That Con fel tree A) hil Lat tor I! Ecatw Mem ters Lt Bod breetin Gee / . ; | 745. oS Onn ar Ww | Essence Medial Researn. Ae pO Tenet hong, 707128 4 - . Jorg © . he Ah oo , Z re nal 4 “f 7 xp le, oy Mp Mare Laem / thm ¢ 108 hay, A ce, rar) ) “prteilin thy Up : WOME IRE -Yrary dl LARP J (Plate ty Wren tif. Maile Prin Wh, 2 OT Chir elim g te ) | 16 Whe a dare hhh fe , il Latnesr yt Cn Liddence of b : a SF A fhitre,, Cste'a Ce F fresh» SP OP = JESS Ret Tr. . “ “aes | z this, LP IE /GRE c, “ | (Leth 7s A strvtrable o hen Lamp leg TN STATE UBRARY AND ARCHIVES COR 18 ork M8 ey vering . bo Not 403 SEVENTH AVE., NORTH ‘ REPRODUCE NASHVILLE,TN 37219 _ WEEHOUT =~ b-f- 3S Bx BK EA WRITEEN PERMTESION a | WLI yp pba: cn de / be Cote he. ye oe iM Skin , Fee... a — o en Soo Cott / — oe ee aon Pr: / - OM - gj he os ee fe OUD Ene of | a ‘ “8 N co ‘ — / PS CL) PLE: aa foo : an \ Joo oe : a t : a Coe be : ’ , ole - eo : f PLS TT ye fh 7 “?, , Jp es eas 1 ” eee ee pg - . * 7 - 7, [ee wot “ / / , ae “7 ts. of “, , / - / vee e a oy . . , Pep a / f ; f } hmmm aS a : ‘ f / t . wv 1 \ / ' e 7 pele ae e . , / a ere g 7 . | ies ave ANS ARCHIVE: | PO Ni AVE. NORTH , REPRODUCB NASHVILLE.TN 37219 . WITHOUT * JRITFEN Permission TN STATE UBRARY AND ARCHIVES 403 SEVENTH AVE., NORTH * _NASHVILLE,TN 37219 MAN AND ZICROBES A Rot 1284 As the subject of my remarks this evening 1 have borrowed the title of a book called Man and liicrobes, written by Dr. Stanhope Bayne—Jones and published in 1932 by Wiliiams and Wilkins as one in a series in connection with the celebration of the Century of Progress Exposition. This book admirably describes the thriiling story of our knowledge of microbiology ana the ways in which this knowledge has influenced the life. of man and changed the courses of civilization. “icrobes are everywhere about us; in the air, in the soil anu in the waters that courses through ana surround the earth, there dwell myriads of microorganisms of diverse kinds invisible to the unaided eye of man. Happiiy, the vast majority of them are peneficent in their action, indeed indispensible to the life of higher plants, animals anu man. For example, microorgarisms fulfill usefui and i.iportant functions in agriculture and in maintaining the fertility of the soil. Working together in cooperation, each according to its kind une ebilities they perform chemical tasks essentiai in the econony of nature; decomposing glent molecules of protedh, fermenting co-aplex carbohydrates anu splitting fats into simpler forms hey salvage from dead organic metter und return into circulétion those elemental substances upon which devend the continuity ana renewal of life. Indeed, as Bayne-Jones has pointed Out, uilcroorgunisus are the original chemical pioneers of the worid. Far frou being the simplest and most primitive form of life, they wer. originally thought to be, they are now know to possess structural complexity ana biochemical organization significant or the operation of co:nmon principles everywhere in aninate nature am indicative of tne chemical unity of iife from microbes to man hinself,. With increasingknowledge of tne biochenicel ectivities of micro-~ Oorganisus, man now uses them as specific agents for the carrying out of WISATEUsRAR: AND ARCHIVE «MS DI. 2a g Re 403 SEVENTH AVE.. NORT! eee NO. 28 NASHVILLE. TN 37076 important agricultural and industrial processes in tne accouplishment of which they exceed the competence and technical skill of the most expert biochemist. The harnessing of :sicrobes in the service of incustry was forccast by Pasteur when he seid, "A cay will come, Ll an convinced, when microorganisms will be utilized in industrial processes on account of their ability to attack organic mutter". That prophesy has long since been fulfilled. Today microorganisms are used as biochemical tools in solving intricate problems in human physiology and in comparative bio~ chemistry. They are employed es selective agents in the bioassay of vitamins and amino ucids. The isolation from soil bacilli of an enzyme capable of attacking creatinine has provided biological methods for the study of protein metabolism. And such of the Lnowledge of the intermediate metabolism of carbohydrate and its significance in the understanding of muscle physiology received its initial impetus from the recognition of the nature of alcoholic fermentstion by yeast. If tine permitted, one might go on citing instance sfter instance of the manifold ways in which microorganisms have contributed to human welfare anc rewarded man in his search for truth. Although it is evident thst microbes are the loyel cllies of man, it is also true thit certain other varieties are his ruthless foes. Just as in the universe in which we live, not all hunen seings are men of good will, so in the microbial world there exists a willfull minority of microorganisms that are harmful and destructive. Some of these are antisociul and antagonistic to the growth anc life of their fellow microbes, while others are the ceusitive sgents of highly infectious diseases of man, . The phenomenon of bacterial entagonism is not new. It hes long been known that when two different species of microorganisns are grow together, one of them may elabori.te and secrete into the medium of their environment a substance which is antagonistic and often fatal to the life of the other MS. DIV. 4 - TW STATE UBRANY ANDARCHIVES 4c. no. 707128 5. 403 SEVENTH AVE., NORT- NASHVILLE. TN 37719 species. In 1877 Pasteur observed in the course of his studies on anthrax that "When a culture of the antnrax bacillus is seeded at the saae tinue with a@ comaon air microorganisa, the anthrax bacilli grow aly poorly and sconer or later die. lt is a reaarkable thing, said he, tiusat the same phenomenon is seen in the body even of those animals :zost susceptible to anthrax, leading to tne astonishing result that anthrax bacilli can be introduced in profusion into an animél which does yet not develop the disease".., He further stated, "These results perhaps justify the highest hopes for therapeutics." But it is only in recent years with further advances in knowledge of the biochemical activities of ulcroorganisas that this principle has been practically applied in the therupy of human infections. From soil bacillus bubos isolsoted the antinicrobial substances, Graaicidin and Lhyrothridin; from a conuon bread nold flening Ciscovered Penicillin; from onothcr species Waksnan derived Surcptomycin. uhese are anong the most beneficient gifts of wicrobiclogical scionce to the welfare of mankind, ‘low let us turn to the greup of microorganisms know as the pavhogenic bacteria. Among these I have chosen to speak of the pneumococci, not because I regard them cs the sost important or thit our knovleage of them is more conplete, but rather because they happen to be the group with Waich 1 am most familar ana the stucy of which hes revealed certsein facts which serve to illustrate the interdepenuence of méclicine End the other sciences of nature. you are all familiar with the fact that the large majority of all cases of bactcriczl pnousonic are caused by the pneumvcoccus, Although 8ll pneumococci grow alike in ertificicl medis, anu closely resemble each otner under the microscope they have, nevertheless, been classified into a number of sharply cefined and specific types. These type differences ere not detectable by the ordinary sethods of bacteriology, »sut require for TN SIAL umn. An ARCHIVES MS. Div. 403 SEVENTH AVE. NORTH AC. No. 707128 «4 4. NASHVILLE. TN 37296 - their demonstration the aore subtle uethods of serology and iimaunology. x The biological classification, based on these methods, was worked out <4 g and clinically applied before there wis any mnouwledge of the chemical nature of the substance upon which type specificity is now imown to depend, The recognition of the existence of pncumcscoccal types made it possible to determine the relative frequency of their occurrence in pneumonia and to recognize differences in the severity and wortalit vy of the infections they producs. A study of the distribition of the various types in health and disease has given a new interpretation of the epidemiology of pneumonia; provided biological methods for trecing the origin gad spread of infection; the mode of dissemination of the ciseuse producing tyyes in the south secretions of convalescent aic normal ancividusels, enc the cetection of the currier stete in pationts and neclthy persons. ihe kuowledge of the classification and the principles involvec vrevealeé the importance of type-specific antibodies in the mechanisn of regovery from pneumonia and ad to the development and clinical use of enti-sneunccoccal E¢rum, the oaly effective and specific form of treatuent prior to the suvent of the sulfenauides and antibiotics. In the course of these stuciies, the yuesticn naturally erese, why should pneumococci, which are cthertise so similar, exhitit such diverse ana sharp aifferences in their serological reacticas? What is the chemical basis of treir biological specificity, and what constituent or structure of the bacterial cell is responsible for these aifferences in behavicr? The answer to these guostions lies in the following facts During growth in an cptimal secdius and especially when growing in the blood and tissues of a susceptible host, pneumccocel elaborate a viscus, gua-like material which surrounds the cell body, forming = structure known és the cell capsule. This capsular substance diffuses into medium of their environnent and in soluble form retains the type Specificity of the organisms Yp P y & TN STATE UBRARY AND ARCHIVES is. py 5 403 SEVENTH AVE. NORTH Ac. po. 707128 © . NASHVILLE.TN 37219 from which it is derived. This soluble specific substance is found, not only in the cell-free, cultured filtrates, but also in the blood and urine of patients during the course of pneumococcal pneumonia. The capsular substance has been isolated from various types of pneumococcus and in aZl instances has proved to be a complex carbohydrate, a polysaccharide, But interestingly enough, the ca,sular polysaccharide from each specific type is chemically different, possessing unique properties that serve to distinguish it from that of all other types. Indeed, the capsular polysaccharides are as chemically distinct as they are serologically specific for each type. Thus, type specificity is determined by the chemical indivicuality of the particular polysaccharide present in the cell capsule, This fact is all the more striking since hitherto only proteins were thought to participate in immunity reactions. While there is no proof thet the capsular polysaccharides are primarily toxic, there is evidence that they can interfere with tne natural processes of recovery from pneumococcal pneumonia. Because of the avidity with which they combine with antibodies in the patient's blood they tend to neutralize these protective agents before they can reach tne focus of infection. Moreover, the capsular polysaccharides are known to inhibit phagocytosis, an important protective mechanism of the body whersby the white blood cells seek to remove and destroy the invauing bacteria. Like other bacteria, pneumococci, growing in an unfavorable environment, can undergo variations, resulting in changes in cellular structure and function. Under these conditions, fully encapsulated and virulent cells give rise to variants which have lost the capacity to form capsules, and with the loss of capsules the cells. also lose their type specificity and Virulence. The variants are referred to as the rough or R forms, indicating merely that the surface of the colonies they produce on solid media are rough in contrast to the smooth colonies of the encavsulated cells which ‘ATE LIBRARY AND ARCHIVES OR ATE SE MENTH AVE., NORTH MS. DIV. — 6. NASHVILLE.TN 37219 ac. wo. 707199 %4 are called smooth or S forms. The change from encapsulated S forms to non-encapsulated R variants is often reversible. In ‘any iostances these variants con be caused to regain their cavsules by animal passage or by serial transfers in 4& modium containing anti - R serum. When reversion occurs, the variant RA cells always revert back to ihe same specific type as that of the encapsulated parent culture from which they originally arose. This reversible change within a single tyve ia quite different from that which invelves the actual transformation of one specific type into snother and wholly different specific type. The phenomenon of transformation of snevmococcal tynes Kas never been observed to occur spontaneously but can be experimentally produced at will in preactermant ways depending upon the type specificity of the subostance used to induce the reaction. The phenomenon was first discovered by Griffith in 1928. A single example will illustrite the method ha originally used and serve to indicate the variety of transfornmotions possible among the known types. For instance, Griffith observed that When &@ small number of living, unencapsulated R variants derived from Type II are added to a suspension of heat killed, encapsulited Type III cells and this mixture is injected into mice, many of the ani:suals so treated died. From the blood of the mice that succumbed he recovered living, encapsulated Type III pneumococci in pure culture. This surprising result is all the more startling since the Type 1II organisms originally injected were dead cells and the living R culture vsed was wholly incapable, by itself, of causing fatal inféction. These experisents rere avequately controlled und the results have since been confirued by other investigators. These experiments were oringinally done in mice, and ell attcmats to induce the resection without the intervention of the animal were unsuccessful. Subsequently, Dawson and Sia succeeded in inducing transformation in vitro. This they accomplished by growing the veriant (R) strain in e fluid mediun TN Slane wenn... ww ARCHIVES 7. 403 SEVENTHAVE..NORTH MS. Div. NAsHEE.TN 37219 AC. NO. 10-128 ig containing enti - R serum ana kliled encapsulated (S) celiss shus they showed that transformation can be inducec in the test tube as well as in the animal becy. Later, Alloway prepcred active extracts of encapsulated pheuiaococed from which ali formed elements and cellular fragments had been renoved by Serkefeldt filtration. Active extructs containing the transforming agent in soluile form are eas effective as are the intact cells from which they were preosred. Pneumococcal extracts are crude mixtures of' complex substances com prising the proteins, polysaccharides, lipids anc other compounds of Which the bacterial cell is composed. 1 will aot burden you with details of the technicel procedures and chemical swethods used in attemsts to isolate the substance upon which the biologieul activity of tne extracts is now known to depend. lt wilt suffice xzcrely to meation that by chemical fractionetion, by the use of specific onzyaes anu serological reauctiolty it was possible to eliminate inart and irrelevant wmiterial and Pinaily to isolate, in highly purified fora, a substance possessing tne specific transforming activity of the original extracts. The data obtained by chemicel enelysis of the active substance, togetner with determinations of its physical properties oy ciectropnoresis, ultracentrifugation and ultraviolet spectroscopy indicate that it contains no demonstreble protein, lipid or serclogically reactive polysaccharide, and that it cousists chiefly, if not exclusively, of a nucleic acid of the desoxyribose type. QGuantative titrations of the biological activity of the substance isolated rroa type lil cells have shown that an usount es small us 9.00¢ HiCrograu, representing & final dilution in the reacting systeu of L pert in 600 miilion, is capable of inducing specific transformation of K cells into type III pneumococci, Further studies have revezsled certain facts which contribute to the understanding of the mechanism of the transformation reaction. For example, AND ARCHIVES. DIV. _ - TST seventh ave, NORTH “ie wo. 707128 "4 8. NASHVILLE, TN 37219 it is known that not all non-encapsulated R cells are responsive to the stimulus of the transforming suostance; sons fail to resyond while others react to minimal amounts. these differences in the physiological response © of R variants are reflected in corresponding difference in certain characteristics of the colonies they produce, This fact makes it possible» to isolate "compet nt" or R strains in pure culture. However, even chese : culturss can undergo spontanecus dissocistion, giving rise to other variants which are wholly incompetent and incapable of reacting with the transformin substance. Furthermore, it was eurly discovered that the addition of blood serum © or serous fhiids to the nutrient médium used in the test is essential, since in its absence transformation doas not occur, lt is evident then, that serun provides certain accessory factors that play an important part in the interaction of competent R cells and the active transforming principle. | Two coiponents of serum have been identified, neither of which is active by itsell, but together they function as well as does the whole serum. One of these serum components is the R antibody protein that agglutinates the growing ® organisms, forming large ogzregates of agglutinated cells: which fall to the bottom of the culture tube where they create, presumably due to thelr reducing action, environmental conditions necessary for trans= formation, The other conponent is the serum albumin which, in its native | state or in crystalline forn, appears to prepave the cells for the ready - uptake of the transforming substance, On the basis of the experimental evidence, 4{t aopears that both the physiological state of the R cells ahd the environmental factors 1 have Just cited determine the biochemical events leading to the interaction vetween the living bacteria and the transforming substance. Once this has taken place the ultimate outcome suggests that the active principle intervenes in tne metabolism-.of the R cells, giving rise to a series of TN STALE Le ee MORTN DIV. 9 er NASHILE.TN 37219 70-128 = es coordinated enzymatic reactions which culminate in the synthesis of a new capsular structure. Whatever the mechanism may be, the experimental findings clearly demonstrate that the induced alterations in cellular Structure and function are not random changes, but are predictable always corresponding in type; specificity to that of the encapsulated cells from which the transforming Substance was isolated. Once transformation has occurred, the newly-acquired characteristics are thereafter transmitted in series through innumerable * transfers in artificial media without any further addition of the transe forming agent. And from the transformed cells themselves, a substance of identical activity can &gain be recovered in amounts far in excess : of that initially used to induce the transformation. These induced changes are not temporary modifications but permanent alterations that persist, provided the cultural conditions are favorable for the continuence of capsular development. lt is evident that the inducing substance, which evokes the synthesis and determines the specificity of the capsular polysaccharide, is itself reduplicated in the daughter cells. it is also Significant that the substance inducing the reection anc in turn the substance produced in response to it are chemically distinct, each belonging to a wholly different class of chemical compounds, The accumilated evidence justifies the conclusion that the fundamental chemical unit of the transforming substance is a nucleic acid of the desoxyribose type. In view of this, nucleic acids of this type must now be regarded, not merely as Structurally important, but as functional active substances determining the bio:hemical activities and biologically specific characteristics of the cells of which they form a part. in closing, I take pleasure in acknowledging, on this occasion, my gratitude and thanks to those colleagues of mine whose expert knowledge and loyal support has made possible the work in which 1 have been me to |«67 07128 +4 10. priviledged to share. lo then 1 am indebted for an association which has : meant much to me personally, and has contributed greatly to the research | problems I have briefly reviewed this evening. And may I again express to the Cirectors of the Foundation ay deep sense of appreciation of a being enrolled as a recipient of the cilstinguisned Passano Award. TN STATE UBkAnY AND ARCHIVES , _, BONOE NASHVILLE. TN 37219 Bee WeEHOUT