[Reprinted from THE JOUILUAL OF EWERIXEKTAL MEIWXNE, October 1, 1959,
          Vol. 110, No. 4, pp. 571~5841
                Printed in U.S.A.

SIMULTANEOUS PRODUCTION OF TWO CAPSULAR POLYSAC-
         CHARIDES BY PNEUMOCOCCUS

I. PROPERTIES OF A PKETJMOCOCCVS MAMFESTING BIKARY
            CAPSUL.4TION*

Blr ROBERT AUSTRIAX, M.D., Ah3 HARRIET P. BERNHEIMER, PH.D.

(From the Departmest of Medicine, Slale Uwiversity of New York College oj Medicine

ot New York City, Brooklyn)

PLATES 32 TO 35

(Received for publication, June `4, 1959)

The concept that a pneumococcal cell may produce more than one capsular
antigen is not new. The possible occurrence of the phenomenon was considered
as early as 1928 by Griffith (l), who described, in his original report of the
transformation reaction, a strain of pneumococcus iagglutinated specifically by
antisera to two different pneumococcal types. In the same year, the serologic
cross-reactivity between pneumococcus Type III ,snd a strain classified later
as pneumococcus Type VIII was recognized (2, 3). It was shown subsequently
by Goebel (4), however, that the immunologic relationship between these two
capsular types resulted from the presence in their respective capsular poly-
saccharides of a common chemical subgroup rather than from the production
by each type of several discrete capsular antigens, one of which was common
to both. Similar immunologic cross-reactivity has been demonstrated among
a number of other pneumococcal types (j), but in no instance has it been shown
conclusively that an organism of this species produces more than one molecular
type of capsular polysaccharide.

In 1953, Leidy, Hahn, and Alexander (6) reported on the production in vitro
of new types of HemophilzGs irzfluensae obtained by transforming capsulated or
non-capsulated organisms of this species with deoxyribonucleates (DNA)
derived from bacterial cells of heterologous capsu'.ar type. Organisms reacting
with each of two unitypic anticapsular sera were isolated and, although deiin-
itive experiments are not described, it is possible that these cells were pro-
ducing capsular polysaccharides of two molecular species. Analogous experi-
ments performed with pneumococcus have led to the isolation of a variety of
phenotypes manifesting binary capsulation. In th:ls and in subsequent reports,
some of their biological, biochemical, and genetic properties will be set forth.

* This investigation x-as supported in part by research grant E-1018(C2) from the In-
stitute of Allergy and Infectious Diseases, Cnited States Public Health Service.
                        571


572          PRODUCTlOX OF C.kPSULAR POLTS.iCCR.iRIDES. I

Materials and Methods

Xonzenclalwe oj Pnezr~r~ococcal l'arianls.--In certain of the pneumococcal strains to be
described, there is lack of complete correspondence between phenotype and genotype. For
this reason, complexities arise in the description of these strains. For the present, only pheno-
typic designations will be employed and terms descriptive of genotype will be introduced
later.

Fully capsulated phenotypes are indicated by the letter S followed by a Roman numeral
designating capsular type; e.~., SIII is a capsulated strain of pneumococcus Type III. Be-
cause of certain biochemical relationships to be described subsequently and for convenience,
non-capsulated variants and intermediate capsular variants agglutinable by antibody to
somatic antigens, including those described by Taylor (7) under the term SIII-1, will be
designated by the expression S-x,, the subscript designating the capsular type from which the
mutant strain was derived and the strain's designation; e.g., S-rrr, is Strain 4 of a non-capsu-
lated phenotype derived from pneumococcus Type III. Phentotypes manilesting binary
capsulation will be indicated by the letter S followed by the two Roman numerals descriptive       .
of the strain's capsular components separated by a hyphen; e.g., SI-III is a pneumococcus
characterized by the production of both Type I and Type III capsular polysaccharides.
S1rair.s of Pneztmococcus.-SI phenotypes-SVI, ID, 141S, IP: strains of the SI genotype
isolated originally from human sources and carried in the laboratory for several years.
SIII phenotype-111466: a strain of the SIII genotype carried in the laboratory for many
years.

S-r11 phenotypes- S-rrr., S-rrrl: strains of a non-capsulated variant of pneumococcus
Type II (R36.1) which carry different mutated Type III genomes acquired by transformation
with DNA from variants of Strain IIIA66.

S-rrrl: the same non-capsulated mutant of pneumococcus Type II transformed with
DN.4 of a variant of the Type III Strain SV3. (The three preceding strains are Ephrussi-
Taylor's SIII-1, SIII-lb, and SIII-lc (8) and were made available to us by her and by Dr.
Arnold Ravin.)
S-rrrI: a non-capsulated variant of strain IIIA66.
Preparation of DNA (Transforning Principles) and Techniqzbe of Transformation Re-
acfionJ.--The methods used were those described by MacLeod and Krauss (9).
Preparation of Anticapszdaar Sera.-Vaccines of unitypic pneumococci and of pneumo-
cocci producing two capsular polysaccharides were prepared from cultures grown at 37oC.
for 12 to 16 hours. After standing for 1 hour at room temperature following the addition of
neutralized formalin to a final concentration of 1 per cent, the organisms were collected
by centrifugation and resuspended in a volume of normal saline solution one-tenth that of the
original culture. The suspension was heated at 63oC. for 30 minutes and stored at 4'C.
Rabbits were immunized by the injection intravenously of 1 cc. of vaccine daily for 5 days
and were bled on the 10th day following each course of vaccine.
Precipilin Tests.-Precipitin tests were carried out in small test tubes. 0.2 cc. of each of
several serial dilutions of antigen was layered over 0.2 cc. of serum. The tubes were incubated
for 1 hour at 37oC. and the reactions read after the tubes had stood overnight at 4oC.
Precipitin reactions in agar gels were carried out according to the technique of Ouchterlony
as modified by Halbert (10).

I'irzllewe and Prelection T&$.-Tests of virulence were performed by the injection intra-
peritoneally into mice of the CFCV strain of 1 cc. of an appropriate dilution of a blood broth
culture of the strain to be tested which had been incubated for 16 hours. Counts of viable bac-
terial units were obtained by dilution and plating. In protection tests, mice were injected
intraperitoneaily with 1 cc. of the serum to be tested 24 hours before being infected with
pneumococci as in tests of vixlence. Control animals received normal rabbit serum. All tests


               R. AUSTRIAN ASD H. P. BERNHEIMER          573

were continued for 10 days and results were scored in terms of survival or death, The cause
of death was determined by culture of the animal's blood at autopsy. In experiments in which
the enzyme which hydrolyzes specifically Type III capsular polysaccharide' was employed,
2 mg. of enzyme were injected intraperitoneally.
Tests of Phagocytosis by Human Polymorphonzdear Leukocytes.--The technique employed
was one described previously (11). When the enzyme hydrolyzing Type III polysaccharide
was used, it was present in a final concentration of 2 mg./cc.

EXPERIMENTAL

Phemtypes Manifesting Binary Capsdation.-When cells of pneumococcal
Strain S-r1r, are transformed in the presence of DNA from any of the Type
I pneumococcal strains listed above, organisms of two different phenotypes
can be recovered: cells of the SI phenotype and cells of the SI-III phenotype.

TABLE I

Relationships of Pneumococcal Strains Participating in Transjormafion Reactions Citing Rise
         to Variants Producing F&o Capsular Polysaccharides

Original capsular ty  of strain
manifesting the SI-II!?phenotype
  followrng transformation

SI

SII

SIII

SV

_-

Parent Type III strain of variant
   with S-III genome

IIIA66

IIIA66
IIISV3

IIIA66

III.466

.-

-

Source of SI genome

SVI

SVI, ID, 141S, IP
WI, ID

SVI, ID, 141S, IP

ID

In similar fashion, 5X-111 strains may be obtained by transforming cells of
lines S-I~I~, S-Irr, and S-IIIJ. From these experiments, it can be seen that
mutant Type III cells, the capsular genomes of which were derived originally
from two different Type III strains, IIIA66 and IIISV3, can be transformed
to the S-111 phenotype with DNA from several SI strains. In addition, it is
evident that non-capsulated cells derived originally from capsulated strains of
different capsular types, i.e. Type II and Type III, may manifest binary capsu-
lation when endowed with the proper genetic factors. It has been possible also
to transform non-capsulated cells derived from Type I and from Type V pneu-
mococci to the SI-III phenotype. The phenomenon of binary capsulation,
therefore, may be expressed by cells derived from a variety of capsular types
when they are endowed with the appropriate genetic factors derived from any
of several Type I and mutant Type III strains (Table I).

1 The enzyme preparation x-as made available through the generosity of Dr. Alan W.
Bemheimer, Department of Microbiology, New York University College of Medicine.


574        PRODUCTION OF CAPSULAR POLYS.ACCH.4RIDES. I

SI-III cells were observed first in experiments in which strain S-rIr4 had
been exposed to DNA from a capsulated Type I strain. In quellung prepara-
tions,2 it was noted that the transformed cells showed capsular swelling when
exposed either to Type I or to Type III antiserum. The size of the refractile
capsular halo in Type III antiserum was approximately that seen about cells
of the normally capsulated Type III phenotype but the refractile zone about
cells in Type I antiserum was distinctly smaller than that about capsulated
Type I cells and at times could be seen only with difficulty. Variation in the
size of the two capsular components in relation to the phase of bacterial growth
was not observed. When the cells manifesting binary capsulation were exposed
to a mixture of Type I and Type III antisera, the capsular swelling was not
recognizably greater than that which followed contact with Type III antiserum
alone. In smears stained by the Gram technique, such cells showed no consistent
variation in size or in shape which permitted their distinction from singly
capsulated variants. SI-III cells isolated from the peritoneal cavity of the white
mouse did show elongated forms often in short chains, morphologic charac-
teristics not manifested by the organism when grown in vitro.

.I

On solid medium, SI-III cells give rise to clones morphologically indis-
tinguishable from those formed by pneumococcus Type III, a not surprising
finding in view of the significant amount of Type III polysaccharide formed
by the SI-III cell.

Eridence Establishing the Existence of the S-III Cell.-The observation that
more than 90 per cent of cells from clones of the SI-III phenotype gave a
positive quellung reaction in both Type I and Type III anticapsular serum
provided strong presumptive evidence for the existence of a cell producing
two capsular polysaccharides or one so constituted chemically as to possess
the serologically reactive groups of Type I and of Type III polysaccharide.

To distinguish between cells manifesting binary capsulation and mixtures of
SI and SIII cells, several types of experiment were performed. Table II shows
the results of growing SI, SIII, mixtures of SI and SIII cells and SI-III cells
in broth containing either Type I or Type III anticapsular serum. Only those
cells producing simultaneously two capsular polysaccharides are agglutinated
by both unitypic antisera and yield cultures with limpid supernatant fluids in
the presence of each antiserum in so called "thread tests."

Similar evidence distinguishing cells with binary capsules from unitypic cells
has been obtained from mouse protection tests. The evidence in Table III
indicates that all animals infected with cells with binary capsules were protected
by either unitypic capsular antiserum whereas none was protected by either

2 Capsular typing sera were made available through the courtesy of Dr. H. D. Piersma,
Lederle Laboratories Division, American Cyanamid Company, and Dr. E. F. Roberts, Wyeth
Laboratories, American Home Products Corporation.


R. AUSTRIAN ASD H. P. BERKHEIMER           575

antiserum when infection was carried out with mixtures of Type I and Type
III cells.

Evidence for the Production of Two Capsular Polysaccharides by Z-III Cells.-
SI-III cells grown by continuous passage in broth containing either Type I or
Type III anticapsular serum give rise to mutant cells producing in amounts
detectable by the quellung reaction only Type III or Type I capsular poly-
saccharide. These mutants derived from SI-III cells suggest that separately
variable factors controlling two capsular components are present in the SI-III
cell but do not exclude the possibility that each mutant is associated with a
qualitative variation in the composition of one of two polysaccharides.

A second type of evidence is derived from the exposure of SI-III cells to the
enzyme which hydrolyzes Type III capsular polysaccharide. After such treat-

                    TABLE II
.4ppearance of Supernatazt Fluid of Pneumococcal Cullzcres Containing Unitypi
             Anlicapsdar Serum

Pneumococcal type or types in culture

         Serum

An ti-Sl              Anti-S111

       SI                       L                  T
       SIII                     T                 L
      SI + 911                T                 T
       SI-III                    L                 L
      SI-III + SI               L                 T
      SI-III + SIII               T                 L

Medium: neopeptone broth plus 10 per cent anticapsular rabbit serum; L = limpid
supematant fluid. T = turbid supernatant fluid.

ment, SI-III cells are no longer agglutinable by Type III anticapsular serum
and they fail to give a quellung reaction with Type III antibody. Their aggluti-
nability by Type I anticapsular serum remains unimpaired, however, and the
treated cells give still a positive quellung reaction with Type I antibody. It
has been shown that the action of the enzyme hydrolyzing Type III pneumo-
coccal polysaccharide is more highly specific than antibody to Type III poly-
saccharide. In addition, the action of this enzyme has been demonstrated
recently to be that of an endoenzyme rather than that of an exoenzyme attack-
ing the end-groups of a macromolecule (12). The experiments suggest strongly,
therefore, that the SI-III cell produces normal Type III polysaccharide or a
substance resembling it very closely and one that is probably distinct from the
Type I component of the capsule.

In precipitin reactions carried out in an agar gel by the modified technique
of Ouchterlony (lo), the precipitates formed when Type III anticapsular


576          PRODUCTIOS OF C.APSUL.kR POLYS.XCCH.\RIDES. I

antibody is allowed to react in the same plate with SI-III cells, SIII cells and
Type III polysaccharide give rise to a line of continuity (Fig. 1); and, in like
manner, a line of continuity is produced when the reactants are Type I anti-
capsular antibody, SI-III cells and Type I polysaccharide (Fig. 2).
Precipitin tests performed with partially purified polysaccharides from
SI-III cells show that the Type III polysaccharide can be precipitated by
Type III anticapsular serum without significant removal of the Type I com-

TABLE III

Proleclicc Ejecl of Type Z and Type ZZZ Antiserum in Mice Znfected with SZ-IZZ and with
             Mixtures of SZ and SIII Pnexmococci

strain

SI-III

Amount of culture injected

Serum injected

cc.
10-E
10-s
10-s
10-G
10-7
10-s

Anti-S1
Anti-SD1
Kormal
None
None
Iione

D/S'

Viable units/cc. (undiluted culture): 4 X 10s

  Strains     Amount of culture injected     Serum injected          D/S'

                 cc.
SI + SIX1        10-s            Anti-S1          6/O
             10-C            Anti-S111         6/O
             10-s            Normal          6/O
             10-s            X'one           6/O
             lo-'            Kane           6/O
             10-8            None            4/z

Viable units/cc. (undiluted culture) : 3 X 108 (equal numbers of SI + SIII)
* D/S = died/survived.

ponent of the capsule and conversely, specific precipitation of the Type I
capsular component is not accompanied by removal of the Type III component
from solution.

A 200 cc. culture of SI-III cells was grown overnight afte: which time glucose was added
to a final concentration of 1 per cent. During an additional 6 hours' incubation, the lactic
acid formed was neutralized by the addition of 3 N NaOH. The cells were then removed by
centrifugation and 1 volume of 93 per cent ethyl alcohol was added to the separated super-
natant fluid. The suspension was stored overnight at 4oC. after which time, the precipitate
formed was collected by centrifugation and dissolved in 10 cc. of normal salt solution. This
solution served as the unfractionated capsular antigen. Aliquots thereof were allowed to


              R. XUSTRIXN AXD H. P. BERNHEIXER           577

react with appropriate amounts of Type I or Type III anticapsular rabbit serum or with
normal rabbit serum. All volumes xere adjusted to give the same quantity of the original
antigen in precipitin tests. The results are shown in Table IV.

On the bases of the independent variability of its capsular components, of
the susceptibility of the Type III capsular component to the enzyme hydro-

TABLE IV

Independent Precipitabilily oj Each Capsular Contponerzt of the SI-III Phenolype by Unilypic

Serum

Anti-S1
Anti-S111
Normal

Serum

-

_-

Anti-S1
Anti-S111
Normal

-

!-

-.

--

Serum

Anti-S1
.4nti-SIII
Piormal

-

Polysaccharide precipitated after treatment with normal rabbit serum

                 Dilution of antigen
1:32 1 1:6+l  1:128 13256 1:512'1:1024'1:20481:40961:8192' C
     pp____-__p--
+++ ++ ++ + + 0 0 0 0 0
t+++ +++ ++ ++ + + , + f 0 0
0,o o/o
Polysaccharide precipitated after treatment with type I anticapsular rabbit serum

Dilution of antigen

1~32 1:6-l
    m--

It     0
t+++/ +++
O I0

I.

-

-
!

1:128  1:256  1:512 1:102~1:20481:4096~1:8192  C
__- -p__---

0    0    0    0 0 0 0    0
t++  ++   +   + f 0 0    0

Polysaccharide precipitated after treatment with type III anticapsular rabbit ~erurn

Dilution of antigen

1:32  '  1:64  1:128! 1:2561 1:512 1:1024'1:2048 1:40961:8192  C

          -pp-----
++    +    +    f    0
0      0     0     0     0  0 ;
                           0    ; ;  ;
0

lyzing Type III pneumococcal polysaccharide, of the serologic cross-reactivity
of its cells with Type I and Type III polysacchsrides in precipitin tests in agar
gels, and of the independent precipitability of its two capsular components,
SI-III cells may be considered to produce two capsular polysaccharides.

Phagocyiosis of SI-III Cells by Human Polymorphonuckar Leukocytes.-In
Table V are shown the results of exposing SI-III cells to human polymorpho-


578        PRODUCTIOK OF C-\PSUL:lR POLTSlCCHARIDES. I

nuclear leukocytes in a smooth-walled system. Several facts of interest are
demonstrable in these experiments. First, the persistence of the antiphagocytic
effect of the Type III capsular component is demonstrable even in the presence
of Type I anticapsular serum. That the Type I antiserum is fully effective is
shown in the last line of the table. Second, the relatively small antiphagocytic
effect of the Type I capsular component becomes apparent when the SI-III
cells are exposed to the enzyme depolymerizing Type III polysaccharide to-
gether with normal rabbit serum. The efficacy of Type I antibody is definitely
demonstrable in the presence of the same enzyme, however, by a further in-
crease in phagocytosis.

The Relative Importance of the Two Capsdar Components to the Virulence of
5'1-111 Pnezinzococci.-Table VI shows the effect of several reagents upon the

TABLE V

Phagocytosis of SI-III Pnezcntococci by Hwnan Polymorpkonuclear Leukocytes in the Presence
    of Various Sera and of the Enzyme Hydrolysing Type III Polysaccharide

Strain

SI-III

SI

Anti-S1
Anti-S111
Normal
Anti-S1
Anti-S111
Normal

Anti-S1

Per cent PMN con- Average No. of
thing pneumococci, mococci per P IX
            gneu-

Absent
  "
  "

Present
  "
  "

Absent

     8         0.36
    100        10.0
     4         0.05
    96         8.2
    96         8.0
     32         0.92
/     85  I     8.0

outcome of infection with SI-III cells in mice. It may be seen that each capsular
polysaccharide contributes to the virulence of the infecting organism. Of
interest is the role played by the Type I capsular component. Although the
number of animals infected is small, it may be noted that the only animal
infected with pneumococci manifesting binary capsulation and not receiving
antiserum to survive was one treated with the enzyme depolymerizing Type
III polysaccharide. Not shown in the table but noteworthy also is the fact
that animals treated with a single injection of enzyme at the time of infection
survived twice as long after inoculation as did untreated controls. Similar
results have been observed in analogous experiments performed with cells of
the binary capsular SIII-V phenotype. These observations are in accord with
those derived from the study of phagocytosis of these strains. They suggest
that the smaller Type I capsular component is able to protect the population
of SI-III cells from complete elimination during the period that the enzyme is
still hydrolyzing effectively the Type III capsular component but is unable by


R. AUSTRIAK XX0 H. P. BERSHEILIER           579

itself to shift the balance of infection decisively in favor of the pneumococcus.
Later, when the enzyme is no longer effective, regeneration of the Type III
capsular component provides more effective protection against phagocytosis
and permits rapid progression of the infection to a fatal outcome.

Properties of Anfiserum Prepared zailh Vaccines of SI-III Pneumococci.--
Rabbits immunized with vaccines of pneumococci manifesting binary capsu-
lation form antibodies to each of the capsular components of such cells. The
relative amount of antibody to each of the capsular antigens may vary during
the course of immunization. Antisera to SI-III cells cause capsular swelling of
SI, SIII, SI-III, and SIII-V cells. Shown in Table VII is the protective effect

TABLE VI

E.ject of the Enzyme Hydrolyzing Type III Polysaccharide and of Unitypic Antisera otl the

Strain

SI-III

SI

SIII

-

--

-

Amount of culture
  injected

cc.
10-G
10-6
10-6
lo-6
10-e
10-1

lo-6

10-G
10-a

Serum injected

None
Anti-S111
Anti-S111
Anti-S1
None
"

`<

Anti-WI

-

.-

-

Enzyme injected

D/S

me.
2
2
None
"
"
"

3/l
014
o/4
o/4
3/o
2/o

2        4/o

2        o/4
None       o/4

\kble units/cc. (undiluted culture): SI-III = 2 X 108; SI = 2 X iv; SIII = 3 X 108.

of such antiserum in mice infected with cells of each of the aforementioned
phenotypes and with mixtures of SI and SIII cells. Recorded also in the table is
the absence of protection against infection with pneumococcus Type V. Ana-
logous results are observed in experiments concerned with the phagocytosis of
pneumococci of several capsular types in the presence of Type I-III antiserum
(Table 1'111).

Precipitin tests in an agar gel (Fig. 3) disclose several facts. First, with Type
I cells and Type I capsular polysaccharide, the anti-SI-III serum forms a
continuous band of precipitate, and with Type III cells and Type III capsular
polysaccharide a second and distinct band is formed. Second, these lines of
precipitate are continuous with those formed by the antibody reacting with
each of the capsular components of the SI-III cell. Third, there is no band of
precipitate formed to indicate the presence of a single species of antibody


Strain or strains

SI-III
SI
SIII
SI + SIII
SIII-v
SV
SIII + sv

SI-III
SI
SIII
SI + SIII
SIII-V
sv
SIII + sv

SI-III
SI
SIII
SI + SIII
SIII-v
sv
SIII + sv

Amount of culture
  injected

CC.
10-e
10-e
10-G
10-e
10-G
10-C
IO-6

10-G
10-e
10-C
lo-6
10-C
lo-6
10-C

10-7
lo-'
lo-'
lo-'
lo-'
lo-'
lo-'

Serum injected

.-lnti-SI-III
   `I
   L`
   ii
   "
   "
   1`

Normal
  `I
  `I

  8,
  6`
  l`
  C`

&Cone
1`
`<
"
`i
"
"

D/S

O/j
O/j
O/j
O/j
O/j
5/O
5/O

Viable units/cc. (undiluted culture): SI-III = 5 X 10'; SI = 3 X 10'; SIII = 2 X 10';
SI + SIII = 2 x 108; SIII-v = 2 x 10s; sv = 2 x 106; SIII + sv = 2 x 103.

                      TABLE VIII
Phagocytosis of Pneunmocci of Sewal Types by fIt~nza~z Polyvzorphonuclear Leukocytes i~z tire
         Presence of Type I-III Anticapsdar Rabbit Serum

Strain

SI-III
SI
SIII
SIII-v
sv

s-111
SI
SIII
SIII-v
sv

,-

Serum

Anti-SI-11
   "
   "
   "
   "

?iormal
  L`
  "
  "
  "

:I

I
I-

PMIN COD taining
pneumococci

per cc-n1
8-l
60
92
72
  0

1

Average No.`of
2neumococci per!PJiN

8.6
9.4
8.1
7.4
0                   .

0
0
0                   i
0
0

580


R. ATJSTRLlS AND H. P. BERNHEIXER           581

reacting with both capsular components or the presence of a capsular com-
ponent reacting with antibody in a fashion different from that of unitypic
cells. The results are consistent with those presented earlier which indicate
that cells manifesting binary capsulation produce tmo species of capsular
polysaccharide each of which is closely similar if not identical with that of the
corresponding unitypic pneumococcus.

In precipitin tests performed in tubes, Type I polysaccharide and Type III
polysaccharide are each precipitated by antiserum prepared with a vaccine of

                    TABLE IX
Independed Precipilabilily of Capsular Antibodies from an Antiserum Prepared with a
            T*accine of SI-III Pna~ntococci

        Polysaccharide precipitated by anti-SI-III serum before absorption

                    Dilution of antigen
             __.

Polysaccharide precipitated by anti-5X-111 serum after absorption with SI cells

               Dilution of antigen

Antigen

Polysaccharide precipitated by anti-SI-III serum after absorption with SIII cells

                       Dilution of antigen
__-

Antigen     1:16/ 1:3?! 1:6+! 1:128
~______

SI
SIII

* Salice solution of purified Type I or Type III capsular palysaccharide in a concentration of 1 mg./cc. diluted
as indicated.

%-III cells. That two distinct anticapsular antibodies are present in such an
antiserum can be demonstrated by the independent precipitability of each.
\Vhen an antiserum prepared Cth a vaccine of SI-III cells is absorbed with
either capsulated Type I or Type III cells, the corresponding anticapsular
antibody is removed without significant alteration of the titer of antibody
precipitating the other capsular component (Table IX). Analogous experiments
in which precipitin reactions were carried out in an agar gel demonstrate the
independent removal of one capsular antibody without alteration in the reaction
of the other with appropriate antigens (Figs. 4 to 6).


582          PRODUCTION OF CAPSULAR POLTS.iCCH.ARIDES. I

Properties of Deoxyribomcleates of P?leunzococci of the SI-III Phenotype.--To
determine the nature of the genetic determinants of the capsular components
of the SI-III cell, DKA was prepared from cultures of such cells in the usual
manner. When cells of a non-capsulated pneumococcus (R36NC) derived
originally from a strain of capsular Type II were exposed in the transforming
system to DNA from SI-III cells, organisms of three different phenotypes were
recovered: S- rrr, SI and SI-III. No SIII cells were observed. The results
indicate that there has been no major alteration in either of the genetic units
concerned with the formation of the two capsular components of the cell mani-
festing binary capsulation during their residence within that cell and that each
is able to engender expression of that character controlled by it when it is
reintroduced as the sole genetic factor concerned with capsular formation
into another pneumococcal cell.

DISCUSSION

Through the agency of the transformation reaction, it has been possible to
isolate pneumococci manifesting binary capsulation of several phenotypes.
Examination of cells of the SI-III phenotype in a number of experimental
systems reveals that organisms of this type produce two distinct capsular
polysaccharides which are closely similar if not identical to those produced by
cells of the corresponding unitypic phenotypes. The evidence available suggests
that the two types of polysaccharide are intermingled in the capsule in a random
fashion. Study of cells of the SIII-V phenotype yields results of a similar nature.

Binary capsulation in pneumococcus provides a new basis for serologic
cross-reactivity in this species. Cells manifesting binary capsulation react with
unitypic antiserum to each of their capsular components and stimulate the
formation of antibodies which will react with cells bearing either of the capsular
antigens produced by cells of the binary capsular phenotype. Whether or not
pneumococci manifesting binary capsulation exist in nature remains an un-
answered question. Examination by genetic and serologic techniques of several
groups of pneumococcal strains the capsular antigens of which show cross-
reactivity has failed to result in the recognition in any of the strains of separable
capsular antigens. It would appear more likely that the serologic interrelation-
ships of these strains are based upon the presence of common chemical sub-
groups in their single capsular polysaccharides rather thanupon their production
of multiple types of capsular polysaccharide. For this reason the nomenclature
of pneumococcal types proposed by Eddy (13) seems preferable to that proposed
by Kauffmann and Lund (14).

These experiments with pneumococcus show many similarities to those
performed with a. irzfEuenzae by Leidy, Hahn, and Alexander (6), and it ap-
pears probable that binary capsulation may be manifested by organisms of the


R. AUSTRIAN AND II. I'. BERNHEIXER           583

latter species. In each, hoxvever, it would appear to represent a product of the
laboratory. This fact notwithstanding, binary capsulation provides a useful
object for the study of a variety of biologic phenomena.

Pneumococci manifesting binary capsulation have been produced through
the agency of transformation reactions. Such cells produce two capsular poly-
saccharides which are closely similar if not identical with those produced by
corresponding singly capsulated strains.
Binary capsulation in pneumococcus provides a new basis for serologic
cross-reactivity in this species.

BIBLIOGRAPHY

1. Griffith, F., Thesignificance of pneumococcal types, J. Byg., 1928, 27, 113.
2. Sugg, J. Y., Gaspari, E. L., Fleming, W. L., and Neil& J. M., Studies on im-
  munological relationships among the pneumococci. I. A virulent strain of
  pneumococcus which is immunologically related to, but not identical with typi-
  cal strains of Type III pneumococci, J. Exp. Med., 1928, 47, 917.
3. Harris, A. L., Sugg, J. Y. and Neill, J. M., Studies on immunological relation-
  ships among the pneumococci. II. A comparison of the antibody responses
  of mice and of rabbits to immunization with typical Type III pneumococci
  and to immunization with a related strain, J. Exp. Med., 1928,47, 933.
4. Goebel, W. F., Chemo-immunological studies on the soluble specific substance
  of pneumococcus. II. The chemical basis for the immunological relationship
  between the capsular polysaccharides of Types III and VIII pneumococcus,
  J. Biol. Ckem., 1935, 110, 391.

5. M@rch, E., Serological studies on the pneumococci, London, Humphrey Mil-
  ford, Oxford University Press, 1953.

6. Leidy, G., Hahn, E., and Alexander, H. E., In vitro production of new types of
  Hemophilus i~q`lz~ensae, J. Eap. Med., 1953, 97, 467.
7. Taylor, H. E., Additive effects of transforming agents from some variants of
  pneumococcus, J. Exp. Med., 1949, 89, 399.
8. Ephrussi-Taylor, H., Transformations allo&es du pneumocoque, Ext. Cell
  Research, 1931, 2, 589.

9. MacLeod, C. M., and Krauss, M. R., Stepxise intratype transformation of
  pneumococcus from R to S by way of a variant intermediate in capsular poly-
  saccharide production, J. Exp. Med., 1947, 86,439.
10. Halbert, S. P., Snick, L., and Sonn, C., The use of precipitin analysis in agar
  for the study of human streptococcal infections. II. Ouchterlony and Oakley
  technics, J. Esp. Med., 19%, 101, 557.
11. Austrian, R., Morphologic variation in pneumococcus. I. An analysis of the
  bases for morphologic variation in pneumococcus and description of a hitherto
  undefined morphologic variant, J. Exp. Med., 19j3,98, 21.
12. Torriani, A. ;2I., personal communication.


58-l        PRODUCTIOS OE C.WSUI.-\I~ l'OI.~S:~CClI.~RID~S. I

13. Eddy, B. E., Nomenclature of pneumococcic types, P'uh. Hcullh Rep., 1941,
  59, 449.

11. Kauffmann, F., and Lund, E., 1\Iemorandum on the nomenclature of the pneu-
  mococcus group, I~lcr~af. HUM. IM. Nomcirclalirre nrd Tarommy, 1951,
   4.125.

PLATE 32

FIG. 1. Reaction oi Type 111 anticnpsular antibody (.A) with Type III cells (B),
Type I-III cells (C), ~tl Tl-pc IlL capwlnr l~olysnccharidc (D).
FIG. 2. Reaction of Type I anticapsular antibody (.I) with Type I cells (B), Type
I-III cells (C), and Tvlx I cal)sular p01>-saccll;kdc (D j.








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.E JOLYRS.\L OF ESPERIMEST.\L 3KDICISE VOL. 110         PL.1TE 3