activity of fections, but the biochemical bases for these guinea pig and mouse leukocytes against bacteria specificities are unknown (116, 121). A differ- (45, 81, 105). Nevertheless, a viral constituent or ential distribution of antiviral mechanisms could product which might reasonably be termed a virus explain some of these specificities; this might be aggressin has yet to be identified. the reason why the susceptibilities of different There seems little doubt that the cytopathic strains of mice to mouse hepatitis virus and West effects of some viruses (poliovirus, influenza virus, Nile virus were paralleled by the susceptibility to Newcastle disease virus, mumps virus, poxviruses, infection of appropriate macrophages (45, 89). and mengovirus) on tissue culture cells are not Differential suitability of cells for growth of the due solely to intracellular growth but to the for- virus might also explain some specificities. This mation of cytotoxic compounds (3, 42, 96, 106, might be a question of the presence or absence of 121, 131). Similar cytopathic effects, which either receptors which allow viruses to penetrate into take place in localized areas of animal hosts (e.g., cells. On the other hand, there may be equal poliovirus in the cells of the anterior horn) or are penetration but the cells of susceptible hosts or more widespread (e.g. poxviruses), are probably tissues may have a biosynthetic apparatus for responsible for many of the pathological effects virus growth that is more favorable than that of of virus diseases (121). Hence, factors responsible other cells. Differential influences of simple fac- for cytotoxic effects are probably virulence fac- tors like temperature (116), pH (116), or low tors and, in certain cases [e.g., Newcastle disease molecular nutrients (10) might also have some virus (6, 131) and mengovirus (3)], virulent strains influence. To investigate the biochemical bases for have been shown to have greater cytopathic effects host and tissue specificities, whole animals or than avirulent strains. The larger plaques pro- organ cultures must be used, because, in contrast duced in cell cultures by the virulent strains were to normal, nondifferentiated, tissue culture cells, due to their higher cytotoxicity and not to a faster organ cultures appear to retain their parent growth rate, the burst population of virulent specificities for viral infection (7, 129). Future strains being lower than that of avirulent strains. studies on the biochemical bases of virus specific- Apart from the neurominidases of influenza and ities should be encouraged by reports of the 178 SMITH BACTERIOL. REV.

transfer of mouse hepatitis virus susceptibility to toxin in the bacterial field: i.e., a toxin, signi- otherwise insusceptible mouse (C3H) macro- ficant in disease but produced outside the phages by extracts of susceptible mouse (P.R.I.) host) could be responsible for the effects of infec- macrophages (45). tion, but the writer is unaware of such a toxin (other than aflatoxin) being unequivocally Fungi demonstrated and purified. Peptidases may be Although quantitative methods are available involved in the toxic action of dermatophytes for comparing the virulence of strains of patho- (22). Hypersensitivity undoubtedly occurs in genic fungi, and virulent and avirulent strains of many fungal diseases and probably explains to a various species exist for comparison, the factors large degree the pathology of some fungal skin responsible for fungal pathogenicity in animals diseases; however, the degree to which hypersen- are largely unknown (33, 55, 68, 100). This is sitivity is implicated in the main pathology of surprising because the dimorphic fungi seem par- deep mycoses is a matter of conjecture (55, 68). ticularly suitable for studies of pathogenicity. The compounds responsible for these hypersen- The yeast forms predominate in vivo and appear sitivities are many (even for one fungus) and are to be more pathogenic and immunogenic than the ill-defined chemically, although some progress mycelial/arthrospore forms which, although they has been made with products of dermatophytes occur, are less prevalent in vivo (55, 68, 100, 116). (9). The two forms are antigenically and chemically Host and tissue specificities occur in fungal different (68, 70, 100), and studies of pathoge- diseases (33, 55), and the explanations for these nicity could benefit from comparing them in specificities may be similar to those discussed appropriate biological tests, and from observa- above for bacterial diseases. For example, the tions of the influence of the products of one on lack of sebaceous glands which excrete mycostatic the behavior of the other. Chemical extraction of fatty acids may allow the growth of Trichophyton any relevant product could follow. Such studies spp. in certain areas, e.g., between the toes (55). would be facilitated by methods that are now Also, the growth of Aspergillus fumigatus, the available for producing some yeast forms in cause of mycotic abortion, in the placenta of vitro (116), although some reservations have been domestic animals could be determined by nutri- expressed regarding their in vivo nature (100). tional factors in a manner similar to the placental Neither the host defense mechanisms against localization of B. abortus. pathogenic fungi nor the fungal aggressins that combat them are clear. The noninvasive, mycelial Protozoa dermatophytes probably lack powerful aggres- Although the pathology of protozoal infections sins, and this may be related to the lack of the has been studied, the microbial factors responsible yeast form (55, 100). Powerful antifungal for the pathological effects on the host have materials in body fluids have not been reported, received little attention (2, 41, 55, 56, 57, 75, 78, and hence possible aggressins that act against 116). Since protozoa can be counted easily and them are a matter of conjecture. Interference with suitable experimental animals exist, quantitative phagocytic ingestion appears important in the comparisons of virulence present no insuperable virulence of some fungi, e.g., Crvptococcuts neo- difficulties. The major difficulty in identifying formans, in which the capsulalr polysaccharide is virulence factors appears to lie in the versatility the aggressin possessed by virulent strains (19). of the protozoa. The different phases of their Mere size may prevent ingestion (68) since some life cycles, their different morphological fungi are larger than phagocytes; hence, rapidity forms, and their antigenic plasticity all influence of growth in host tissues may be a factor in patho- pathogenicity (26, 41, 55, 75, 116). Keeping one genicity. Some fungi, e.g., Histophisinia capsulatum strain in one form for repeated experiments on a and Coccidioides inmmitis, appear to survive and particulair problem in pathogenicity appears to grow within phagocytes (68); hence, aggressins be a major operation, and valid comparisons comparable to those described previously for between corresponding forms of different strains B. abortus and which combat intracellular killing are even more difficult, if not impossible. Perhaps may be produced. Such intracellular aggressins this is an area in studies of microbial patho- could be connected with various ill-defined pro- genicity where comparison between strains of teins, carbohydrates, and protein/carbohydrate different virulence may not be a good approach. complexes isolated from fungi that appear to Observations of a single strain and the changes produce immunity to the appropriate disease (68). in its biochemical behavior that affect pathoge- The toxic mechanisms of pathogenic fungi are nicity may be more rewarding. obscure. Clearly, toxins such as aflatoxin (which Microbial nutrition plays a role in protozoal in the fungal field is comparable to botulinum pathogenicity; e.g., p-aminobenzoic acid has VOL. 32, 1968 MICROBIAL PATHOGENICITY 179 been shown to influence malarial infections (55). ency of cancer cells to grow better in some tissues A nutritional influence could also determine some than others, and the production of active cases ofhost and tissue specificities, e.g., the invas- immunity by malignant cells. There is even a ion of red blood cells by the malarial parasite possibility that cancer cells produce toxic factors (55). In the host defense mechanisms against (126). The analogy must not be taken too far, as protozoal attack, humoral factors, particularly cancer cells are more similar to host cells than are lysis by antibody and complement, appear to be invading microbes, and the differences between important in some infections [e.g., trypanoso- animal cells which determine malignancy are miasis (26, 55)] and phagocytes are important in probably even more subtle than those which deter- others [e.g., malaria and leishmaniasis (2, 56)]. mine virulence in microorganisms. Nevertheless, Thus, virulent protozoa must produce aggressins the analogy suggests an approach to studies of which interfere with extracellular lysis, ingestion malignancy by the microbial methods outlined by phagocytes, or intracellular killing [e.g., for in this review. Leishmania spp. (2)]. No protozoal aggressin Sublines of differing malignancy were prepared seems to have been identified, although enta- from two tumors, a carcinogen-induced rat tumor moebae appear to produce factors which kill and a spontaneous lymphoma in AKR mice, by leukocytes (58). Some protozoa avoid extra- serial passage in isogenic animals. Tumor progres- cellular lysis by antibody and complement by sion occurred, and, as for microbial virulence, changing their surface antigens periodically cells from late passage generations were more (26). malignant than cells from early passage genera- The toxic effects ofprotozoa on their hosts have tions (117). The two sublines of the ascitic rat been investigated (55), with special attention tumor showed differences in intraperitoneal given to Plasmodium spp., which cause red cell growth and blood and visceral invasion. In par- destruction, anaemia, and shock involving circu- ticular, when compared with the less malignant latory and renal failure (41, 56, 78). However, no subline, the more malignant subline showed a protozoal toxin has yet been recognized as being greater initial decrease in free tumor cells in the unequivocally responsible for the main patho- peritoneal cavity (possibly indicating a greater logical effects of a disease. A malarial toxin capacity to invade) and subsequently a greater appears likely but has yet to be demonstrated increase. Furthermore, the rats having the more (41); the lytic effects of Entamoeba spp. on tissues malignant tumor, which died before those receiv- are known, but the microbial enzymes responsible ing the same dose of the less malignant tumor, for them have not been isolated (55); and, al- contained far less tumor than the latter rats; thus, though toxic products of trypanosomes (55, 75) the toxic activity of the more malignant cells was and toxoplasmas (57) have been investigated, greater than that of the less malignant cells their importance in disease is still not clear. Un- (135). Similar results were obtained with the two doubtedly, hypersensitivity and auto-allergic strains of the lymphoma in AKR mice (73). The phenomena occur in protozoal infections (2, 41, continued investigation of these sublines will be a 55), and may be responsible for important patho- small contribution from microbiologists to the logical effects in some cases, e.g., in the destruc- vast effort of others in the cancer field. tion of normal red blood cells by phagocytosis after opsonization by antibodies to antigens WHY STUDY BIOCHEMICAL MECHANISMS OF changed by the red blood cell parasitization (41). MICROBIAL PATHOGENICITY? This review ends with an answer to the question Cancer Cells of why biochemical mechanisms of microbial The existence of common features between the pathogenicity should be studied when so many behavior of malignant cells and pathogenic infectious diseases can be controlled satisfactorily microbes has been noted (39, 104). Malignancy, without the information such studies yield. The i.e., the ability of cancer cells to invade and kill a reasons are twofold. First, there is the intellectual host, like microbial virulence, appears to vary satisfaction in studying the interactions of the and to depend on genetic and environmental microbe and host at the biochemical level by use factors. Thus, cancer "strains" of differing of the rapidly developing techniques of biochem- malignancy exist, and malignancy can be istry, microbiology, and experimental pathology increased by animal passage and decreased by which offer increasing chances of success in such culture in vitro (39, 104, 117). Additional similari- studies. This review has indicated some areas ties (117) between cancer and infectious disease requiring attention. Second, there is a practical include the importance of nutrition [e.g., the reason for such studies. Infectious diseases are effect of asparagine on leukemias (14)] and host dangerous and, despite public health measures, defense mechanisms in tumor growth, the tend- outbreaks still occur. An effective chemotherapy 180 SMITH BACTERIOL. REV. of virus diseases is lacking, and drug resistance of proteins of normal tissues. J. Bacteriol. bacteria, protozoa, and fungi is increasing. Many 64:855-858. vaccines remain unsatisfactory, either because of 14. Boyse, E. A., L. J. Old, H. A. Campbell, and L. T. Mashburn. 1967. Suppression of murine incomplete immunogenicity or because of the leukemias by 1-asparginase. J. Exptl. Med. hazard of injecting live microorganisms. Thus, 125:17-31. new measures for attacking microbial disease are 15. Brezina, O., I. Drobnicova, and L. Drobnica. needed, and such measures may arise from an 1967. Proliferation of Mycobacterium thibercla- increasing knowledge of the biochemical mecha- losis from mouse lung tissue on various carbon nisms whereby these microbes invade and damage sources. Nature 214:1036-1037. the tissues of a host. The biochemical challenge of 16. Braude, A. I., and J. Siemienski. 1960. Role of microbial pathogenicity should be accepted. bacterial urease in experimental pyelonephri- tis. J. Bacteriol. 80:171-179. ACKNOWLEDGMENTS 17. Braun, W., and D. V. Siva Sankar. 1960. Bio- chemical aspects of microbial pathogenicity. In preparing this review, I benefited from many dis- Ann. N.Y. Acad. Sci. 88:1021-1318. cussions with J. H. Pearce, J. Stephen, A. E. Williams, 18. Buddingh, G. J. 1965. Pathogenesis and pathol- 0. Basarab, K. R. Wood, and other colleagues in ology of viral infections, p. 339-355. In Viral the Department of Microbiology, University of Bir- and rickettsial infections of man, 4th ed. mingham. J. B. Lippincott Co., Philadelphia. LITERATURE CITED 19. Bulmer, G. S., and M. D. Sans. 1968. Crvpto- cocclus lieoormnans. III. Inhibition of phago- 1. Adlam, C., J. H. Pearce, and H. Smith. 1968. cytosis. J. Bacteriol. 95:5-8. Virulence attributes of staphylococci grown 20. Burrin, D. H., J. Keppie, and H. Smith. 1966. in vivo. Nature, 219:641-642. 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Diseases 114:203-216. 52. Holmes, B., P. G. Quie, D. B. Windhorst, B. 36. Finkelstein, R. A., P. Atthasampunna, M. Pollara, and R. A. Good. 1966. Protection of Chulasamaya, and P. Charunmethee. 1966. phagocytized bacteria from the killing action Pathogenesis of experimental cholera: Biologic of antibiotics. Nature 210:1131-1132. activities of purified procholeragen A. J. 53. Hotchin, J. 1962. The biology of lymphocytic Immunol. 96:440-449. choriomeningitis infection: virus-induced im- 37. Fitzgeorge, R. B., J. Keppie, and H. Smith. mune disease. Cold Spring Harbor Symp. 1965. The relation between resistance to hy- Quant. Biol. 27:479-499. drogen peroxide and virulence in Brucellae. 54. Housewright, R. D., et al. 1960. The 1959 Fort J. Pathol. Bacteriol. 89:745-747. Detrick symposium on nonspecific resistance 38. Fitzgeorge, R. B., and H. Smith. 1966. The to infection. Bacteriol. Rev. 24:1-200. chemical basis of the virulence of Brucella 55. Howie, J. W., and A. J. O'Hea. 1955. 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