128

Report of Dr. Avery (assisted by Drs. Adams, Curnen, Goebal,

Dorsfall, McCarty, rvf:zcLeod, Irlirick and Stillman)

      A study of bacterial virulence as manifested in infections pro-

ducec by pneumccocci (Avery, Mucieod, Horsfall and McCarty). The intro-

duction of a given microorganism into the tissues of a particular &nir;nLl

species initiates a series of intcrretxticjns which may progress in differ-

ent ways.  The puasite my f&i1 to survive or multiply in :he host; it

mty survive and multiply for u vtiryinc period of time;  or it mky survive and

persist throughout the life of the host. Yhc result is depondent upon

the presence or cbscnce of two interrelated characteristics; one posscsscd

by the parasite and termed virulence, the other possessed by the host and

designated as resist.Lnce.  The dqpce of virulencti of the plrssitc is neccs-

sarily defined in  term s of a particular host and similarly resistance or

its absence on the part of the host is r3foriiblo to a particular pzasits.

      Individual animals of ti single species differ somewhat among thom-

selves as to susceptibility to infection by a bxtcrial pathogen although

US gL-eater genetic homogeneity is achieved individual differences become Iess

obvious.  That there mny be muked gonetic diffcroncos in susceptibility

h&s been demonstrtLted by the selective breeding of offspring from common

pu%nts and the event&l estr:blisI-gent of rtlcas ahich show wide vcriutions

in their responses to stantid test infections.

     More marked, however, are the differences in virulence beixeen

strains of z single b&cterisl species when teated in a host species, the

individual members of which are of closely similar susceptibility.  :ilor c-

over, the morphological variants which irise from an indivic&J strain of

bacteria whether they dcvelo~ ES i; result of unknown conditions or in

rcspbnsc to experimental alterations in environment usually possess varying


. .

      The pncmococcus c:.psule is curn~~usc~ 1c:rl;el.y of pt&ysr,cchmidc,

which in each tj~pc h&s n distinct cheraia.1 structure.  kre the type spucific

c&pmlcr +lysacchi-ride wholly respmsible for the propertq of virulence,

till strrtins c;f the 8a.m type should pcissovs ititmticd virulence if the

qumtitiab cif scrly ssccliiide eli,borztec! were equcl.  It 1~s been rsps;,txQ~

iiemnstrutod tbt this is not the C&SC.  Numr~us gru~ations in virulenca

me fuu.d amng different strdns uf L sir&c type althc>ugh, $0 far ~6 is

kmwn, their cspsuL.r constituents ;,r? identicsl. Even wre striking zc

the aif'fexwxes in viruleuce ketwcen stn;ins cf 2 single type whai taste&

in m\;rtz -t.hzn one hcst species.  For tix~mple, tm stmins uf 'Q{pa III gneu-

~~tic~ccu~ t~try ~wsess r-ppruximctely dp,l virulcnco when tesiek in mice but


130

When tested. ir: rabbits one mzy Oe highly viruient and the other Tiholly

~virulent.  Similarly, T*yye XIV >neumococcus slthough it is often tssociated

idith severe pnerunoni=r in huz:z~ beings, knd my therefore SC thought of as

virulent for man, is Llmost wholly without virulence when tested in mice

tin:: i.,obits.

    In a nun~*r of inst:.nccs it is possible by repcatco rapid pas-

~,i.gc:, throu,gh L susceptible host to enhmce markkijr the vi.ru'Lcnce of en-

ctpsulatcd $neuniococci for t&is p2rticuk.r species.  There ap+nr to be

thrw possible oxplan:-fions for t!k3 phenomenon.  (1)  'The incrckse in

viruicnce mny be Co result of f!~ progra5sive kla@tion of the b2ctcrit.

to the: onvircnrwnt provii.ed by the host.  (2) The incrase in vi:ulenw

LILY be the result of the selection of intiividu:l br.ctc;ricl cells which

&JOsSef5s initiiLly the grtiztobt virulcrlce:.  (31  'i'ha incrc;~so in virulence

nay be the rssult of mutation of the D;ctcritt followed 01 the seiecticn of

calls yosbussing the grater virukzce.  Ghichuvcr of those cx+nations

may prove corract, in the cast3 of gnaumococci it should IX pointed out that
virulence can be erknced without sny dcnonstrabl& :.lcert:tion in the cap-
sulk or in the chouiclA constitution of the ctrpsular @ysrcchari&e.

    'The considerations s&ted above suggast th;t th6 property of

virulence possessed by pncumococci, although only manifest in the presence

of the intwt capsule, is also dependent upon some other ccllukr function.

As L hypothesis, subject to experimental trial, it 'SJES consitkred possible
that this second basic factor for virulence might be present, although
inapp'zent, in the non-encspsuixkd or so-ct-:llcC k cell.

    It is possible to derive non-encepsu&.ted (k) pneumococci from

cncapaukttid (S) ~311s 'oy means of 3 vurictiJ of ~zxpcrimcntal procedures.

Cne of the nost rogultrly offoctive techniquas c'kpcnds upon th?: fu.ct thl*t


131

enczpsulkted (S) pneumococci grown in the presence of homologous type spec-
ific t;ntiserum eventually become non-encapsulated (ii) and subsequently re-

main in this ~Jhilse even though grown iri the nbsence of antisera. As has

been stated, the induced i-t variants are wholly tievoid of manifest virulence
irrespective of the virulence of iha L cells from which they were derived.

    It is also possible to derive enc;psuli.tcd (S) pneumococci from
non-encapsulated (R) varitnts by means of ono or :noth!.r of sevtzsl experi-

mental techniques.  One of tix least complic?.tcd proccdurcs is dependent
upon the observation th&t when grown in the presence of :;nti ii. s;orum induced
K vzrjtmts revert to the cncapsuktcd (S) forms of the same type as th&L

of the parent cells.  EklC~i~SU.i&td CCIilS derived from in vrlrisnts in this

mznncr subsequently rcmain type specific even though grown in the absence

of 6n tiserum.  Gore important, however, is the finding tklt on rcvcrsion

from n to 2~ the latter cells manifest virulence in a tiegee comparable to

that of thu parent S cells.  Thus, when encapsulsteci Type III pneumococci
viruient both for mice and for rabbits ;se converted to the avirulent non-
encapsulated (kt) forms the latter cells still possess the virulence factor
in a latent form since upon reversion to encapsukted (S) cells unaltered

virulence for both these animal species reappears.
    Another and more critic&l approach to the problem of virulence
is provided by means of the phenomenon of controlled transformation of

pneumococcus types.  It will be recalled that non-encapsulated (R) variants
derived from one type of encapsulated (S) pneumococci can be caused to

revert to S cells of the original type.  Of much grcator significance is
tho fact that under certain special conditions non-encapsulated (H) viLriL;nts
can be seicctively transformed into cncapstiated (S) cells of any desired

type.  'This c:,n be ;Icconipiished in vitro during the 24 horn growth of a
                                    --


                                            132

singlti CULLUIY of' H ceils by r1lO:ins of the ciirwtion:A influawc of L. puri-

fied ceil-free cxtrzct obt;ined from encr.p,suLtcd (L,) calls of clny solecteci

type.  'l'hus, in the presence of a>proprikte extr&ctti, non-encspsulkted (IL)

caiis clcrived from TaFpti f pneunoccrcci ctin be c~:used to produce onczptiulttod

(L) pnowococci of 'i;rpes II, 111, 2tc.

        IL' tht l'l:ctor 1'ur viruiuncc rrcre :,ct>z;liy present t!iGwh l~ttint

in tht: non-znc,psul.;:tzd (rt) vsriats, viruknct: should ki;':in uccome msmi-

i'a:,t vrhcn th,?sc cells trc! trc-nst'urrad to cncr.psu1uL.d (:J) pncUlococci uf G

h~terologcus type.  This h:s {been found to bc the CLS~~.  Thus, t9o ztrciins

tif Type III pneuwcoccus were chusen ant; crf which :'):.s \!iru.lcnt ;ar:d the uthar

aviruL;nt fcr rr;bbits.  Non-encr.prjulz..ted (kc) v;riacj iiere obtained from

eah of these twcr str:-ins.  Then bclth H v:lrialts \/eri: transformed by clans

ci i sterile and specific cxtrr~ct prcp:2r& frc~~:; tinc::psukted Yyp'c- II yncu-

Llbcucci.  'The trto :;trAns of `Iype II pneuwcocci. thus tierived \iere then

touted for virulence in rtibbits.  The strain cf type II trrasformud frum

the rabbit &virulent 'iype III strain YES found tb be zvirulcnt for r&bbits.

Conversely, the &ruin of P~pc II sLli1::rl.y obt;ined by transformation from

the rabbit virulent '@pe III strain was eqwlly ts virulent for rabbits QS

was the parent culture from which it VW uerived.  These results lend

support tc, the view that the '&sic attribute responsible for virulence in LL

paAicul.:z host species is prastint though Ltcnt in the non-enckpsuktod

(a) cell and t&t if this fz;ctor is present in'the p&rent strain it cbn be

curried thrilugh the intsrmcdizte h form anu will resippoar when these cells

tire txnsfcrneb to encqmtiateb f'mls of L v;hdlQ different type. The

results of these uxperilaents ise grtiphickll;J presentod in T!sxt figures

1 r-n< 2.

      Althtiugh the uvikence suggests that the f:.ctw for virulence


Cellular Factor in Vixwlence

         8-
I  511
@ +


       + = Rabbit vixwlent

                -=      II    avirulent
     Fig. 1

Capsular Factor, in Virulence

411             R

   -=.     II

Fig. 2

vifwlent
avirulent


.

                                      134

resides in the K celi there is ~,dtiitior&. evidence chich indicates thr1.t the

(;lieiiiicul nkiu*e of i?,a c~~sul;z poly LiiCC1iiml`itie of b CellS m2y be Of con-

sir:erLble importance in cetermining the extent to which potential virulence

rxq be mi:nir'est .  Type XIV i,neurkococci L,re not:lbl;r Livirulent for both

rhi bbii,a and mice .

     As Sready mentioned, Type XIV pneumococci, although frequently

oncountcr~~ in hum:in infection 6:t-A thcrsf'ore evicientiji virulent :'or man, iarc

without virulence for mice.  An iit.tempt w&s made by the use of transforming

methods to oetormine whcthcr the Lck of virulence for mice is due to a

peculi;rit.jr of the cripsular polysjucch:,riic r::thcr tk:n to the :,bsonce of

that ccl1ula.r factor C.S ir, the exanrplo illust.r~,:tod in Text figure i. To

dcterminc this point, i strain of Type II pn~ULi0COCcUS `:Lghly virulent for

mice VILS chosen for study.  (See 'l'oxt figure 2). li cells o;erivtA from this

strain wore tr;nsformcd into T;-pti III, Typo XIV ::nti Yypc I.  'i'hut the fr,.c-

tor for mouse virulence WLS pres:nt in the; K cell is evident fron the fact

                                                                           ,
that the enc:lpsulLt~d strains of Type I ana Tfp;: III derived therefrom were

uquaii;r 2s virulent for mica as the p&rent strain of Type II from which

they were derived. However, when thtisc YPCW basic K cells were aused to

proriuce the Yype XIV capsular m&terial, the S cell3 thus derived wore no

longer mznifostly virulent.

for virulence is present in

self is the limiting factor

This feet shows th&t even though the.f&ctor

the cell body, the nuture of the cupsulo it-

in this puticular insknce. To prove tbt

under these conditions the cellula factor is retained !!ithin the cell body,

un K strain was derived from those amo Type XIV cells :nd trclnsformed into

Type III encapsul&ted cells vlith the roappokrancc of virulencs.

      Finr;lly, it has been possible in a single stop to derive from

Type I pneumococci of maximum virulence cells Trhich although still enczp-


135

sultrted nevcrthciess htve lost com+otely their virulence for both iiniul

zgecies.  This e~cb ..cconpiiuhod by grovring Type I cells in ii solid mditlld

in the prwence of a~:11 qtl;.ntities of sulf;*thi;.lzole.  c;clls c!crived in

this wnner ~1`;;: fuil;; tiiic+LpSjul:,tid, mor,A-,ol.ogictlky t,nd ir.mlunologicLlly

inuistin&shablc fror,l i,he parent cclis, and tiepcnding upon the expcrinttnt~l

conditions uscci m;Ly or iky not possess the; property of  ttfii sinl;ssil to this

cirug ,  Lcspitti the tjosscssion of ct.psulcs these Typo I sirzins msy be CL-

tirely &virulent.  Since the c:,p~ulkr synthesizing functkn 2nd thcreforc

the prwauction of type spucific cclpaul:.r polysaccha5de appears: to be un-

iAtcrc;d in strains dcrivcd in this mhnnc'r it SC'CLIS probable that the loss

of viruluncc is thu result of dtLrati:Jns in netbolic systems i;ithin the

ceil bail not w&ted to the czlpsti~~ s~nthcsizing system.  Scri:A sub-

cuiture 4 those strsins ha not rLsult4 in c-ny ak@7r;lenktion in virulcnco,

nor su f&r as tlit: sxperinats h.kvc pragresscd, hzs r&pic; serir-1 @~sszgc in

nurwlly susceptibic ho 3 t s incre:.sed their virulmce.  It ShotiLd be men-

tioned that the rcyetted cultivtition of enccpsuisted (LB) pncumococci in
liquid radii* containing grU&lly increasing ccnccntrztias of sulfonamiie
h&s resulted in the develc+mcnt of so-c&lied sulfonamide llfastnessn. Cells
tckptod in this mttnner to growth in the presence of mlfomkides have re-
tained both their type specificity and their virulence in umltored degree.

The results of these experiments afford additional evidence in

favor of the hypothesis that some of thabzsic frctors responsible for

virulence reside in the cell body md me unroltted to the capsular system

or the chemical structure of the t;olysucc~irl~ protiuc&.

     The avsiit;ble svicience a@,pears i;c i&ic&te thr;t thr: mmifest

virulence of enckpsuktcd pnewiococci f5r k pa-ticuiar host species is

cepentient upun kt lo;st bid tiistinct :-ni SC;ihIYtt.~ prtipcrtias Lf the


                                        136

bacterial cell.  One factor seems to reside in the cell body and its in-

fluence is not, cienlonstratiie in the absmce of a cuysuie. The other factor


Lppears to ije j.ntir~~te1.y &sbocic;ted witi1 the ca,jsuie, if incieed it iS not

actually the Wpsule itseli', znd its influence is without effect in the

t,bsence of the cellulz f&ctor.  iiith Tao intcracpendont i':.ctor*s responsiSlo

for either of the two exwemtib of virul<%ce,  th:;t is full virulence ilr~ci

tot&l tLiWetLCC 01' viruiencc:, it shoulJ IX possible thtiorctic&iJr to o:>tclin
1.0~~ c!istil;ctly Gif'ferGnt variccntr; tram `.ni t;~p,e of pm~.~~ococ~us of known

virulunct: i'or L given sPJi-ciGs.  'ih~1.c: is ovicierice indict ting thzlt this cr;.n

Lx riccorn~lishtri.  If tile c,+uikr l'tLctor is Covign,t& ;.s 3, thi: cei;.uL~.r


l';lctor :.s B irnc2 the p.~2~~1ice 3i' L.G~;J~~CI of' citkr incSc:Ltfk bJC + i:tl(: - sigs

rtispLLtivuly, :: t;blti illwtr:tinc tilr: the0rati.ci.l prX55lL~ilities era TV con-


L;tructm.  In cdch inst;inc;: till: cxivtcncc of t,hG pr&ic?cd v::riLntu bhoL'n

in 'I'LL bli: i h&s been colLiil*mk cxG.;l.inmt: II)-. CIf thti four va-i;.nt.s only
#l, the cell in which both ftctors wsrc: &Xkmstrtblc vi~'.s found to D3 viru-

Icmt.  V:Lyitints #2 ;.nrj #3, though wch W;LS shown to posscat; oniy on2 or ihc

othtir of the two fsctors, wrs r:virultinC,.

     2resenco of Ccrpsulhr tin d (;cJ:ukr Virulcnc~ F~.ctcrs in

         V;wi:.nts &rived frum T;rp3 I Pnournococ~
t ; Viruknce R:cturs 1 iwnifust  ]
V<Lriknt i     ii       B i  viruioncs  1 ExperimcntLl Corifizwition
                                                ,
                          I
    1  i
     i      +      + ;      +     Type I, s cells
     I            i
    2   I      -I-               ,     t Type I, S cells dwivui from
    1        / j$l in su~.ftithir:zole
3 i _       + :      1 i-i ccl15 mrivi:c! frcm kl in
I                       i  iati 2 swum
I                            i
   L                  j fi cells clerivoi from #2 by
\        -./ -   1 spmk,nekus dissoci~tiun

H = cc.psul:~r f<,ctzr itir virulcrtcz;  i3 z cul1uL.r fa2t.m r'a virulence


3

137

      It is hoged that additional invcstig~tion may -be. helpful in the

probiem of eiuciciating the nature of the factcrs concerned with bacterial

virulence.

       The heterophilc anticenof pneUmococcus  (Goebel and Adams).

Heterophile antigens uerived Srom Gram negative microorganisms have been

aescri';red by several investit;ators,  but the isolation i;nd identification of

such subsshnces f'rom k-am positive bacteria has hitherto not been achievtid.

L'uring tht: past two an& &. half yca:*s b;z bvc conductad un extemive in-

vtistigstion on methods of isolat,ion, ths chemical nature, bnd immunological

properties of the hcterophile anti&Ton of pneumococcus.  \5e arc now ;iblc to

report that the nature of this substance hiIs bwn fully revealed.

       Tho hctoroyiLlt; untijen of pnewococcus has ueen isolated and

cbr&cter-i- '.
      yL~ tls a lipo-csrbohjrirato intimote?ljr rolatud to the intracellu-

lar rlC" poqsaccharidc.  It c,if'fc:rs from the kttor in that it contains a

lipid bound in firm chemical union to the carbohyd;ato moiety. The comyiox

has beon isolhtcd in its entirety from autolyscd pneumococci by an intricate

series of chemical fructionations.  The substance comprises about one per
cent by weight of the bacteriai cells and from 0.5 to 0.7 gm. are obtained

from the microorganisms from 500 lit&s of bacterial culture.

      The heterophilc antigen is a water- soluble non-diffusable macro-

~loleculc constituted from tin wctyluted amino hexose, a second hexose, phos-

phoric acid 2nd i; lipid.  Solutions of the heterophilu antigen foam on

shaking.  The antigen is not precipi-kted from solution by the salts of

heavy metals, nor do such solutions give :ny of the us&i protein tests.

Lpcctrbbctigic cx&minrticn revc~ls no gross ctintt:.minE;tion of the htitcrtphile

;ntlgen with nucleic tLcid or prutcin, inciccd, when ext.mincd by olectro-

phorosis thr: iipb-carbohytir;te i;ppctirs to bc quit2 homo~onoous.  'The htiteru-


138

Ghile entigen is not tiesiroyed by dige:;tion ;;it;l l~ibocucickse, IapCn, $,eg-

8in, iryjjsin, ck~-il;otr)r&ri, or by iJi?C6piliitbbt:S cerivcd from various sources.

Cior is the hr:iigen uestroyed by lii)HWY or bJr tiie :.utolytic ferment 01% the


pneumozoccus.  'I'he buwtknce shor;s i* rel:lhrl.;:ale stabiiity to the action of

!i urd `3ii ions st i&P aetvlee:i pH i.8 tncl ,j.!j.   hutside these limits the

ljioiogicul activity of the snt.iL;en is ulo~ly uestr`oysci and this tiestruction

is ticcomwnied bjr rrn hyczolysis ot' the carbohycir::te component tind the

liber:ation of the 1ipiG constituent.  lhuu, i.t is not possibic to sever the

                ?. .

union between ~lpici ;nd csLrbohyur*lAte by zny simzie chemic?,l procedure. The

union rewins firm and the biologicz;l aztivity intact when th;: atigen is

subjoctcci to anything but clrtistic chemictl trc:.taat.

      l'he intiinnta chemicL1 rolntionshi> bctwen the pnamococcus

hcti-rophiie untigcn (F) iAnti the c ~aliultr c;rbohytirkio ("C") VJC,S ckscriocd in

the report of l;st year.  FurthGr chemiclil sttiics hzvt. furnishtid aAiit.ional

tivicience of this close rcktionship.  2tudies on the kiwztics of hydrolysis

of the LVIO subsknccs as mo~=a.uw.i by thit inci'c:;Sc of {;j reducing sugws,

b) amino (giucosaminc) nitrogen, md c) phosphoric i+cid give proof of the

iacntity of the common xroohydrate moiety.  Hydrolysis of the haterophilc

wrbohydrata with mineral ucid rwiLts in L dsstruction of the carbohydr~ts

constituent accompdeci by ;5 liberation of the lipid component.  The lbtter

h;is been isotitod and found to contain neither nitrogon nor phosphorus tnd

is probably a ftitty scid of kiigh moloculnr weight.

     The immunological proptztius of the heterophilc utigen (I?) of

pneumococcus hzive bocn extensively sttiidd ~.nd corqxred with those of the

ccliulxr (C) cmbohyaxtc.  The hetarophilc substance hcs been found to be

fuily ::ntigaic nhcn injwzted into rabbits.  The purified F lipo-c:Lrboh,ydrktc

gives rise to Lntibodios which not only firccipitztc the homologous mtigen


but lielnolyse red blood cells in the prmence of conplenent.  The i; Cui.bO-

hydrL te , on the other :znd, f2ifs to f'unction cntigenic:Jiy in r!.bbit,s.

Thus it his Leen shoxn thr:t the incbrpor;tion of c simple iipid within the

ctrbohydr;te ~~olecule endows Lt;e co~.l~lex thus forlxd aith new iu~inunolcgictl

propatii3s.  Antibotiies tivokdd bj the heterophiie (F) atigcn likewise

prtxipiLte the C cr.rboll;rrlrci.,c;, hut to 2 L:sscr ticgrec.

      Various stzins of pntaqocztcci htve been invcstil:;rtsd for thcil

contsnt of thti C i.na F pol.ysxchzides.  One B strs.in corived from Type III

pneumococcus contains fl C @ysErcchr;riGc tissontially i2anticr:l with that

obtained from the ii varisnt cjf Tyye I.  Huwvcr , the hctcrciphilc anti&tins of

the two R strtins hzvo been found to lx, quite different; thrit of the R

strain from pneuwcoccus '@pe I cvckes kicwlysins vihich :~re not neutxlizcd

by the F rintigcn r;f the III H varirnt. The ic:tter r;ntigcn, cn the other

hznd , gives rise tc hemvlysins which tlrc ncutralizcd by the F hntigcn crf

tiithtir strain.  Thus it has been riem,,nstrxtcd that tiiffcrcnt sixAn tif i!.

vcrixits irrcspxtivc tii' tjrpe GxivJr;tiljn  CL:J cat-in sntigcns, which are

choniCi<ll;r rclt~tai ;jut ir:u;~un~i~,gic*,ll~ iistinct.

In csnclusi;r,, it UL~ be +intcd \jut th;;i; the lipti-cz.rb;hyWLtt:


                                          49

(F) ;'rom pnemococcus shozs co toxic c;l'i'ects ahen injected into mice or

rabbits.  in this ;-espect the StibstLnce is quite unlike the unsiogous sub-

stances obtained from the Gram negative group oi' microorganisms which show


iI v cry high order of toxicity in 'both these species of ,rnim;ils.

      'The capstiar s;rnthesizing enzymes of pncumococcu.Goebel and

ical structurti of tile c~psultti* ~ly~;:~cch::ridc of Type III pnou;nococcus.

In the: light of this knowlcdgc we ire noX in i. position to inVestigi.te t11i:

enzymatic mcchzism ;.lhich i)ri.ngs ccbout its chtimiccl s:;nt?lusis.  Guring thr:

course of this year preliminq~ stuciies h;-vc bcun snide with tiiib end in


viovr.  The problam has ken appronchcd from;: two :.r@r~s. The chcmic;l

synthesis of certain sugar cieriv~,tivos iM.ch at: consider& GS possible

substrattis in the enzymatic system has bean undcrtakcn and the !xhsvior

of thaw dcrivhtivus in the srcscncc of the kxxtorikl onzymcs studied.

Diffcrenccs in th;;: rxtzbolism of vixious &rc:ins of pnuuaococci of the

SUE md different types cs well cs the nckbolisn of diffcrmt R va+..nts

derived thwcfron arc also being investigated.  This stu&~ ha ~lrezdy

;riulucd c;;ta on curbohyar:Ltu mctr..bolis:.r of ~xwumococci vlhich it is hopod

~,y prove: vLLEibI.2 in &Lining L:n insight into thz :ac-chanism of the enzy-

mcltic synthesis of the c~~psulrs &ysLcchErrid.zs.


f
Q

      Immme response of hunan kings tre::ted pkrenterally with the

gzyne tyrosinase (lidiims).  An xcount of the production in rabbits of pre-

cipitating antibodies to the enzyme tyrosinzse YIBS included in the report

of last year.  The serz of rabbits actively immunized with tyrosinnse were

found to prticipita te tyrosin.l-
                 GLIe qu&ntitatively from soiution.  The immune

precipitates rrere found, hov;ever, to possess s~I.1 the camlytic crctivity of

tht; tTFrosinzsc contained thsrcin
     i                              , thus demonstrtiting th:..t the antibody to

thu enzyme could prccipit~tti the latt,-r without ncutrulizing its physiolog-

iczl r-ctivity.

      The ~,rentt;r::l &i~inistrstion of Qrosinase to ,mtients (Lchroti-

der, report 1342) Wit:; esst:ntiCi L$p~rtonsion il&s made it possible to study

the ix::uno roLponse of human beings to the injection of tyrosincsc.

Sxx obtnincd from trtilltcd Ftitnts varied widely in their antibody content

some having no prccipitins for tyrosinasc.  The precipitin titers of the

human immune sex did not corrcktc with the dosage of t:yrosinase, or the

duration of treatment.  However, the pracipitating antibodies when present

reacted rfith the enzyme tyrosinase in a manner antllogous to the ret;ction of

antityrosinasc rabbit sex-c.  The enzyme activity was completely precipitated

from solution by active immune human sern, but these sera h=d no neutralizing

effect upon the catalytic activity of the enzyme.  This observation was of

importance in assaying the results of tyrosinase troatment in wpertcnsion.

Although patients did develop antibodies to tyrosinase during treatklent

these antibodies did not alter the catalytic Ectivity of the enzyme in vitro
                                                                           --

and hence should not nffcct the physiological CActivity of the same enzyme

in vivo.

      The suif;in+ide rcsisknce of strnins of nneumocticci isolated

_from patients (ICrick).  It has been rcper;tcdly demunstod that gnoume-

cocci, following prul,ngcd exposure to the suiftinnmide drugs, may xquire


increttsed resistznce to the bzcteriostntic action of these &gents.  k toch-

niauo hzi bean pravlo~til~ described for the study in vitro of the suscep-
  A                                                     --

tibility of +cumococci to the sulfonsmides.  'R-As tcchniqut; employs fresh

liver infusion madium vlhich, unlike ordinary pcptonc broth, contnins no

substance inhibitmy tir the bacteriosthtic :` ctim of the sulfonx~idti dariv:.-


tives.  This technique hils been :igplied to CA study of s'xnins of pncumo-

cocci freshly isoltitcd from +tionts.

      It ms considered quite tjossiblc that nny variations in the

sulfaxdde resistance of differunt struins i;f ~IIMJLUXXC~ might tcke plsce

through processes of ni;turLl scloction rsthor than LS the result of a&p-

t.5tion.  For this reason it sczmed importi:nt to perform the bactcriostatic

tests sn the ltirgost pmibli: s~nplo uf the bxtc?rinl populatitin in o.

cuiturc freshly isolated from the pticnt.  Attention MS p&id to this

princi.@lc both in the isolsltion of strains r^raa ~tionts t;nd in czrrying out

the in vitro bocteriost;ltic tests, vrhcre rcll<tivcly lrzge inoculrr of
    --

5,000 to 20,000 cells were cqAoyad.

      Eighty-seven strains of pncumococci have been obtnined from 57

ptitients with pnoumococcal infections, ad tested in the liver infusion

medium for scnvitivity to the bactcriostatic effect of sulfapyridine.

Thirty-tight of the strains were isolated from eticnts before the ndminis-

triition of any sulfonticlc drug.  Fmty-nine of the strains were obttiincd

t;ftcr the patient hsd received sulfonxnide theralry. The bactcriostatic

tests shovrod that there is considerublc vkriction in the resistance to

sulfatlyridine of different freshly isoltlted strains of 2neumococci.  On the

whole, however, it was clenr that the strains isolated after zdequc+te sul-

fontimide thcr;fl ware mtire resistant tv sulfz.l~ridinc thsn xere those iso-

Lted before the ~tient hzd received Culy drug trcatuent. Only ant: of the


                                              14%

38 strains collected before treatment gr*:w  readily in the special iivcr

z1ediu3 cont&iriin& conticntrLLii.ns ns great t:: lit to 20 m&L. leer cent of

suifapCrricine rrhercxs 13 out of 49, or 26 per cent of the cuAtures isoL?.ed

ilr'te1-  treatment , grew rer;diQ in tk,esa conconkxtions GI' the-: drug.

       In 2~) inL;t;ltlces, 1.~43 streins isoluix from the tmr: patient have

ncen comphrcd. In i;C cis;i;s the first culture w;~s ohtnint-d ber'ore kny

suifonmias tirug htid txcr. ci.lren ULC: thi: otlztir seven ct.Stis hLd rxxaived o~ziy

:A. 1'ew QOSUS.  The scconc str;in i'rom th2se 2'9 pkticnts LILA isolhtea aft0

t11c. c~u-:,~ 01' suif'anr;nliati tiiurtrpj hti !xun comrk2tLa.  In l'ift~on 01' the 29



CiiS3b the second strain bhow~d crc:.t.x rxsistincc to sulfCLi~yriiinw thr,n

dia the origintii cultxc.  In tWCiVl: mbthnccb,  tkxx wks no demonstrkblc

tiil'fcrtincc in tht: tv:o ~t~~*::i.ns.  in ihc 1~ci:i~ining two insknces, the swond


strt.ln u'~s r,ctuCl,I~ more suscdptiblo to sltift`i.pJV:`iuinc: th;-n VJLS the origins1

strsin.  &t,h of these USC k,cttiri;zzzic p;&tir;nts, one evcnt~Lly dying of

'b~cteri:~l endoc.xditis.

      It bus of ton bc:Ln suggtstd th::t Lhe occ;tslon::l unsiltisfkctary

response of ir p;,siont i7itii TV pn9x1ococc~L1 infaction to yropcr sulfonaiticio

tharqy iu due to rtisistanct: of the infecting struin ko thr3 sulfonamide

(il.rugs.

       An sttempt lx1.s been ~itic in 20 ripproprititc casas to correlkte
the wtientls clinical response to chwwtjier;ipjT with the aIiLfori~mic.Ae resis-    '


tance uf the infecting pneumwoccus.  The initkl strains of pneumococci

isol;teb. 'before trei;t,mcnt from six ~ticnts who lctcr shoh'ed ic poor chento-
therlLpeutic response, were no more resialxnt tu stitipyridine thkn were the

initir-I str~:ins from U. pkiients vlko subsequently responded aoll to drug

tharLpy.  On the other h:..na, the str:,ins of >neumococci isCk.t& r:fter

sulfu-na;kk tjlwxpy from +tients xhf, shiracd L pwr raspmso to the treu.t-


mei-i t were, on the whole, aeflniteiy more resisknt to suifrpyridine thiin

~e~`t: str:Lns obttined after therbpx from ;j&,ients nho showed t. gcod thert:-

peutic response.  It sbculu be noted.
                         I          , hotiJever, t1lc.t the pz,tient who 5howeci

L 1~oor rc.sporlse receivea LAX~ i.Iore of thG sulforicnide prepration tnd for iL

ionger period 01' tine.  AS it l*GSUlt, thi;se sir;.ins of pncumococci ku:d i:

grater opportunity to develop stifonarAde resistmcc thx those strt.ins

isoktsa l'ro!.~ p2t.knts who received the shorter course of trc:ltmcnt.

3

      it is well known thilt mica infcctcd rrith "drur: !':st" struins of

pneumococci respond poorly to suifoncmidu trwtncnt.  Up to this time no

"dIxl& fast" strain of pntumococcus h& been isoltted from 5~ cztie of hur;lLn

infection prior to sulfonzckiac tri;:.tmcnt.  T!'ho oci;urrenc:u of ihitiaily

r~slsknt strains of pnoumococci or of 5tIYiin5 c~,p.~ble of rl&pidly dev~luping

"f&stn:3ss" hove not so f:.r beei? isolittc;a from ~2.5~s of pneumunit-.   Con-

seqwntly, it has not ;;een prissitile to rokte thi: occ;.sion;i poor rcspons5

to thertipy cithor to initiki resist+,nce or :.cquireti f;Lstness (,I the invading

micrcurg;lnism.  Huwt`ver , thti cu~.City of the infecting strilin of pneumwi;ccus

to cievelop sulfcn~micie resisknce auring th t: course of trc~tment n:~y hi;ve

stirno influence upon the ultiwte cffectivcness of drug thcmpy.

      Further studios on tl soil bacillus ctipr:blc af destrwing wx-

aminubenzuic acid (r&rick). A suil bacillus IX-X been prwiously aescribcd

which CLL~ prtiuce.a&ptivc enzymes ctcp;ble of so modifying p&r&-minobcnzuic

ccid (hereimftcr rofcrrcd tc; as PAB) thr,t it nc, longer C'-ES il. diem rc+

;cti;zn iind is nc longer active as ,an inhibitur of the sulfon~tmide drugs.

ldtrwwtric actermin~ti~ns hzve slum tkt t&en one mdeculo uf PAB is de-

strgod by the t;ctivc bwtorial tolls, &bout 13.4 at.>ns uf tixygen are n'+

scrbod.  This is b qaxntity sufficient to ;,ccmnt for nwrly ccmpitite cxi-

tlaticn "f the c;;mpound tc; carbon dioxiae,  WL:t,cr rnc :LmmuniL.


                                     L. -. .-+3&k Jr r(r-. -
                                          L* `.   :>q&., ,,.&&&,









        .  R tee,hnique haas been developed to measure the specific enzymatic

xtl.2A~y oi b s*;;i;pchsion CL resting bacterial cells.  The number of cells as

determined by nephclometric methods, is plotted against the amount of PAB

oxidized in 30 minutes at 37%.  Within the limits studied, there is a

line;lr relationship between the number of %ctcriai cells, whatever their

activity, and the quantity of qnbstrata oxidized. This technique has been

used to investigate the phenomenon of specific activation.  The bacterial

cells, even when grown on a synthetic medium containing no P&3, possess a

certain small basal capaciQ to oxidize PAE3.  'l'hc enzyme activity may bo

greatly increased if PAB is present in the medium during growth. This

specific activity of the cells is a function of the concentration of sub-

strate p&sent during their growth.   k concentration of 1'3 mg. per cent

causes the bacterial cells to be &bout fifty times mom active thm the



oxidizing enzymes can be demonstrated in bacilli tested after they had




PAB.           ." '..,".,,









in definite activation of the cells.



stances chemically related to P.B has been tested both with respect to the     '


ability of the bacterial cells to


c

LS to the slmLi:.neous ad specific ztiv~,Licc of  these stme cells to cxi-


ciize PiEi.

       "ihe aiino grc;ap in the p2-2 position on t'ne ijenzene ring ~eerm to

be the particklkr structure  !;;hich sti:mL:ies s pecific tictivz.tion of the


enzyme sysian.  Both the Saj;i ~nci ;c~pted ceils ire equi;ily tctive in

attacking substsnces such LS benzoic acid, pr::-hyuroxybenzoic md PUS-

toluic kcids, which do not POSSOSS an uiliino group.  b:orcovcr, none of thcso

three substances uctivstc the: spc.cific enzymes c;pr;Sle of oxidizing PliB.

Pmilinu, XhiCh possesses kn aaino group, but not in thC ;prr, position, is

noi; ~tta5m.l and dots not &etizLt;! tho specific sJstcn.  Both ihc baSl!l :lnc;

wtivstk9i ccllv qu~lly ~rici rapidly split iiwt?-lattid -'LB ;nti glycyl PAB to

the free fom of PAB.

        i.iome o',hcr rclzted subtaces i.rc s1ov;l.y dustroycd oy the ?~~il-

lus when incorpo YiiteCi in thti PC~~IAI, ad they all probi!lCy pas, during the

I)rocess, throwh the form of MB itself'. 'They UY. ~11 found to :;ctiv;tc

specificclly the PAB s~uten.  These compounds tire:  pc;rt;-nitro:%:nzoic ;icid,

which other b;lcterir kve been shotjrn to reduce to PAB, the methyl ester of

PAB tind novocaine, which probably undergo initial hydrolysis, and puru-

aninophanyl acetic Gcid.

      Thi: ortho-isomer of PAE3 is rcadikJ destroyed by the growing

bacilli but, unlike thrt other amino compounds studied, does not tictivatc

the paa enzyme systems.  tin the other knd, 'bacilli so grova.wwerc found to

possess cnzymcs Directed specifically qainst the ortho compound while

maintaining only their baa1 activity qninst PAB itself.  "I&so two sys-

tcws zire quite indepenrient iinci r~r;y bc tiithcr sepriltcly ~1' 2k3.ultu.neously

mtivated.



                                       U8

nevel* `oeen kdeq-a.teiy explored.  Use CL.I; ije lktie of the soil tmilius and


cl' tiit? te~lli~i;/uj d~;ilcrit;t~d UC . Ice:ltiCji iile vtly s2E.i 1 quantities of

cilLzotiaestile ccupounds exount~red in na+,kre.
-. .


       By applicst ion of Lhese iecnt-+ues it hiis 'ueen possibie to z.c-

quire SjYide unuerstsr~cin~; of  the nslurc of iniiuced rcsisiauce to the sulfon-


tiidicc2 'Lrilgs, acquired Sy cl strciin of pnemococcus.  T!E soil. baciili, groan

in thu iiver infmion which is free of sLlfonami.de inhibitor shcw no acti-

v&tion of Lhe PfiB oxidative enLjVtie SySte:i:.  If ir straifi of Ij*pc I pnemo-

coccus is grown in tkis mdiw, a sulfonmide inhibitor a;~pars which ha

the soiu'niiitics of TAB am! :dso civcs the: di:izo rcmtion.  nlorcovor, the

s*Jlfbgyridine-fast derivr:tive of this strain of pneu!iococclS produces kt;out

tm times as wch suLfor,mide inhibitor tati dir~zotizet.bla mtcrial cs thr:

puent strai.n or about one ga.mm per 25 craI;ti of liver.  F'inalky, this

subskncu is thought Eictually to b)e Yh'3 becnus it iS rapidkJr CioStrOyed by

the soil kcilli which havt$ been spccificsll;{ r:d+tcd to oxiaize PA!! ad is

only very slowly Lttzcked 'u2r cells which h;;ve !:ot L'oen so nctimted. Further-

nore, the sulfonaiac inhibiting subsknce prociuced in iivcr infusion by

pneumococci will itself specificully activate the pza enzyme system of

the soil bcrcil1u.s.  An extrcct of pncumococcal cells yields none of this

substmce.  Therefore, the PAB wust be either synthesized by the pnemococci

or enzjjm~tically relet:sed from a bound form initklly present in the liver

medium.

      The discovery of Y&3 in animal tiss3ues is suggestive evidence

that this coiilpound my have a IAcie biologic&i significmce.  NaLritioml


studies tire contcc@ttcd in r:Lts, freeicg t,:ie diet fror;: al'1 PAI3 by use of

tho soil bzciiius in rmch the s:aae manner          . ?.
                                   tlut :iv~am hr<s t~:de wssible the

stud;r of the S;W~~~GIIW in ri.ts C~UC to u tic;'icicnc;~ of biotin in their diet.


      '~hc presence of 2 pOlyS~LCCh.Gride in the blocd during acute infec-
                                                                       --

e 5'rj;.zs (durhe:: and I,;l:ick) .  EECi.j* in Ll,is c2ntiCy seversl observers rcgwteti

that growth of gneuzococci in the serh of patients viith pneur~onih rem13

i,i-iz scr:. of Fiicnts with tt variot.y of infcctiouv diL;mszS ;s ~~11 LS in

stzi from cbses of ncutc rkUJ;titic fcviir, rxphritis 2nd urexiix.  He con-

cludod that "there is......some substance vkich makes its appcamnce in the:

blood strum under certain conditions and from which the j~~m.mococcu~ is

cepable of forming 1t;rgc quantitiw of r;cid."  Ho indicated that the sub-

stance is not glucose, but implied that it might be carbohydrate in nature.

In 1339, Friedemann and Sutliff demonstrated quantittLtivcly that the in-

creased ticid formation is more than can bc sccountcd for on the basis of

the free glucose present and concluded that "the phenomenon is due to the

presence of abnorntri qumtitios of 9 @ljrs&cch:&de which supports rapid

growth anti which is rwdily l*eri;lcnl;cd by the pnoumococcus."

      These obscrv;tions suggcstod th:;t pnoumococci possess an ax;;me

c~~ptble of uttwking the ~aticuicr poiys;.cchr.rido present in blood tiurir,g

ini'ection.  &2yzr, Dubos, r:ncZ Smayth hr,w isolatuci from pnwwcocci an enzyme


153

which specifically decomposes hyaluronide, L'. polyszccharide found in the

tissTacj of iii;;:, o_~*~;i*~;S :4yc ir, certz;ifl :T;;LCi;iA.i,%l ;;&7s.  Consequently, a

purified preparu.tion of byaluronidase Otis  iaol ~teci from pneumococci by the

method of Meyer and its action tested in normal and 'pneumonic' serum.  The

enzyme Freserved in dry stale as the protein flavi:nzte WLS found to be

highly active in dcpolymcrizing authentic samples of waluronidc of both

human Lind streptococcii origin.  The specificity of the enzyme prop:iration

used in the present study is shown by the f,-.ct th;t it does not ::ttLck any

other of ths polysaccharides thus fzr tcated includkg those of pneiunococcnl

origin.

      The enzyme wt!s used exptzrimcntailjr in the follov!ing manner. The

reducing vr.A~s of deprotcinized scruw fi!. I
                                  t ztes wore determined by the

method of Hag:ldorn and Jensen.  The results wore cxprcsstid :.s mg. per cent

of reducing substance calculated as gLucose.  Any increase in the reducing

vsluo zs conparcd with th,zt of the control KLS considered as due to

activitjj of the hyaluronidcse upon the serum polysaccharidc.  Ii simiiar

method WGS employod for the detection of' tho pclysaccheridc in chest fluid

and in urine.
     w

      The results obtained thus far indiccte tti:t e polyszccharidc

susceptible to hydrolysis by pneumococccl hyaluronidase is present in the

scra of patients with pneumonia.  The reducing value after snzymatic

hydrolysis of sera obtained from pneuntonia cases tended to reach a maximum

during the acute stage of illness and to decline to lower levels thereafter.

The serum of individual patients, hwevcr,  showed considerable differences

in polysaccharide content.  Too few normA xtirc h~vo been tested thus far to

establish tho normal range and limiting vailucs in huc.lthy individuals.

      The polys:icci;ari& hau bzcn detcctwi by a similar method in the


                                              151

urine of &bout one-third of the pneumoniri pktiente tested.  Correlation of

tiii: poQ;aick.ri~~ values iii ~cr;ili? xd urine obL:intid from patients on the
si'ric achy indicx,tc that when the serum values i~rc high, the urine vAuus
  -1



i.ro lov, vrhcrti:t;L; when the urine vzluc: s are high little cr no polysacckride

ctn bc detxtcd in tht: scrur~.

      A f2w preliminr-;ry invc;tig2tions in animals h&ve; revealed high

l,~vels of poly:;ucch:Lridc in the; sex of normal rkibbits.  In this connection

it is of interest to recall the originul observntion of Dr. Longcopc that

the substanct! from which pncumococci produce acid during growth was not

dctectablt in !lorsc and tuef serum but was present in c:;lf itnd rabbit serum.

       The chemical naturt; of the serum ~olys~ccharide is indicated by

the following cvidcnce.  In the: present study, it has Seen found that when
i highly specific prepcrrcltion of pneumococcr.1 hysluronidaso is &llowcd to
:,ct on scrulll containing tha polysaccharide reducing substnncos 3x-e liberated

Meyer and his associates hLve shoxn that this enzyme: hydrolyzxs hysluronic
acid into its components, glucuronic ocicl :ind acetyl glucosamina, both of

which ;Lri: reducing substances.  In the prf2sent study the color reaction for

acctyl glucosamine is invariably positive in sera treatod with active enzyme
and negative in the corresponding controls. Finally, it has been shown that
yeast which specifically destroys any free glucose present in the serum has
no effect upon pure ucetyl glucosamine or glucuronic acid nor upon the split
products of the serum polysuccharide after hydrolysis b,, pneumococcrl

hyaluronidase.  These findings strongly support the vie= that polysacchariCe
present in abnormal q~tntitics during acute disease is either hyczluronic

acid or a closely rolatcd chemical substance.  The acidity noted by earlier
invcstigztors in serum during growth of pneumococci prokbly arises frcm
the primury hydrolysis Gf the polysticchariic bar the bactcrixl hph.IronidaSe


and the subsequent formation of lactic acid by the bacteria from the glu-


col;;u,,;r*G tiibs 1. :-
          AV`Li ated.

     Factors effecting reversion of R to S pneumococci in mice (Still-

;:lan) .  It has been suggested that pneumococcus infection may be initiated by

the degraded R forms of pneumococci changing in the body into virulent

smooth organisms which are associated with pneumonia.  ks alcohol is known

to render mice more susceptible to invasion b-; pneumococci, the effect of

injecting the &virulent Ir pneumococci into intoxicated r:lice was tried.

In the case of the h variants derived from Types I and II pncumococci no

evidence of reversion to tht: corresponding encapsulated cells was observed

to occur under these experimental conditions.  But in the cast of an R

strain derived from Type III tneumococci, virlllent cultures of the homolo-

gous type were rccovcred following the injection of degraded avirulent H

forms into intoxicated mice.  The smooth forms were recovarcd from mice

2 - 5 days after inoculation.

      In view of the incidence of respiratory

with the presence of H. influenzae as a primnAry or

infections sssociated

secondary invader, an

intensive study of this organism has been undertaken.  Serum is being pre-

pared for the 6 specific types so far differentiated so as to be able to

classify the various strains for epidemiological studies immediately after

recovery.

AdtLms, M. H., Heevcs, H. E.,
                       _ -

and Goebel, W. F., The synthesis of 2,4-

dimet~l-B-netiusl-elu~osidc, J. Biol. Chern., 1941, l&l, 653.

Publications

Curncn, E. C., and tiaclsad, C. M., The effect of sulffipyridine upon the
       development of immunity to pneumococcus in mbbits, J. EXP.
       Med., 194.2, ?J, 77.


Horsfall, I'. L. Jr., Recent studies in influenza, km. J. Public Heaith, 1541,
         2, 2.275.

heeves, fi. E., ond Soebel, E. F., Chemoirmunofogical studies on thz soluble
          specific substtince of pneumococcus. V.  The structure of the
         Type III polymccharide, J. Biol. them., 1941, 139, Sli.

Ltiliklan, E. ct., k. comparison of antibody production by rabbits following in-
        jection of pneunoco:cus vaccines heated at 60o C. or mto-
          claved,  J. Imnunol., 1341, LJ, 343.

Stillman, E. G., The preservation of' pneumococcus I${ freeztilg end dying,
        J. &xt., 1941, 42, 689.