Effect of culture medium composition and pH on the production of M protein and proteinase by group A Streptococci.

Abstract

The effects of pH, yeast extract, and neopeptone on the production of extracellular proteinase and M protein by group A streptococci were studied with a type 1 strain capable of producing both M protein and proteinase. The strain DS 2036-66 grew moderately well in a semisynthetic broth. M protein was produced without adding peptides to the medium. When added to a medium with 1% glucose, yeast extract (0.1%) was found to stimulate both growth and proteinase formation. Limiting the glucose to 0.25% prevented a drop in pH below 6.7 and prevented proteinase formation. Although less growth occurred with limited glucose, M protein of high specific activity was produced with an actual increase in acid-extractable M protein during the stationary phase of growth. When the medium was buffered at pH 7.85 with tris(hydroxymethyl)aminomethane buffer, 0.5% neopeptone prevented proteinase formation. This was true even in the presence of 1% glucose and 0.1% yeast extract, which resulted in a fall in pH to about 4.8 by 48 hr. Growth was greater than in Todd Hewitt broth, but the specific activity of M protein was considerably less than that found in the medium with glucose limited to 0.25%. Neopeptone was found to have little direct action on crude streptococcal proteinase. Instead, the evidence suggested that neopeptone somehow prevents proteinase elaboration. Yeast extract, on the other hand, appears to stimulate proteinase elaboration. To prevent proteinase formation, neopeptone must be added early, during the logarithmic phase of growth or at the start. In contrast, when yeast extract was added as late as 24 hr, it resulted in the elaboration of extracellular proteinase and in the decline of M protein. When 38 M nontypable strains from the diagnostic laboratory were tested for proteinase activity under conditions similar to those used in the diagnostic laboratory, only six produced much proteinase.

Full text

Page 1

JOURNAL OF BACTERIOLOGY, Sept. 1969, p. 737-744
Copyright © 1969
American Society for Microbiology
Vol. 99, No. 3
Printed In U.S.A.
Eflect of Culture Medium Composition and pH on
the Production of M Protein and Proteinase
by Group A Streptococci
JAY 0. COHEN
Health Services and Mental Health Adninistration, National Communicable
Disease Center, Atlanta, Georgia 30333
Received for publication 26 May 1969
The effects ofpH, yeast extract, and neopeptone on the production of extracellular
proteinase and M protein by group A streptococci were studied with a type 1 strain
capable of producing both M protein and proteinase. The strain DS 2036-66 grew
moderately well in a semisynthetic broth. M protein was produced without adding
peptides to the medium. When added to a medium with 1% glucose, yeast extract
(0.1 %) was found to stimulate both growth and proteinase formation. Limiting the
glucose to 0.25% prevented a drop inpH below 6.7 and prevented proteinase forma-
tion. Although less growth occurred with limited glucose, M protein of high specific
activity was produced with an actual increase in acid-extractable M protein during
the stationary phase of growth. When the medium was buffered at pH 7.85 with
tris(hydroxymethyl)aminomethane buffer, 0.5% neopeptone prevented proteinase
formation. This was true even in the presence of 1% glucose and 0.1% yeast extract,
which resulted in a fall inpH to about 4.8 by 48 hr. Growth was greater than in Todd
Hewitt broth, but the specific activity of M protein was considerably less than that
found in the medium with glucose limited to 0.25%. Neopeptone was found to have
little direct action on crude streptococcal proteinase. Instead, the evidence suggested
that neopeptone somehow prevents proteinase elaboration. Yeast extract, on the
other hand, appears to stimulate proteinase elaboration. To prevent proteinase for-
mation, neopeptone must be added early, during the logarithmic phase of growth or
at the start. In contrast, when yeast extract was added as late as 24 hr, it resulted in
the elaboration of extracellular proteinase and in the decline of M protein. When
38 M nontypable strains from the diagnostic laboratory were tested for proteinase
activity under conditions similar to those used in the diagnostic laboratory, only six
produced much proteinase.
For a number of years, various laboratories
have experienced difficulties in preparing antisera
to be used in M protein precipitin tests for a
number of types of group A streptococci. Basic
to this problem is the maintenance of the ability
of strains of streptococci to produce M protein.
Also, the M antigen must remain intact after it
has been synthesized. Failure of a strain to react
with antisera in the precipitin test may be due to
its inability to produce M protein or to the de-
struction ofM protein after it has been produced.
Since 1945 when Elliott (5) reported the produc-
tion of extracellular proteinase by Streptococcus
pyogenes and its effect on M protein, no detailed
studies have been reported on the elaboration of
proteinase during the various stages ofgrowth.
Elliott stated that neopeptone
hydrolyzed complex protein material) interfered
with streptococcal proteinase, but it was not clear
whether
inhibited proteinase directly. Todd Hewitt broth
is the medium used predominantly for growing
streptococci for M typing. This medium, with
2% neopeptone as the main nitrogen source and
only 0.2% glucose, was designed to support M
protein synthesis and to inhibit proteinase. De-
spite these features of Todd Hewitt broth, many
strains of group A streptococci fail to produce M
protein when grown in it. This includes some
strains from infections as well as cultures isolated
from carriers. This paper describes experiments
with a type 1 strain ofS. pyogenes which produces
both M protein and proteinase. Factors ofgrowth
conditions, pH, and medium composition are
737
it prevented proteinase synthesis or
(a partially

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J. BACrERIOL.
shown to influence greatly the amount of acid-
extractable M protein per milligram (dry wt) of
cells and to control the elaboration ofproteinase.
MATERIALS AND METHODS
Cultures. Strain DS 2036-66 and other strains of S.
pyogenes group A were supplied by Joseph Padula
and Caroline Baker of the Streptococcus
National Communicable Disease Center. Strain DS
2036-66 produced both type
cellular proteinase.
Antisera.
Streptococcus M
were supplied by the Biological Reagent Section of
the National Communicable Disease Center. Type
serum (lot 5) was used for most of the work.
Culture
media
components.
hydrolyzed casein was purchased from Nutritional
Biochemicals Corp., Cleveland,
defined reagents,
were purchased from a variety of suppliers, and no
evidence of any significant differences dependent upon
source were observed for the purposes of this study.
The formula for the semisynthetic medium is shown in
Table
membrane
filtration.
Complex
sterilized by autoclaving for 12 min at 118 C. Hutton
D. Slade advised the author on a synthetic medium
from which the semisynthetic medium was devised.
Trypsin inhibitors. Ovomucoid trypsin inhibitor was
prepared by Leo
Pine,
Disease Center, according to the method of Fredercq
and Deutsch as restated by Laskowski (9). Soy bean
trypsin inhibitor was purchased from Worthington
Biochemical Corp., Freehold, N.J.
Growth. Growth was expressed in terms of milli-
grams (dry wt). This was determined by measuring
the optical density (OD) at 655 nm on a Spectronic
20 colorimeter (Bausch & Lombe, Inc., Rochester,
N.Y.) and converting from OD to a dry-weight curve
prepared from a sampleof a washed culture of strepto-
coccal strain DS 2036-66.
Quantitative M determinations. These were done by
the quantitative precipitin procedure of Cohen and
Pine (3).
Proteinase. Proteinase was measured by the hy-
drolysis of casein, followed by precipitation of un-
hydrolyzed casein by 5% trichloroacetic acid. The
amount of proteinase was determined by a difference
in OD at 280 nm between zero-time controls and the
incubated experiments. The method is essentially that
of Kunitz for trypsin as reported by Laskowski (8).
Neutralized glutathione (final concentration 0.01 M)
was added to the reaction as an activator of strepto-
coccal proteinase. When the proteinase activity of su-
pernatant fluids was measured without prior concen-
tration, incubation times of 6 or 18 hr were used; to
prevent bacterial growth, 5,000 units of penicillin per
ml and 50 ug of chloramphenicol per ml were added
to the 1% casein solution.
Inocula. Growth-curve experiments reported
this paper for DS 2036-66 were started with frozen
streptococci
kept
at
-65 C. The
distributed in 1-ml amounts. A vial was thawed at
room temperature just before use as inoculum. Prior
Unit,
1 M protein and extra-
precipitin
antisera
1
Vitamin-free,
acid-
Ohio.
Chemically
e.g., amino acids and vitamins,
1. Semisynthetic medium was sterilized by
supplements
were
National Communicable
in
inocula
were
to freezing, the cells from Todd Hewitt broth were
washed once with 0.05% neutralized cysteine and then
resuspended
in 0.05% neutralized cysteine.
inocula remained viable and relatively uniform for at
least 1 year.) Usually, 0.04 ml of inoculum was used.
The cell suspensions for inocula contained between
108 and 109 colony-forming units per ml.
Growth curves. Duplicate tubes, each containing
10 ml of broth, were set up. The OD values were read
and the tubes were harvested at selected intervals.
The cultures were then refrigerated at 3 to 5 C and
held in the refrigerator until the growth part of the
experiment was completed. They were then assayed
together by various methods. For M determinations,
the growth from two tubes (20 ml) was harvested by
centrifugation, and the entire sediment was extracted
with 0.25 or 0.3 ml of 0.2 N HCl.
(The
RESULTS
Preliminary experiments were performed with
strains DS 2036-66, type 1; DS 2092-66, type 1;
and DS 2051-66, type 6. The streptococci were
grown in semisynthetic broth (Table 1) with 1%
glucose and phosphate buffer (pH 7.45). At both
37 and 30 C, all three strains produced their re-
spectiveM proteins from both semisynthetic broth
and Todd Hewitt broth, as measured by the quali-
TABLE 1. Composition of semisynthetic mediuma
Prepn
Amt per liter
Vitamin-free acid-hydrolyzed
casein (10%).....................
Glucose.....
Sodium acetate.10 g
Sodium bicarbonate.1 g
L-Cysteine.0.1 g
L-Glutamine.0.1 g
L-Glycine.0.1 g
L-Tyrosine.0.1 g
L-Tryptophan.0.1 g
Adenine sulphate.0.010 g
Uracil.
Guanine hydrochloride.
K2HPO4.2.5g
KH2PO4.
MgS47H20..
FeSO47H2 .0.02 g
MnSO4 H20.
Riboflavin.........................
Folic acid.25 g
Biotin.g
Paramino benzoic acid.1 mg
Pyridoxine hydrochloride.1 mg
Thiamine hydrochloride.1 mg
Nicotinic acid.1 mg
Calcium pantothenate.1 mg
100 ml
b
.010 g
.010 g
2.5 g
0.4 g
0.02g
500 ,Ag
10
g
aAdd either 80 ml of K2HPO4-KH2PO4 buffer
(1 M) or Tris buffer (final concentration 0.15 M, pH
7.85).
bVariable from 0.25 to 1.0% depending on ex-
periment.
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VOL. 99, 1969
M PROTEIN AND PROTEINASE
tative M protein precipitin test (11). Growth was
under anaerobic conditions with the cultures un-
der a pyrogallol seal (10). Under aerobic condi-
tions, there was considerable lag (several days) in
semisynthetic broth. Significantly, the production
ofM protein by strains DS 2036-66 and DS 2092-
66 was strong in the absence ofadded peptides.
Growth in the semisynthetic medium
limited and maximal OD of about 0.50 was
reached by DS 2036-66. In contrast, the growth
reached a maximal OD of about 0.85 in Todd
Hewitt broth. In terms of dry weight per milli-
gram, this was twice as much growth (0.33 as
compared with 0.17 mg/ml). Attempting to im-
prove growth yields and to study effects on M
protein production, experiments were initiated
with yeast extract and neopeptone supplementa-
tion ofthe semisynthetic medium.
When 0.1% yeast extract was added to the
semisynthetic broth, there was little lag before
vigorous logarithmic growth occurred. The final
OD reached was 0.65 (0.22 mg/ml, dry wt) in the
case of DS 2036-66. This was a considerable
improvement over the growth attained without
yeast extract; yet, little M protein was obtained
in the presence of yeast extract. A similar effect
did not occur with strains DS 2051-66 (type 6)
or with DS 2092-66 (type 1). Yeast extract had a
detrimental effect on M production by 2036-66 at
30 C, but the effect was strongest at 37 C. It was
postulated that strain 2036-66 was a potential
extracellular proteinase producer, and that yeast
extract stimulated proteinase production as well
as cellular growth.
Time studies were initiated to correlate growth
and M protein production at various points in the
growth curve. At first, the production of M pro-
tein was roughly approximated according to the
method previously described by Brock (2). When
0.1% yeast extract was added to the semisynthetic
broth initially, after 8 to 12 hr of incubation
(37 C) there was ample M protein to produce a
strong reaction in the qualitative test. By 24 hr,
however, little M protein remained. The 48-hr
cultures
were shown to contain considerable
amounts of extracellular proteinase. In contrast,
when yeast extract was left out, M protein was
demonstrated at 24, 48, and 72 hr. After about an
18-hr lag, rapid logarithmic growth took place.
When 0.25% neopeptone was added in addition
to the 0.1 % yeast extract, there was rapid growth,
reaching a maximum comparable with that ob-
tained with Todd Hewitt broth. Strong M protein
reactions were observed early and at 24, 48, and
72 hr.
Test for direct antiproteinase activity of neopep-
tone. An experiment was designed to test for
direct activity of neopepto.ie against already
was
formed proteinase. Concentrated crude proteinase
obtained from stationary-phase cultures was used.
The enzyme was added to acid-extracted crude M
protein; previously neutralized glutathione with
a final concentration of 0.01 M was included as an
activator. The protocol called for four different
combinations (Table 2). These were (i) no enzyme
control,
(iii) neopeptone supplementation, and (iv) pro-
teose-peptone supplementation. Enzyme activity
was stopped by immersing the tubes in a boiling-
water bath for 5 min. This procedure did not
destroy the M protein, as observed in the "no
enzyme control."
The capillary precipitin test reported by Cohen
and Pine (3) was used to assay M protein in these
experiments (Table 2). In the absence of pro-
teinase, after incubation for 1 hr, the "no enzyme
control" showed considerable M protein. By
contrast, the enzyme without supplement was
almost devoid of M protein. Neither neopeptone
nor proteose-peptone protected the M from
decay, and about 75 and 85% of the respective
activities were lost. These results indicate that
neopeptone
did
not
inactivate
proteinase or irreversibly inhibit
although some competitive effect was observed.
This experiment suggests the alternative, preven-
tion ofpriLeinase elaboration of synthesis.
Growth experiments: pH effect. Figures 1 and 2
illustrate the effects of initial pH on growth, M
protein, and proteinase in a semisynthetic medium
to which 0.25% neopeptone had been added.
When the initial pH of the semisynthetic medium
(ii) enzyme without supplementation,
streptococcal
its activity,
TABLE 2. Direct effect ofneopeptone and proteose
peptone on streptococcal extracellular M protein
activity for preformed M protein
Total units
of X pro-
tein a ter I
hr at 37 C
Test condition
No enzyme controla ...................... 675
Enzyme without supplementsb ............
Neopeptonec......................
Proteose-peptoned......................
40
168
82
aConsisted of 0.8 ml of M protein acid extract
and 1.2 ml of buffered saline (pH 7.4).
bConsisted of 0.8 ml ofM protein acid extract,
0.2 ml of 0.1 M neutralized glutathione, 0.3 ml of
concentrated crude proteinase, and 0.7 ml of
buffered saline.
cConsisted of 0.8 ml of M protein acid extract,
0.5 ml of 2% neopeptone, 0.3 ml of enzyme, 0.2 ml
of0.1 M glutathione, and 0.2 ml of buffered saline.
dConsisted of 0.8 ml ofM protein acid extract,
0.5 ml of 2% proteose peptone, 0.3 ml of enzyme, 0.2
ml of 0.1 M glutathione, and 0.2 ml of buffered saline.
OF GROUP A STREPTOCOCCI
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J. BACrERIOL.
10
2030
40
50
HOURS
FIG. 1. Semisynthetic broth with 1% glucose, 0.1%
yeast
extract, 0.25%
neopeptone,
buffer (initial pH 7.45). The total M protein is from
the extraction of the cells from the entire 20 ml of
culture at 10, 24, and 48 hr. Proteinase readings
represent the difference in optical density between 18-hr
incubated samples and their zero-time controls after
precipitation of residual casein with 3 ml of 5%
trichloroacetic
acid.
The
contained 0.5 ml of broth supernatant fluid, 1.0 ml of
1% casein solution in phosphate buffer, 0.2 ml of 0.1
m neutralized
glutathione, and 0.3 ml of pH 7.6
phosphate buffer (0.1 m). The dry weight for this and
all subsequent figures is that for a volume of20 ml.
and phosphate
proteinase
test
mixture
was 7.45 in the presence of 0.25% neopeptone,
proteinase production was delayed for 24 hr
(Fig. 1). As a result, M protein was highest at
24 hr. When this experiment was run with 0.1%
yeast extract but no neopeptone, at 24 hr there
was very little M protein and considerable pro-
teinase was already present in the medium. The
amount of neopeptone which could be added to
the semisynthetic broth with phosphate buffer
was limited because a precipitate was formed in
the medium at levels higher than 0.25%. (Later
tris(hydroxymethyl)aminomethane (Tris) buffer
was substituted for phosphate buffer, and the
level of neopeptone increased to 0.5%. Under
these conditions, as will be seen later, no pro-
teinase was formed.) The effect of neopeptone in
the medium with phosphate buffer was found to
depend on the initialpH.
Growth was rapid with initial pH 7, reaching a
final maximum greater than that obtained when
the initial pH was 7.45 (Fig. 2). At 24 hr, how-
ever, very little M protein could be extracted from
the cells, and considerable proteinase had been
released in the medium. Elliott (6) reported that
the precursor of proteinase was not formed at a
neutral or slightly alkaline pH. He found slightly
acidpH to be conducive to proteinase production.
The discovery of these pH effects led to experi-
ments in which the glucose was limited to 0.25%
in the semisynthetic medium with phosphate
buffer.
Experiment with phosphate buffer, 0.1% yeast
extract, and 0.25% glucose. Figure 3 shows the
results obtained when glucose was limited and
only yeast extract was added to the semisynthetic
medium. First, when glucose was limiting and the
pH was kept near neutral, no extracellular pro-
teinase was released between 10 and 48 hr. Al-
though growth was less than that obtained in
Todd Hewitt broth, which contains 0.2% glucose,
M protein production was much greater. Growth
reached a maximum at 6 hr, after which there was
a significant decline in OD; M protein continued
to increase to 48 hr. Later experiments showed
that after 48 hr the M production did not increase
and the OD leveled off. Gram stains showed a
shift from apparently normal gram-positive cells
at 10 hr to pleomorphic gram-negative cells at 48
hr. The increase in total M and M specific activity
may represent a real net synthesis of M protein
during unbalanced growth of metabolism after
HOURS
FIG. 2. Same as Fig. I except initial pH was 7.0
(semi-synthetic broth with 1% glucose, 0.1% yeast
extract, 0.25% neopeptone, and phosphate buffer).
Total M and proteinase were determined as for Fig. 1,
except that the residual protein-like material soluble in
trichloroacetic acid was measured by the method of
Lowry et al. (9a).
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VOL. 99, 1969
M PROTEIN AND PROTEINASE OF GROUP A STREPTOCCIOC
the glucose is used up. This increase would not
necessarily occur in the presence of 1.0% glucose
because, in this case, glucose is not the limiting
factor, pH declines to near 5, and the acid pH
itself may shut off further growth and synthesis.
However, in the absence of any method which
will give total M at all times, one must be cautious
in accepting this experiment as evidence of net
synthesis during the stationary phase. The change
in surface properties ofthe streptococci and prob-
able leakage or partial autolysis, reflected in the
drop in OD, may increase the extractability of M
protein, providing an apparent, but not neces-
sarily absolute, increase.
Experiment with 0.25% glucose, 0.1% yeast
extract, and 0.25% neopeptone. Here, the semi-
synthetic medium was augmented with 0.25%
neopeptone as well as yeast extract (Fig. 4). The
results were similar to those with only yeast
extract, except that the initial growth was greater
by about 20%. Strangely, the additional growth
did not appear to lead to increased M production
over the yeast-extract-only experiment. Proteinase
was kept at a low, insignificant level; pH did not
decline below 6.7, and a fall in optical density was
contrasted by a rise in extractableM protein.
Todd Hewitt broth with anaerobic seals. The
results obtained with Todd Hewitt broth are
6
10
2024
HOURS
30
40
48
FIG. 3. Semisynthetic broth with 0.25%
0.1% yeast extract, and phosphate buffer (initial pH
7.45). Total M and proteinase
described for Fig. 1. The M specific activity [Munitsl
mg (dry wt)] was calculated by estimating the dry
weightfrom a curve, OD versus dry weight, anddividing
the total M units by the total dry weight.
glucose,
were determined as
HOURS
FIG. 4. Semisynthetic broth with 0.25% glucose,
0.1% yeast extract, 0.25% neopeptone, and phosphate
buffer (initial pH 7.45). Total M, proteinase, and M
specific activity were determined as described under
Fig. 1, 3.
shown in Fig. 5. Glucose is limiting in Todd
Hewitt broth as in the low glucose semisynthetic
broth, and, as a result, the pH does not fall below
6.7. The growth curve is somewhat similar to
those shown in Fig. 3 and 4. However, the M
protein curve is quite different. M protein (ex-
tractable) did not increase after 10 hr and, in fact,
declined after 24 hr to a much lower value than
that seen earlier. This was not due to proteinase
formation as none was detected at 10, 24, and 48
hr. It was postulated that the supematant fluid
might contain the missing M. The supematant
fluids were dialized against dilute phosphate
buffer and were concentrated by mild heat and
forced air (about 45 to 50 C). M protein is not
appreciably inactivated by exposure for several
hours to these temperatures. Some of the M
protein was indeed found in the supematant
fluid; perhaps even more was originally present
but was lost in our dialysis and concentration
efforts. The M loss here may have been due to
release from the surface during the partial autoly-
sis or leakage.
Experiments substituting Tris buffer for phos-
phate buffer. In the prior experiments, M protein
enhancement and inhibition of proteinase forma-
tion occurred only at the expense of growth. Since
our primary objective was production of M
antigens, a method was sought that would lead
both to increased M protein and increased
growth. To increase the amount of neopeptone
in the medium without a concurrent precipitation
of other ingredients (probably heavy metal ions),
0.15 M Tris buffer was used in preparing the semi-
synthetic medium. This is not enough buffering
capacity to neutralize the lactic acid produced
741