THE ELABORATION OF EXTRACELLULAR CAPSULAR POLYSACCHARIDE BY KLEBSIELLA PNEUMONIAE AND ITS RELATIONSHIP TO VIRULENCE By PHILIP DOMENICO, B.A. DISSERTATION IN MICROBIOLOGY Presented to the Graduate Faculty of Texas Tech University Health Sciences Center in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved Acc'epted December, 1983
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THE ELABORATION OF EXTRACELLULAR CAPSULAR POLYSACCHARIDE BY
KLEBSIELLA PNEUMONIAE AND ITS RELATIONSHIP TO VIRULENCE
By
PHILIP DOMENICO, B.A.
DISSERTATION
IN
MICROBIOLOGY
Presented to the Graduate Faculty of Texas Tech University Health Sciences Center
in Partial Fulfillment of the Requirements for
the Degree of
DOCTOR OF PHILOSOPHY
Approved
Acc'epted
December, 1983
f^C-'^'^ . ACKNOWLEDGEMENTS
I extend my gratitude to all who have made this dissertation
possible:
To my supervising professor, Dr. David C. Straus, who
encouraged me when I was frustrated and frustrated me when I was
encouraged.
To Dr. Dana L. Diedrich, who enriched my scientific repertoire
with his insight into microbial biochemistry and physiology, and
who burned the late-night candle with me on numerous occassions.
To Dr. Charles W. Garner for his guidance and his knowledge of
chemical phenomena.
To Dr. Rial D. Rolfe and Dr. David J. Hentges for their
assistance and constructive criticisms.
To Cathy Portnoy Duran who put up with my mess for two years.
To Linda Froelich, who inspired me and helped me through a
variety of difficulties during this period.
And finally to my parents who taught me the gift of
perseverence.
n
I-V"*.
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS i i
LIST OF TABLES vi
LIST OF FIGURES x
LIST OF ABBREVIATIONS xiii
I. INTRODUCTION AND LITERATURE REVIEW 1
II. MATERIALS AND METHODS 11
Bacterial Strains 11
Media and Growth Conditions 11
Purification of the Extracellular Polysaccharides
of K_. pneumoniae 12
Preparation of Rabbit Anti-Type-Specific Antiserum.. 16
Rocket Immunoelectrophoresis 17
Opsonophagocytic Assay and Serum Sensitivity 18
Assay for Virulence of l<. pneumoniae in a Mouse
Model 20
Assay for the Production of Lobar Pneumonia in a
Rat Model 21
Assay for Characterization of Outer Membrane
Proteins of j<. pneumoniae 23
Electrodialysis of Extracellular
Polysaccharides from l<. pneumoniae 24
m
Page
Determination of Capsule Size of J<. pneumoniae 26
In Vitro Quantitation of Extracellular
Polysaccharides Produced by K. pneumoniae 26
Electron Microscopy 27
Gel Diffusion Method for Immunological Analysis 27
Saponification of K. pneumoniae Polysaccharides
and Quantitation of Fatty Acids 28
Hydrofluoric Acid Treatment 29
Statistical Analysis 29
III. RESULTS 30
Strain Variation and the Production of Apparent
Isogenic Sets 30
The Establishment of a Chronic Lobar Pneumonia by J<.
pneumoniae in a Rat Model 32
J<. pneumoniae Virulence in a Mouse Model 59
In Vitro Quantitation of Extracellular
Polysaccharides Produced by J<. pneumoniae 61
Serum Sensitivities and Opsonophagocytic Assays
for J<. pneumoniae 75
Purification of the EPS of J<. pneumoniae 80
Effect of Purified Extracellular Products from K_.
pneumoniae on Virulence in a Mouse Model 126
Structural Studies on the EPS Produced by
J<. pneumoniae 146
Gel Immunodiffusion Studies for Identification and
Quantitation of ECPS Produced by K_. pneumoniae.. 186
iv
Page
Survey of the Outer Membrane Proteins of
J<. pneumoniae 194
IV. DISCUSSION 200
LITERATURE CITED 225
LIST OF TABLES
Page
1. Capsule size of K. pneumoniae 31
2. Establishment of a chronic Lobar Pneumonia in Rats 37
3. Effect of Dosage on the Ability of KPl to Produce
Pneumonia in Rats 51
4. Establishment of a Chronic Lobar Pneumonia in Rats
Emp 1 oyi ng KP1 -0 54
5. KPl-T in the Rat Lung Model 55
6. KP2-0 in the Rat Lung Model 57
7. KP2 2-70 in the Rat Lung Model 58
8. LDcn Values in Mice and ID^n Values in Rats for Strains
50 50
of J<. pneumoniae 60
9. ECPS Production by Strains of K_. pneumoniae Serotype 1 at
Various Intervals of Incubation 62
10. ECPS Production by Strains of j<. pneumoniae Serotype 2 at
Various Intervals of Incubation 68
11. Production of ELPS by K. pneumoniae Serotypes 1 and 2
after 48h of Culture in Defined Medium 71
12. Comparison of ECPS, ELPS, Capsule Size and Virulence of
J<. pneumoniae Serotypes 1 and 2 73
13. Correlations Between Polysaccharide Production and
Virulence in the Mouse Model 74
14. Serum Sensitivity of K. pneumoniae 76
15. Opsonophagocytic Assay 78
16. Effect of the Addition of EPS on the OPA 79
VI
Page
17. Elution Ionic Strength and Apparent Molecular Weights of
the ECPS from Various Strains of KPl and KP2 82
18. Elution Volumes for Dextran Calibration Standards on
Sepharose 2B (S-2B) 103
19. The Extracellular Products Found in Ethanol Fractionated
Supernatants of K_. pneumoniae 104
20. Comparison of the ECPS and ELPS Content in the Neutral
(N) and Acidic (A) Fractions from DEAE-Sephacel 106
21. The Extracellular Products Found in KPl and KP2 EPS after
Purification 107
22. Percent Yield Obtained from ECPS Purification for Two KPl
Strains 109
23. Pur i f i ca t ion of KPl-0 (EtOH) EPS by ED, cetavlon and Gel
Fi 1 t r a t i on I l l
24. Pur i f i ca t ion of KPl-0 (EtOH) EPS by ED, Cetavlon and Gel
F i l t r a t i o n : Percent Contamination wi th ELPS and
Protei n 115
25. Purification of KP2-0 EPS by ED and Cetavlon 121
26. Purification of KP2 2-70 EPS by ED and Cetavlon 123
27. Effect of KPl EPS on KPl-T Virulence in the Mouse Model.. 127
28. Probability Matrix Comparing the Virulence Enhancing
Potentials for all EPS Fractions Co-injected with
the KPl-T Strain in the Mouse Model 129
29. Effect of KP2 EPS on KPl-T Virulence in the Mouse Model.. 132
v n
-y^mmfia
Page
30. Effect of KPl or KP2 EPS on the Virulence of KP2-0 in
the Mouse Model 134
31. Effect of ED on the Virulence Enhancement of KPl-T by
KP2 2-70 EPS in the Mouse Model 137
32. Effect of ED on the Virulence Enhancement of KPl-T by
KP2-0 EPS in the Mouse Model 138
33. Effect of Saponification on the Virulence Enhancement
of KPl-T and KP2-0 by KPl-0 (N) EPS
in the Mouse Model 140
34. Virulence Enhancement of KPl-T in the Mouse Model:
Comparison to the Dosage of ELPS in KPl EPS
Sampl es 141
35. Virulence Enhancement of KPl-T in the Mouse Model:
Comparison to the Dosage of ELPS in KP2 EPS
Sampl es 142
36. Effect of an Al ternat ive Pur i f i ca t ion of KPl-0 EPS on
the Virulence Enhancement of KPl-T in the
Mouse Model 145
37. The pH D i f fe ren t ia l of the Anode and Cathode Chambers
During ED 161
38. Quantitation of Ions Retrieved from the Cathode and the
Anode Chambers During ED of KP2 2-70 (EtOH) EPS 163
39. Effect of ED on the Quantity of Divalent Cations Found
in KP2 EPS 164
vm
IV
Page
40. Effect of ED on the Quantity of Phosphate Found in the
KP2 EPS 165
41. Quantitation of Fatty Acid Methyl Ester (FAME) Released
from EPS after Saponification 181
42. Quantitation of FAME Released from the EPS of KPl-0
and KPl-T Obtained from Gel Filtration 182
43. Standard Curve for the Rocket Immunoelectrophoresis
(RIE) of KPl-0 HMW and KPl-0 LMW EPS 187
44. RIE of Standard Concentrations of KP2 2-70 ECPS and
Serum from an Infected Rat 189
45. Quantitation of KP2 2-70 EPS in the Serum of an
Infected Rat by Radial Immunodiffusion (RID) 193
IX
LIST OF FIGURES
Page
l a . Transmission Electron Micrograph (TEM) of KP2-0 and i t s
Capsule 34
36 lb. TEM of KP2-T and its Capsule
2. Photomicrocrographs of H&E Stained Lung Tissue Sections
During J<. pneumoniae Infection in Rats 39
2a. Normal Rat Lung Section 41
2b. Rat Lung Section at 24h Post-inoculation 44
2c. Rat Lung Section at 3 days Post-inoculation 46
2d. Rat Lung Section at 6 days Post-inoculation 48
2e. Rat Lung Section at 9 days Post-inoculation 50
3. Comparison of the Rate of Production of ECPS by KPl-0 and
KPl-T at Various Intervals of Incubation 64
4. TEM of KPl 2-70: Example of Capsule Sloughing
5. Comparison of the Rate of Production of ECPS by KP2-0
and KP2 2-70 at Various Intervals of Incubation.
66
6.
7.
8.
9.
10.
11.
12.
13.
Elution
Elution
Elution
Elution
Elution
^ Elution
Elution
Elution
Profi
Profi
Profi
Profi
Profi
Profi
Profi
Profi
le fo r KPl-T EPS on DEAE-Sephacel
le fo r KPl-0 (A) EPS on Sepharose 2B (S-2B)
le fo r KPl-0 (N) EPS on S-2B
le fo r KPl-T (A) EPS on S-2B
le fo r KPl-T (N) EPS on S-2B
le fo r KP2-0 (A) EPS on S-2B
le fo r KP2-0 (N) EPS on S-2B
le for KP2 2-70 (A) EPS on S-2B
70
84
87
89
91
93
95
97
99
Page
14. Elution Profile for Dextran Calibration Standards
on S-2B 101
15. Elut ion Pro f i le fo r KPl-0 F r I I on S-2B 114
16. Elut ion Pro f i le for KPl-0 Fr I I I on P-300 118
17. Elut ion Pro f i le fo r KP2-0 F r I I on S-2B 120
18. Effect of an Al ternat ive Pur i f i ca t ion on the Elut ion
Pro f i le for KP2 2-70 EPS on S-2B 125
19. Effect of ED on the Elut ion Pro f i le for KP2 2-70 EPS on
BGA-150m 148
20. Effect of ED on the RID pro f i les of KP2 2-70 EPS 151
21. Effect of ED on the Elution Pro f i le fo r KP2 2-70 LMW
EPS on BGA-150m 154
22. Effect of ED on the Elution Pro f i le for KP2-0 EPS
on S-2B 157
23. Effect of ED on the Elution Profile for KP2-0 EPS on
S-2B: Effect of a Small Sample Volume 159
24. Histogram of the Effect of ED on the [PO^"^] in the
KP2 2-70 EPS 167
25. Effect of Sodium Dodecyl Sulfate (SDS) on the Elut ion
Pro f i le fo r Electrodialyzed KP2-0 EPS on S-2B 171
26. Effect of ED on the Elut ion Pro f i le for KPl-0 EPS
on S-2B 173
27. Effect of Hydrofluoric acid (HF) on the Elut ion
Pro f i l e fo r KPl-0 EPS on S-2B 177
XT
Page
28. Effect of Saponification on the Elution Profi le for
KPl-0 EPS on S-2B 179
29. Elution Profile for KP2 2-70 (EtOH) EPS Obtained from
Growth in DMH on BGA-150m 185
30. Standard Curve for RIE of KP2 2-70 ECPS 192
31. Outer Membrane Protein Profiles for KPl Strains 196
32. Outer Membrane Protein Profiles for KP2 Strains 198
33. Hypothetical Model for the Electrophilic Associations
Capsule size of bacteria from DW medium was determined by the
method of Duguid (20) using India ink preparations. Capsule
production was expressed as the transverse diameter (TD), which is
a measurement of both the width of the bacillus and the width of
the capsule on either side of the bacillus. One hundred bacilli
were randomly selected under oil immersion, measured with an ocular
micrometer, and the average capsule size was calculated.
In Vitro Quantitation of Extracellular
Polysaccharides Produced by K. pneumoniae
Bacteria were grown in a defined medium as described in
Section B of Materials and Methods. Samples (100 ml) of growing
cultures were taken at 18, 24, 36 and 48 h and the organisms
pelleted by centrifugation at 12,700 x g for 30 min. The
supernatant obtained was dialyzed 3 times in 8L cold DH2O overnight
while stirring, and assayed for uronic acid. Colony forming units
per ml culture at these time periods were also determined on TSA.
The production of ECPS for each organism was expressed as yg ECPS
27
ml cell (xlO ). ECPS was calculated by dividing the uronic
acid determinations by 0.3098 (for KPl) or by 0.2643 (for KP2),
which reflects the proportion of ECPS which is uronic acid (30,64).
The production of extracellular lipopolysaccharide (ELPS) for each
strain was also monitored at these time periods by the method of
Osborn et al. (60). The ELPS data were expressed as the yg ECPS
ml cell (x 10 ). ELPS data were further characterized in some
of the studies by the Limulus Amoebocyte Lysate (LAL) Assay
(Pyrotell Associates of Cape Cod, Inc., Woods Hole, Mass.) as
described by Levin (50). Both assays utilized the purified LPS
from Escherichia coli 055:B5 as the standard and the ELPS units are
expressed as yg of E.. coli LPS equivalents.
Electron Microscopy
Electromicroscopy for the visualization of the capsular
substances of J<. pneumoniae was performed according to the method
of Cassone and Garaci (12) on early log phase cultures grown in DW
medium. Preparations were observed and photographed with a Hitachi
H-600 Transmission Electron Microscope. These studies were kindly
performed by Dr. Jack Yee of the Department of Anatomy, Texas Tech
University Health Sciences Center, Lubbock, Texas.
Gel Diffusion Method for Immunological Analysis
The immunological characterization of the various fractions of
EPS from KPl and KP2 strains were performed by the method of
Ouchterlony (62). Ion agar (0.5 to 1.0% solutions) (Difco) in DH^O
were brought to boiling to dissolve the agar and cooled to about
50°C. Twenty-five ml were then poured on 3 1 / 4 x 4 inch glass
28
plates and allowed to solidify. Holes of 2 mm in diameter and 0.5
cm apart in a circular fashion were made in the gel and 5 yl of EPS
at various concentrations were placed in the outside wells.
Another well was cut in the middle of the circular wells and 5 yl
of KPl or KP2 rabbit antiserum was added to this middle well. The
reaction took place at room temperature overnight in a humidifying
chamber. The gels were then washed three times in 200 ml
physiological saline at 4°C and once with 200 ml DH2O at 4°C. The
gels were then stained with 0.2% Coomassive blue in methanol,
acetic acid and DH2O (5:1:5, by volume), and then destained in the
same solution in the absence of stain.
Radial immunodiffusion studies were also performed to
•quantitate the ECPS found in the serum of infected rats, and to
follow the effect of electrodialysis on EPS samples. A 0.5%
agarose solution in DH2O was heated to boiling and allowed to cool
to 50°C. One to two ml of type-specific antiserum was then added
to 23 or 24 ml of the agarose slurry, and poured onto 3 1 / 4 x 4
inch glass plates or in 150 mm petri plates and allowed to
solidify. Hole were made in the gel (2 mm) and 5 to 10 yl of
sample was placed in the wells. The gel plates were incubated for
18-24 h at room temperature. Zone diameters of the precipitin
reaction within the gel were measured and compared to known
quantities of ECPS tested in the same manner.
Saponification of K. pneumoniae
Polysaccharides and Quantitation
of Fatty Acids
In some studies the EPS, that was partially purified up to the
29
by EPS after saponification. No attempt was made to identify the
various methyl ester fractions.
Hydrofluoric Acid Treatment
Various fractions of EPS were weighed out (20 mg) and placed
in 1 ml 60% hydrofluoric acid (HF) for 3 h at 0°C with occasional
mixing. NaOH (4M) was then added to pH 12 and the sample was
centrifuged at 12,700 x g for 10 min to remove debris. The
supernatanat fluid was collected and tested for serological
activity with the appropriate antiserum. The supernatant was then
placed on a gel filtration column to characterize the effect of HF
on the molecular weight fractions of EPS.
Statistical Analysis
All statistical analyses performed in these studies utilized
the student's t test for unpaired samples (73).
CHAPTER III
RESULTS
Strain Variation and the Production of
Apparent Isogenic Sets
India ink preparations of a number of strains of K,. pneumoniae
revealed that not all bacilli of the same strain possessed a
similar size capsule. Two predominant capsule sizes co-existed
within many of the strains. A closer inspection of isolated
colonies on TSA revealed that the two basic colony types within a
given strain corresponded to the capsule size differences seen
under India ink. In particular, for KPl ATCC 8047 and for KP2 ATCC
29011, there existed an opaque (0) and a translucent (T) colony
type. Thus the co-variants in the KPl population were labelled
KPl-0 and KPl-T, and those in KP2 were designated KP2-0 and KP2-T.
In both cases the opaque variant possessed the larger capsule.
KPl-0 possessed a capsule with an average transverse diameter (TD)
of 5.6 ym, while KPl-T exhibited a TD of 2.5 ym. The capsules of
KP2-0 and KP2-T had TD of 2.5 ym and 1.5 ym respectively. The data
for capsule sizes of all the strains used in this study are given
in Table 1. Biochemical and serological typing were performed on
all variants to confirm species and serotype. All strains of K,.
pneumoniae used in these studies had an API 20E (Analytab) code of
5215773 except for KPl 2-70 which coded out as 5005773.
It was much more difficult to obtain large and small capsule
variants for the KPl CDC 2-70. Under India ink KPl 2-70 had a TD
of 2.2 ym. A large encapsulated organism (TD=5.6 ym) was
occassionally seen under India ink, though these were rare. The
30
31
Table 1 . Capsule Size of K. pneumoniae
OiaMlim.^ Tp^(ym) Range(ym)^
KPl-0 5.6 3.9 - 8.6
KPl-T 2.5 2.2 - 3.0
KPl 2-70 2.2 1.8 - 2.5
KP2-0 2.5 1.9 - 3.0
KP2-T 1.5 1 . 4 - 1 . 6
KP2 2-70 3.0 2.5 - 5.0
Bacteria were grown in defined medium for 18h.
TD; Transverse diameter as measured under India ink, calculated as the average (mode) of 100 random determinations.
^The range in TD re f lec ts the smallest and largest TD seen in a s ingle preparat ion.
32
large capsuled variant was finally isolated by enriching the
population by passage through mice.
After several attempts, a sub-population of KP2 CDC 2-70
differing in capsule size could not be isolated. Under India ink
the average TD for KP2 2-70 was calculated to be 3.0 ym. However
the variance in capsule size in this strain ranged from 2.5 to 5.0
ym. A population rich in large encapsulated variants of KP2 2-70
was obtained by passage through mice. However, upon subculture,
the majority of large variants were no longer present in the
population. Furthermore there appeared to be an increase in the
presence of the larger encapsulated variants during the stationary
phase of growth, with an average TD of 2.75 ym at 24h growth, 3.0
ym at 36h growth and 3.3 ym at 48h. This is the only strain among
those utilized in these studies that had the propensity to change
its average capsular diameter at various stages of culture. Figure
la and lb show transmission electron micrographs (TEM) of KP2-0 and
KP2-T, respectively, as examples of the variance in the dimension
of capsule size within a given population.
The Establishment of a Chronic Lobar
Pneumonia by K. pneumoniae
in a Rat Model
Early studies employed KPl ATCC 8047 in chronicity studies, as
described in Materials and Methods. Results of lung bacterial
concentration are shown in Table 2. The total viable bacterial
count (TBC) is expressed as the log-iQ of the average total lung
bacterial concentration for the experimental animals in each group.
As can be seen, the log-j^TBC remained elevated (range of 4.11 to
33
34
36
37
Table 2. Establishment of Chronic KPl Pneumonia in Rats
Group Number of animals Day
dead/total Sacrificed
Log 10
TBC" (CFU)
(Range)
1
2
3
4
5
6
7
8
Control
0/4
0/4
0/4
0/4
0/4
0/4
1/4
2/4
0/8
1
3
6
9
7
14
21
28
3, 6, 7, 9,
14, 21, 28
6.51
7.02
6.32
6.91
9.47
6.20
3.84
3.19
ND^
(5.96-6.90)
(5.67-8.46)
(4.11-8.51)
(4.60-8.95)
(9.05-10.16)
(5.86-6.65)
(2.00-5.00)
(ND^-6.38)
^Rats were inoculated transtracheally with 5 x 10 CFU of KPl in 0.05 ml sterile PBS and sacrificed on the days indicated. Controls received 0.05 ml sterile PBS in the same manner. All surviving rats (except the four used for histological processing on days 1, 3, 6 and 9) were used for bacterial quantitation of lung tissue.
^TBC; viable bacterial count per whole lung expressed in log.|Q units from surviving rats.
^ND; none detected at 10' dilution of lung homogenate.
38
10.16) throughout the first fourteen days of the study, while no
KPl were detected in the lungs of the control animals. After day
14 it became difficult to obtain a statistically sound estimate of
the TBC in the rat lungs due to deaths occurring in the 21 and 28
day groups. Only one of the eight experimental rats in the 21 and
28 day groups cleared KPl from its lungs, while the four remaining
animals had log^^TBC of between 2.00 and 6.38. Mortality for the
entire population was 5 per cent (3/60), but for those rats that
were sacrificed on or after 21 days, 37.5% (3/8) of the animals
died before the time of sacrifice.
By day 2 post-infection virtually all experimental rats
appeared acutely ill. Mucous secretions exuded from their eyes and
most exhibited short and rapid breathing. As the infection
progressed, their coats became shabby and considerable weight loss
was obvious. Gross examination of the lungs showed involvement of
one or more lobes, often affecting the entire lobe in a typical
lobar distribution. The involvement was characteristically massive
and voluminous, presenting as dull, greyish regions that released
copious amounts of purulent exudate upon sectioning.
Histological examination also supported the establishment of a
lobar pneumonia in this rat lung model. Figure 2 (a-e) are
photomicrographs of H&E stained sections of rat lung tissue showing
the progressive development of a confluent pneumonia. Figure 2a
depicts normal lung tissue, representative of all control animals.
The integrity of the alveolar and bronchiolar structures can be
easily visualized. In marked contrast, lung tissue typical of 24h,
39
post-exposure rats (Fig. 2b) shows a phagocytic infiltrate
consisting primarily of polymorphonuclear leukocytes (PMN) filling
the alveolar spaces. By day 3 post-infection (Fig. 2c) a confluent
pneumonia had developed. The structural integrity of the
bronchiolar, columnar epithelium had been compromised, and signs of
necrosis and early abscess formation were evident. Large abscesses
and liquefication of structural walls were characteristic of
infection by day 6 (Fig. 2d). By day 9, foci of chronic abscess
formation were evident (Fig. 2e) with collagen fibers visibly
forming a wall to contain the abscess. This process of progressive
destruction of lung tissue continued up to day 28 when the study
was terminated.
The next set of experiments was performed to determine the 50
per cent infective dose (IDCQ) in the rat model for the KPl 8047
strain, before this strain was separated into its capsular o
variants. KPl was grown to a concentration of 1.55 x 10 CFU/ml in
TSB, washed twice and resuspended in cold PBS. Serial 10-fold
dilutions were then made in cold, sterile PBS and the organisms
were kept on ice until inoculation. Thirty rats were employed in
these studies and were divided into five groups of six animals
each. Group 1 received 0.05 ml of the undiluted KPl suspension
(7.76 X 10^) transtracheally into the left lower lobe of the lung.
Group 2 received the same volume of the first 10-fold dilution r 2
(7.76 X 10 CFU) and so on to group 5 which received 7.76 x 10 CFU
of KPl. All rats were sacrificed on the sixth day after KPl
administration. Table 3 summarizes the results obtained. Twenty
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52
per cent (6/30) of the rats died during the course of this
experiment with half of these belonging to group 1. Lung weight
was determined to document the marked increase in lung size in
infected rats. Serum lysozyme levels were also examined in this
study because these values have been shown to covary with the
extent of infection (4). As can be seen in Table 3, the serum
lysozyme levels of all groups of animals receiving KPl were
elevated with respect to control values, achieving significance at
the p < 0.01 level in two of the groups. Due to the marked
swelling during the infectious process, the weight of the lungs
increased up to more than three times that of normal. The average
lung weight for the infected rats in this study was 5.0 grams (3.1
grams above the control mean lung weight). Rats were considered
infected if they either succumbed to the KPl-induced pneumonia or
if a TBC of at least 5 x 10^ Oog^Q TBC=4.7) was found in the whole
lung of those rats harboring the organism. An ID^Q for KPl of 1.55
X 10 CFU was thus obtained.
In order to test the effect of the medium in which the
bacteria were grown as contributing to the virulence of the
organism, a defined medium was used and the effect of dosage of KPl
was repeated in the same manner as above. The ID^Q obtained using 5
the defined medium was found to be 2.22 x 10 CFU, which does not
differ significantly from the ID^Q value obtained using TSB.
Therefore, the comparative effect of growing l<. pneumoniae in two
different media on the pathogenicity of KPl in the rat lung model
appears to be negligible.
53
The remainder of the bacterial strains and their subvariants,
with the exception of the KP2-T and KPl CDC 2-70 strains were then
examined in the rat lung model. All organisms were grown in
defined medium and harvested as described in materials and methods.
Table 4 shows the results obtained when various concentrations of
KPl-0 were inoculated transtracheally into the lungs of normal
rats. With an initial inoculum of 5.0 x 10^ CFU all rats became
infected, three died, and the one remaining rat harbored a TBC of
1.43 X 10^ CFU at the time of sacrifice. All rats receiving 5.01 x
10^ CFU or 5.01 x 10^ CFU of KPl-0 also became infected. One rat
died in each of these two groups, while lung weight and serum
lysozyme were elevated. In groups 4 and 5, which received 5.01 x
10^ and 5.01 x 10^ CFU of KPl-0 respectively, three of the four
rats in each group were infected, one rat in each group died and
serum lysozyme as well as lung weight was elevated. Finally, a
dose of 5.01 X 10^ CFU of KPl-0 or less did not result in an
infection of any rats.
Table 5 shows the results obtained when various concentrations
of KPl-T were inoculated transtracehally into the lungs of healthy
rats. An initial inoculum of 7.07 x 10^ CFU of KPl-T resulted in
the death of three of the four rats in the first group. The one
remaining rat effectively cleared this massive inoculum of KPl-T
organisms placed in its lungs and showed no overt signs of
pathology. Only one of the four rats in the second group, which
received 7.07 x 10^ CFU of KPl-T, succumbed to the infection, while
the remaining three rats showed no signs of infection at the time
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56
of sacrifice. One of four rats in group 3, which received 7.07 x
5 A 10 CFU of KPl-T, showed signs of infection (greater than 5 x 10
CFU per whole lung). Finally, all rats receiving 7.07 x 10^ CFU or
less of KPl-T (groups 4-6) did not become infected.
Table 6 shows the results obtained when various concentrations
of KP2-0 were inoculated transtracheally into the lungs of normal
rats. Doses of 7.2 x 10^ CFU did not result in infection in three
of the four animals in the first group. The fourth rat had a TBC 4
of 5.25 X 10 CFU with a lung weight of 3.2 grams and was
considered infected. No rats in group 2, which received 7.2 x 10
CFU of KP2-0 were considered infected using our criteria. One of 5
the four rats in group 3, which received 7.2 x 10 CFU of KP2-0 was
infected, but all other rats in this group and in the ensuing
groups (groups 4-6) had cleared K_. pneumoniae from their lungs.
Serum lysozyme was not significantly elevated in any group within
either the KPl-T or the KP2-0 study.
Table 7 shows the effect of various doses of KP2 2-70 4
inoculated into the lungs of rats. Doses of 6.5 x 10 CFU per rat
resulted in the infection of 6 of 8 animals at 7 days 3
post-inoculation. Group 2 received 6.5 x 10 CFU and showed 3 of 8
rats infected. Three animals from group 3 died and 1 of the
remaining 5 were infected. Group 4 rats which received 6.5 x 10
CFU revealed 5 of 8 animals having a TBC above the threshold for
infection. Finally group 5 rats, which received 6.5 x 10 CFU per
rat, showed 3 of the rats infected by day 7 post-inoculation. Both
lung weight and serum lysozyme were elevated in these rats, but
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59
showed a gradual decline to near control values with increasing
10-fold dilutions of inoculum. Seven rats inoculated with 6.46 x 3
10 CFU KP2 2-70 (as in group 2) were sacrificed at 14 days
post-inoculation. Of these rats one died on day 3 and one had a
9 A TBC of 4.7 X 10 . Three rats had a TBC of above 1 x 10 but not
above the threshold of 5 x 10 . The remaining two rats had TBC of
3 3
1.6 X 10 and 3.6 x 10 . Therefore none of the rats had cleared
KP2 2-70 from their lungs though only 2 of 7 rats exhibited all the
signs of infection at day 14 post-inoculation.
The IDcQ in rats for each of the five J<. pneumoniae strains
employed in these studies can be seen in Table 8 along with the
LDcQ values for each strain in mice. The data are analysed in the
following section.
K. pneumoniae Virulence in a Mouse Model
All J<. pneumoniae serotype 1 and serotype 2 strains and their
variants were employed in standard virulence assays as described in
Materials and Methods. Result of these studies are compiled in
Table 8 together with the data obtained from studies of virulence
in the rat lung model. These data show that KPl-0, which exhibited
the largest capsule, was more virulent than its covariant, KPl-T,
by 4 or more log-jQ units. Similarly KP2-0 exhibited a larger
capsule than its covariant, KP2-T, and proved to be more virulent
in the mouse model. The TD of KPl-T was slightly larger than the
TD of KPl 2-70 and was more virulent than KPl 2-70 by more than 2
log-ip, units. Therefore, a direct correlation between two distinct
60
Table 8. LDHQ Values in Mice and ID^Q Values in Rats for Strains of l<. pneumoniae Serotypes 1 and 2.
Organism LD^Q (CFU)^ ID^Q (CFU)*^
KPl (mixed) 1.92 x 10 ^ 1.55 x 10^
KPl-0 4.99 X 10^^ 3.41 x 10^
KPl-T 6.03 X 10^^ 1.53 x 10^
KP2 2-70 1.00 X 10° 4.70 x 10^
KP2 (mixed) 4.29 x 10^ NP'
KP2-0 1.78 X 10^ >7.3 x 10^
KP2-T >6.2 X 10^ NP^
^Five groups of f i ve mice each were inoculated IP with ser ia l 10-fold d i l u t i ons of the appropriate K,. pneumoniae s t ra in in 1.0 ml of s t e r i l e PBS and observed for a 72 h period. LD^Q values were calculated by the method of Reed and Muench (65) ana represent at least two determinations for each organism.
^Rats were considered to be infected i f they succumbed to the i r pneumonia or i f the TBC was 5 x 10 CFU or greater.
^The IDrr. values for KPl-0 and KPl-T were shown to be s ign i f i can t l y d i f f e ren t (p<0.025).
°NP; not performed.
61
populations within a serotype regarding the relationship between
virulence and capsule size appears to exist.
In Vitro Quantitation of Extracellular
Polysaccharides Produced by
K. pneumoniae
Bacteria were grown in a defined medium (DW) and harvested as
described in Materials and Methods. The ECPS production by KPl-0,
KPl-T and KPl 2-70 are summarized in Table 9. With regard to the
apparent isogenic covariants, the results indicate that KPl-0
produces ECPS to a much greater extent than KPl-T at all stages of
culture. -The expression of ECPS by both strains is linear with
respect to time over the entire period of analysis. The KPl-0
organism produced 0.37 yg ECPS ml" cell" (x 10 ) per hour of
culture, while the KPl-T strain produced 0.067 yg ECPS ml" cell"
(x 10^) per hour in this same period between 18 and 48h of growth
in DW. The overall ratio of the rate of production of ECPS between
KPl-0 and KPl-T is estimated to be 5.52:1. These data are
illustrated in Fig 3. The difference between the means of the
production of ECPS by these two bacteria is significant at p <
0.005. The production of ECPS by KPl 2-70 was intermediate between
that of KPl-0 and KPl-T and a rate of ECPS production of 0.15 yg
ECPS* ml"^ cell"^ x 10'^ per hour was characteristic of this
organism. Figure 4 shows a TEM of KPl 2-70, depicting both the
cell-associated and the extracellular capsular material.
62
Table 9. ECPS Production by Strains of 1<. pneumoniae Serotype 1 at Various Intervals of Incubation
Organisms Incubation Period (h) ECPS^ CFU/ml
5.50+3.68 1.17x10^
8.49+3.71 1.17x10^
10.75+3.70 5.64x10^
18.24+1.32 2.00x10^
0.67+0.03 3.7x10^
1.09+0.11 3.7x10^
1.63+0.03 2.67x10^
2.56+0.09 1.82x10'' p<0.001^
0.52+0.24 3.8x10^
2.12+1.55 4.0x10^
9.49+p.OO 3.0x10^
10.01+.2.48 1.1x10^ p<0.01^
p<0.05^
^Cultures were grown at 37°C in defined medium at 200 rpm.
^ECPS; in yg ml"^ c e l l " ^ (xlO^) as quantitated by the method of Blumencrantz and Asboe-Hansen (7) .
^S ta t i s t i ca l analysis comparing ECPS ml"^ ce l l " ^ production of KPl-0 to KPl-T or to KPl 2-70 at 48 h incubation.
^Comparison of KPl-T to KPl 2-70 as in footnote C.
KPl-0
KPl-0
KPl-0
KPl-0
KPl-T
KPl-T
KPl-T
KPl-T
KPl 2-
KPl 2-
KPl 2-
KPl 2-
•70
•70
•70
•70
18
24
36
48
18
24
36
48
18
24
36
48
63
64
UJ
(Q.OI^)J|a3J^-Scd03 E'h'
65
66
67
Table 10 summarizes the ECPS production of the various KP2
serotypes. As can be seen, KP2-0 produces more ECPS than its
co-variant, KP2-T, which produces ECPS at near non-detectable
levels in the supernatant fluid at all intervals of incubation. A
comparison of ECPS production between KP2-0 and KP2 2-70 shows a
difference in kinetics, as illustrated in Figure 5. KP2-0 begins
to produce ECPS much earlier in culture than KP2 2-70, but by 36h,
KP2 2-70 has surpassed KP2-0 in total ECPS production and has
produced twice as much ECPS by 48 h. These differences are
reflected in the rate of ECPS production within this time period.
At up to 18h of culture, the rate of production for KP2 2-70 is
essentially negligible, but it increased significantly at
approximately this time period. Production is linear between 18
and 36 h for both strains, but the rates of production differ
markedly within this time frame, with KP2 2-70 producing 0.078 yg
ECPS ml"^ cell"^ (xlO"^) per hour and KP2-0 producing 0.027 yg ECPS
ml" cell" (x 10" ) per hour. Therefore, in this particular
medium, KP2 2-70 produces nearly three times as much of ECPS as
does KP2-0 within the linear phase of production.
Extracellular LPS (ELPS) production was also examined during
these time periods and the data are shown in Table 11. This table
shows the amounts of ELPS produced by all strains at 48 h of growth
in DW as measured by both the KDO and the LAL assays. Colony
forming units at the highest concentration during cultural growth
are also listed in Table 11. The ELPS is expressed as the yg ELPS
ml' cell' (x 10" ). It can be seen from this table that the
68
Table 10. ECPS Production by Strains of J<. pneumoniae Serotype 2 at Various Intervals of Incubation
Organisms Incubation Period (h) ECPS CFU/ml
KP2-0
KP2-0
KP2-0
KP2-0
18
24
36
48
0.48+0.20
0.59+0.11
0.90+0.001
1.30+0.13
9.22x10'
1.1x10^
9.2x10^
8.6x10^
8
KP2-T
KP2-T
KP2-T
KP2-T
18
24
36
48
0.002'
ND'
1.1x10-
0.003+0.002 9.9x10 8
1.1x10"
0.005+0.0002 1.0x10-
KP2 2-70
KP2 2-70
KP2 2-70
KP2 2-70
18
24
36
48
0.06+0.03
1.66+0.16
2.13+0.11
4.0x10^ p<0.20^
8 0.38+0.02 4.2x10 p<0.10
3.9x10^ p<0.025
3.9x10^ p<0.020
^Cul tures were grown a t 37°C i n def ined medium at 200 rpm.
^ECPS; i n yg ml""" c e l l " ^ ( x l O ' ^ ) .
^ND; none de tec ted .
^ S t a t i s t i c a l ana lys is comparing ECPS ml"^ c e l l " product ion of KP2-0 and KP2 2-70 a t the same stage of incuba t ion .
^Only one determinat ion made.
69
70
24 TIME(h)
71
_ Dtypes 1 and ; at 48h of Culture in Defined Medium
Table 11. Production of ELPS^ by J<. pneumoniae Serotypes 1 and 2
Organism
KPl-0
KPl-T
KPl 2-70
KP2-0
KP2-T
KP2 2-70
CFU/ml
1.17x10^
3.70x10^
4.00x10^
9.48x10^
1.05x10^
4.25x10^
(xlO"^)(KDO)^
1.43+0.04
0.38+0.14
0.54+0.03
0.03+p.OOl
0.01+0.002
0.07+0.007
yg ELPS mr^cell"^ yg ELPS ml'^ceir^
(xlO"^)(LAL)^
1.37 - 2.74
0.22 - 0.43
0.21 - 0.42
0.008-0.017
0.004-0.008
0.04 - 0.08
^Ext racel lu lar l ipopolysaccharide (N=2).
KDO; Thiobarbi tur ic acid assay for ketodeoxyoctanate (57).
^LAL; Limulus Amoebocyte Lysate assay. Values are presented as the range in which the quant i ty of ELPS in the samples is l im i ted (47).
72
KPl-0 organism produces the greatest quantity of ELPS per ml per
cell of all the strains, whereas the KP2 2-70 organism is the
highest ELPS producer among the KP2 strains.
Comparisons of ECPS, ELPS and capsule size at 48h growth for
all serotype 1 and 2 strains as well as virulence in both the rat
lung model and the mouse model can be seen in Table 12. First of
all, there exists a strong postive correlation (r=0.97) between the
production of ECPS and ELPS, per ml per cell. The two serotypes
differ in this regard in that the KPl strains produced
approximately 59 yg of ELPS for each mg of ECPS produced, while the
KP2 strains produced about 30 yg of ELPS per mg of ECPS. Therefore
the correlation between the production of ECPS and ELPS is
strongest within serotypes.
The relationship between ECPS and the capsule size of all
strains shows a direct positive correlation (r = 0.95), which is
even of greater magnitude when comparing strains within serotype
for both the KPl and KP2 strains. Therefore, in general, all of
these organisms seem to reflect their ability to produce
extracellular capsular polysaccharide by the size of their
capsules. A notable exception is the KPl 2-70 strain which seems
to have a TD less than that of KPl-T but produces more ECPS per
cell than KPl-T.
Finally in the comparison between the three parameters of
polysaccharide production (TD, ECPS, and ELPS per cell at 48h
incubation) and the virulence studies in the standard mouse
virulence model, an inverse correlation was noted. Table 13
73
Table 12. Comparison of ECPS, ELPS, Capsule Size and Virulence of J<. pneumoniae Serotypes 1 and 2
Organism
KPl-0
KPl-T
KPl 2-70
KP2-0
KP2-T
KP2 2-70
ECPS
18.24
2.56
3.73
1.30
0.005
2.13
ELPS^
1.43
0.38
0.54
0.03
0.01
0.07
TD^
5.6
2.5
2.2
2.5
1.5
3.0
LD ^
4.9x10^
5.34x10^
7.30x10^
1.78x10^
>6.2xl0^
1.0x10°
^^50'
3.41x10^
1.53X10'
NP^
>7.3xlO'^
NP
4.7x10^
a - 1 - 6 ECPS yg ml" cell"(xl0 ) in dialyzed supernatants of 48h growth at 37 C in defined medium.
h - 1 - 1 fi
ELPS yg ml" cell" (xlO ) in dialyzed supernatants of 48h growth at 37 C in defined medium.
^transverse diameter.
LDrni 50% lethal dose, obtained from IP injections in mice. bU
^IDj-^; 50% infectious dose, obtained from transtracheal inoculations into the lungs of rats.
Not performed.
74
Table 13. Correlations Between Polysaccharide Production and Virulence in the Mouse Model
ECPS/cell^ ELPS/cell^ TD°
Serotype 1 ( L D ^ Q ) ^ -0.46 -0.37 -0.50
Serotype 2 ( L D ^ Q ) ^ -0.82 -0.72 -0.96
All strains (I-D^Q)^ -0.31 -0.15 -0.55
^Quantity of ECPS per cell in dialyzed supernatants at 48h growth in defined medium at 37 C.
Quantity of ELPS per cell as in footnote a.
^Transverse diameter of the capsule.
^The LDnn values obtained from 3 serotype 1 strains. bU
^The LDc^ values obtained from 3 serotype 2 strains. bU
^The LDj-f. values obtained from all 6 strains of type 1 and type 2 K_. pneumonVae.
75
represents the correlations obtained between the polysaccharide
parameters and the virulence data. The data from Table 13 show
that capsule size may be the best indicator of the ability of a
particular strain to be pathogenic, especially when the serotype is
unknown. However, the production of ECPS per cell correlates
nearly as well as does capsule size with these virulence
parameters. Finally the production of ELPS per cell gives the
least amount of information as to the virulence potential of a
given strain, though it still correlates with virulence. Again,
stronger correlations are found within serotype than in grouping
the two serotypes together, which is especially true for the KP2
strains where nearly perfect correlations were obtained.
Serum Sensitivities and Opsonophagocytic
Assays for K. pneumoniae
Of the six strains used in these studies, only one of the
strains (KP2-T) showed inhibition of growth in the presence of 90%
rabbit serum over a 60 min period. Data for all strains can be
seen in Table 14, which shows the change in the log-jQ CFU between
time 0 and 60 min of incubation in serum. The virulent KPl-0 and
KP2 2-70 strains seem to grow most favorably in 90% serum, but
their growth is not significantly different than those of the other
strains, with the exception of KP2-T.
The ability of these organisms to grow in the presence of
human leukocytes (WBC), type-specific antiserum (AB), or a
complement source (C) was then tested, as described in materials
76
Table 14. Serum Sens i t i v i t y of K_. pneumoniae
Strain^ ^^O^^Q C F U ^
KPl-0 + 0.97
KPl-T + 0.54
KPl 2-70 + 0.50
KP2-0 + 0.42
KP2-T - 0.08
KP2 2-70 + 0.64
^Log phase organisms were washed 3 times and resuspended in PBS in various concentrations and added to 9 parts normal rabbi t serum.
^The change in the number of colony-forming units (CFU) in log,Q uni ts a f te r 60 min incubation in 90% normal rabbi t serum at 3/ C.
77
and methods under Opsonophagocytic Assays (OPA). Table 15
summarizes the results after normalizing the data for comparative
purposes. Normalization of the data was as follows: 1) Colony
counts in log-jQ CFU that were obtained for each strain at 0 min
were subtracted from the log-jQ CFU at 60 min incubation in the OPA.
The net change in the log^^ CFU (Alog,Q CFU) was thus obtained. 2)
Secondly the Alog.Q CFU for the control assay, containing only
heat-inactivated serum (without AB, WBC or C), was subtracted from
the Alog-jQ CFU of all other assays within a given strain. The
final value then shows the Alog-.^ CFU from 0 to 60 min compared to
the serum controls and allows for comparisons among the strains
tested. The results in Table 15 show that the most important
variables which affect the Alog,Q CFU is the presence of WBC and
type-specific AB in the assay mixture. The effect of a complement
source did not seem to affect the net log-jQ CFU in these assays.
Also it seems that WBC do not significantly alter the net
log-,p. CFU of any of the strains in question in the absence of AB.
One notable exception to this conclusion involves the KPl-T strain
and its ability to be more readily phagocytosed in the presence of
complement (C-E) than in the absence of complement (D-E), while in
the absence of AB. But also for the KPl-T strain, type specific
antiserum enhances the ability of WBC to ingest KPl-T moreso than
does complement, though this was not a significant difference.
Table 16 shows the results obtained when KPl 2-70 or KP2-0 was
tested in the OPA in the presence of EPS from either KPl or KP2,
containing both ECPS and ELPS. When the EPS from a type 1 strain
78
Table 15. Opsonophagocytic Assay^
(AB+WBC+C) (ABC+WBC) (WBC+C) (WBC)
Strain Control Control^ Control^ Control^
KPl-0 -2.08^ -1.49 -0.10 -0.12
KPl-T -0.27 -0.27 -0.16 +0.16
KPl 2-70 -0.39 -0.42 -0.02 -0.03
KP2-0 -0.19 -0.30 -0.09 -0.13
KP2-T -0.40 -0.22 -0.01 -0.07
KP2 2-70 -0.81 -0.79 +0.37 +0.05
Log-phase organisms were washed in PBS and various concentrations were placed in 3 parts normal rabbit serum containing combinations of the following components: 1) type specific antiserum (AB)-and 2) Human peripheral white blood cells (WBC). The serum was heat-inactivated in some of the trials to test for the effect of a complement (C) source. The assay was performed at 37 C for 60 min and the net CFU in log-iQ units was determined. See appendix 4 through 9.
The net log-,p. CFU obtained from the assay in which AB, WBC and C were present, minus the net log-jQ CFU obtained from the control assay where none of these components were present (heat-inactivated normal rat serum only).
^The net log-.^ CFU obtained from the assay in which AB and WBC but no C source were present, minus the net log-jQ CFU from the control assay.
^The net log-jp, CFU obtained from the assay in which WBC and C but no AB were firesent, minus the net log-jQ CFU obtained from the control assay.
^The net log-.^ CFU obtained from the assay in which WBC but no AB or C were fDresent, minus the net log^Q CFU obtained from the control assay.
79
Table 16. Effect of the Addition of EPS on the OPA^
Strain EPS Alog^^ CFU^
KPl 2-70 PBS 0.56
KPl^ 1.19 p<0.0l3
KP2^ 0.67
KP2-0 PBS 0.41
KPl^ 0.35
KP2^ 0.68 p<0.005^
The OPA contained the following components in equal volumes: Human peripheral white blood cells at 1 x 10 gper ml in normal rabbit serum; J<. pneumoniae strains at 1 x 10 to 1 x 10 CFU/ml in normal rabbit serum; Rabbit antiserum against the homologous serotype, and either KPl or KP2 EPS in PBS or PBS alone. A total serum concentration of 75% was used.
Change in CFU from 0 to 60 min incubation at 37°C in log units.
^KPl EPS from dialyzed supernatants of KPl-0 at 339 yg ECPS/ml and 20 yg ELPS/ml.
^KP2 EPS from the neutral fraction of KP2-0 at 850 yg ECPS/ml and 25 yg ELPS/ml.
^KPl EPS from the LMW fraction of KPl-0 at 331 yg ECPS/ml and 23 yg ELPS/ml.
^KP2 EPS from the ethanol extracted fraction of KP2-0 at 489 yg ECPS/ml and 16 yg ELPS/ml.
^Statistical analysis comparing the Alog.« CFU after treatment with homologous EPS compared to treatment witn either PBS or heterologous EPS.
80
was added to the KPl 2-70 OPA mixture at 339 yg ECPS/ml and 20 yg
ELPS/ml there was a significant difference in the Alog,Q CFU from
the same mixture without ECPS present. However, when KP2-0 EPS,
containing 850 yg ECPS and 25 yg ELPS per ml was added to the same
OPA, no significant difference was seen compared to the non-ECPS
control. In the reverse experiment the KP2-0 strain with the
addition of KP2 EPS at 489 yg ECPS/ml and 16 y ELPS/ml grew
significantly better than both the antiserum control (PBS treated)
or the KPl ECPS treated trials (331 yg ECPS and 23 yg ELPS per ml).
Therefore EPS from a type-specific strain allowed for enhanced
growth of KPl 2-70 over controls in the presence AB, whereas EPS
from a heterologous serotype did not.
Purification of the EPS of K. pneumoniae
Organisms were grown in the defined medium at 37 C for 48h
while shaking at 200 rpm for these studies. The purification
protocol, as described in Materials and Methods, involved ethanol
fractionation, DEAE-Sephacel ion exchange and gel filtration
chromatography. Fractionation with ethanol produced a hygroscopic,
white and fluffy material which was not easily resuspended in
hydrophilic solutions and was precipi table in non-polar solvents
(i.e., methanol, ethanol, chloroform, hexanes). When suspended in
DHpO these materials produced highly viscous solutions, especially
at a concentration of 2 mg dry weight or more per ml. These
solutions were characteristically opalescent and, for certain
preparations, a white precipitate was noted. Removal of the
precipitate by centrifugation caused as much as a 25% loss of ECPS
81
from KP2 preparations but no detectable loss of ECPS from KPl
preparations. The KP2 preparations in general, dissolved less
easily in DH2O than the KPl preparations, but seemed to dissolve
much better in alkaline solutions above a pH of 11. A Tris buffer
was then used (0.01 M Tris, pH 12) in many of the subsequent
purification steps, especially to avoid the cessation of a column
run due to aggregates of KP2 EPS clogging the column filters.
Ethanol fractionated EPS was resuspended in 0.01 M Tris, pH
12, or in another low ionic strength buffer [i.e., 0.02 M
(NH4)2C02], and placed on a DEAE-Sephacel column equilibrated with
the same buffer. The column was eluted with approximately 200 ml
of buffer before the salt gradient was applied. It was found that,
for all of the ethanol fractionated samples from the strains used
in this study, yielded a fraction of uronic acid containing
material eluting from the column at this time. This fraction was
labeled the neutral (N) EPS fraction. When a salt gradient was
applied [up to IM concentrations of either NaCl or (NH^)2C02],
a second uronic acid and hexose containing fraction eluted. An
example of an elution profile on DEAE Sephacel can be seen in
Figure 6 for the KPl-T ECPS. Table 17 shows the ionic strength of
the buffer at which two KPl and two KP2 EPS samples were eluted.
The acidic (A) fraction of EPS for the various samples eluted
between 0.2 and 0.4 M (NH^)2C03.
The acidic and the neutral fractions, after dialysis and
lyophilization, were resuspended in the appropriate column buffer
(O.OIM Tris, pH 12, or 0.5M NaCl were commonly used) and placed on
82
Table 17. Elution Ionic Strength and Apparent Molecular Weights of the ECPS from Various Strains of KPl and KP2
Elution
ECPS
KPl-O(N)^
KPl-O(A)^
KPl-T(N)
KPl-T(A)
KP2-0(N)
KP2-0(A)
KP2-270(N)
KP2 2-70(A)
HMW^
>3xl0^
>3xl0^
>,3xlO^
>^3xlO^
>3xl0^
>^3xlO^
>3xl0^
>3xl0^
%total
15
10
52
53
60
70
100
100
LMW^
8.9x10^
5.0x10^
9.4x10^
1.1x10^
2.3x10^
2.0x10^
—
_ — —
%total
85
90
48
46
40
30
--
_—
Ionic strength
0.02
0.42
0.02
0.21
0.02
0.23
0.02
0.17
^HMW; the high molecular weight or void volume fraction of EPS eluted from S-2B gel filtration.
^LMW; the low molecular weight fraction of EPS found within the, inclusion capabilities of the gel filtration column.
^Elution Ionic Strength; the salt gradient molarity of (NH^)2C03 at which the various EPS fractions eluted from DEAE-Sephacel.
*^(N); neutral fraction.
^(A); acidic fraction.
83
84
20 30 40 50 60 70 80
Fraction Numbtr 90
85
gel filtration (S-2B or BGA-150m) columns. Examples of elution
and KP2 2-70(A) EPS on S-2B can be seen in Figures 7 through 13.
For all strains utilized in these studies, the acidic and the
neutral fraction of EPS produced a high molecular weight (HMW),
hexose and uronic acid containing fraction at the void volume of
the gel filtration profile. For the KP2 strains the vast majority
of the polysaccharide material eluted in this HMW fraction, whereas
only 10 to 15 percent of the acidic or neutral EPS from KPl-0 and
52 to 53 percent from KPl-T eluted at this HMW fraction. The
remainder of the hexose and uronic acid containing material for all
strains was within the inclusion capabilities of the gel filtration
column, and was typically contained in one fraction. This fraction
was labelled the low molecular weight (LMW) fraction. For the
KPl-0 and the KPl-T strains this fraction accounted for 85-90% and
46-48% of all hexose and uronic acid containing material,
respectively. In contrast, it was the lesser of the two fractions
for the KP2 strains, with the KP2-0 strains producing a LMW
fraction which accounted for 30 to 40% of the polysaccharide in the
sample, and the KP2 2-70 strain apparently producing no LMW
material. It was found later that the KP2 2-70 strain does produce
a LMW component which appeared after ethanol extracted material was
placed on a BGA 150 m column equilibrated with 0.01 M Tris, pH 12,
but did not appear when 0.5 M NaCl was used with the acidic or the
neutral EPS. Table 17 displays the percentages of EPS that were in
86
87
Uronic Add/ ig /ml(o) o o o o o o o o o o o o
-r T
.a E
u o
« ^ ""—'—'—'—r
(•)|UJ/B7y980X9H
88
89
Uronic Acid / ig /ml (o)
(•) IUJ/BT/ OSOXOH
90
91
Uronic Acid/ig/mKo)
w — o a> l O CSJ —
(•) I U I / C T / 9S0X9H
92
93
Uronic Acid/ig/mI(o)
o Q o o o ^ (O (\j _
o o o O 0> OD
P o o o f «o «
( • ) | U J / 6 T / 9 « O X 9 H
94
95
10 15 20 25 30
FRACTION NUMBER
35 40
96
97
T
LU CO
o X UJ X
15 20 25
FRACTION NUMBER
40
98
99
Uronic Acid /ig/ml(o)
o o O O o - <D iO -t <Si
- I — I t . i
o <0
- o 'J-
«>
E 3
O o
o CVJ ro
O O fO
O cn CM
( • ) I U J / B T / 980X8H
100
101
250
200-
3 ' 50 UJ
o X Ul X
100-
60
FRACTION NUMBER
102
the HMW and the LMW peaks as well as the apparent molecular weights
calculated from dextran calibration standards (Fig. 14 and Table
18). The HMW fractions from all strains were outside the range of
the column and were calculated to be at least 3 x 10^ daltons in
molecular weight. The LMW fractions were, however, retained by the
column and gave molecular weights as seen in Table 17. For the KPl r c
LMW EPS the molecular weight range is between 5 x 10 and 1 x 10
daltons, while for the KP2-0 LMW EPS a molecular weight of around 2
X 10 daltons was estimated.
As purification proceeded, fractions containing hexose and
uronic acid were pooled, dialyzed and lyophilized. Dried materials
were then- brought up in 1 mg/ml or 2 mg/ml solutions in DHpO and
tested for their content of ECPS, ELPS and protein. Table 19 shows
the percentage of these components to the total dry weight of the
samples. It can be seen that nearly 50% of the KPl ethanol
extracted [KPl (EtOH) EPS] material was in the form of ECPS, while
approximately 5 to 7% of the dry weight was accounted for by ELPS,
and about 4 to 7% was protein. For the KP2 strains, 78 to 84% of
the dry weight of the ethanol extracted [KP2 (EtOH) EPS] material
was ECPS, 2.4 to 2.5% was ELPS and 1 to 1.7% was protein. Between
37 and 45% of the dry weight of KPl (EtOH) EPS and between 12 and
18% of the dry weight of KP2 (EtOH) EPS was unaccounted for in
these preparations by the methods used.
A second study of this type was then performed on the uronic
acid and hexose containing materials that were purified by DEAE-
103
Table 18. Elution Volumes for Dextran Calibration Standards on
S-2B
Dextran^ V ° K ^ av
5-40 X 10^ 159.8 0.0
2.0 X 10^. 250.0 0.27
5.0 X 10^ 379.8 0.67
2.3 X 10^ 396.7 0.72
1.7 X 10^ 413.6 0.76
8.1 X 10^ 415.5 0.77
9.4 X 10^ 453.08 0.89
^Dextran standards in average molecular weight (daltons)
V ; Elution volume in ml at peak of fraction.
''K ; determined by the equation av
V^ - V^ V^=159.8 ml e 0 0
^av
V. - V„ V =490 ml t o t
104
Table 19. The Extracellular Products Found in the Ethanol Fractionated Supernatants of K_. pneumoniae
Strain
KPl-0
KPl-T
KP2-0
KP2 2-70
ECPS^
52.2
42.6
78.2
84.1
ELPS^
6.5
5.2
2.5
2.4
Protein^
3.9
•7.0
1.0
1.7
Other^
37.4
45.2
18.3
11.8
Values of ECPS, ELPS and protein are expressed as the percentage of the dry weight of the sample. The ECPS was calculated from the uronic acid determinations which were performed by the method of Blumencrantz et al. (7). The ELPS was determined by the method of Osborn et al. (57). Protein was determined by the procedure of Lowry et al. (51).
Other; the percentage of unidentified components contributing to the dry weight of ethanol extracted materials.
105
Sephacel ion exchange chromatography. Table 20 shows the amounts
of ECPS and ELPS contained in 2 mg/ml solutions and the percent of
the total dry weight of samples from two KPl and one KP2 strains.
The EPS from KPl-0 exhibited the least amount of ECPS per mg of dry
weight sample, with the KPl-0 (A) EPS being 12.8% ECPS by weight
and KPl-O(N) EPS 20.2% by weight. The figures for KPl-T (A) and
KPl-T (N) were somewhat greater, as seen in Table 20. The KP2 2-70
(N) and KP2 2-70(A) EPS were the most highly purified ECPS samples
at this stage of purification (70 to 76% of the total dry weight).
The amount of ELPS contained in these samples was seen to correlate
directly with the amounts of ECPS present for both the KPl EPS
samples, and it was determined that 1.6 yg of ELPS was present for
eyery 10 yg of ECPS. For the KP2 2-70 EPS, which was a much more
highly purified preparation, 2.5 yg of ELPS was present for ewery
100 yg of ECPS. From 64 to 86% of the total dry weight for the KPl
EPS samples and between 22 and 28% of the KP2 2-70 EPS was left
unaccounted for by these measures.
The extracellular products found in KPl-0 and KP2 2-70 EPS
after gel filtration and the percentages of the total dry weight
that these products comprise are listed in Table 21 . It can be
seen that in 1 mg of the HMW fraction from KPl-0, about 30% was
ECPS, 8% was ELPS and about 9% was protein, and for the LMW
fraction 53% was ECPS, about 4% was ELPS and 3% was protein. Thus
for KPl-0 EPS the fraction most enriched with ECPS and containing
the least amount of ELPS and protein was the LMW fraction (with
106
Table 20. Comparison of the ECPS and ELPS content in the Neutral (N) and Acidic (A) Fractions from DEAE-Sephacel
Sample ECPS (N=2)
KPl-O(N)^ 403.1+45.1
KPl-O(A)^ 255.4+24.7
KPl-T(N) 608.3^51.8
KPl-T(A) 284.2+13.0
KP2 2-70(N) 1400.1+87.3
KP2 2-70(A) 1515.3+66.3
Quantity of component in yg contained in 2 mg of dried sample.
Percentage of component contained in the dried sample.
^(N); The neutral fraction of hexose-containing material obtained from DEAE Sephacel.
(A); The acidic fraction as in footnote c.
%total^
20.2
12.8
30.4
14.2
70.0
75.8
ELPS^ (N=2)
79.5+7.5
29.3+3.7
105.3+18.2
69.3+10.3
36.1+0.8
38.1+1.3
%total^
4.0
1.4
5.3
3.5
1.8
1.9
107
Table 21. The Extracellular Products Foynd in KPl and KP2 EPS After Purification
Strain
KPl-0
KPl-0
KP2 2-70
KP2 2-70
Fraction
HMW^
LMW^
HMW
LMW
ECPS'^
29.6
53.0
80.7
83.9
ELPS'^
7.9
3.7
2.8
1.6
n ^ . b Protein
8.7
3.0
4.9
3.4
Other^
53.8
40.3
11.6
11.1
^Purification procedures include ethanol extraction, DEAE-Sephacel and gel filtration chromatography.
^Values of ECPS, ELPS, and protein are expressed as percentage of the weight of the samples.
^Other; the percentage of unidentified components contributing to the dry weight of the samples.
* HMW; the high molecular weight fraction.
^LMW; the low molecular weight fraction.
108
approximately 7 yg of ELPS and 6 yg of protein per 100 yg of ECPS).
The KPl-0 HMW EPS contained much more of these components
(approximately 27 yg ELPS and 29 yg protein per 100 yg ECPS). The
HMW and LMW fractions from KP2 2-70 contained far less quantities
of ELPS and protein, with the HMW fraction containing about 2 yg
ELPS and 6 yg of protein per 100 yg ECPS and the LMW fraction
containing 0.3 yg ELPS and 4 yg protein per 100 yg ECPS. The ECPS
accounted for the vast majority of the dry weight of KP2 2-70 ECPS
samples at this stage of purification (between 81 and 84%).
Finally, Table 22 shows the percent yield obtained for KPl-0
and KPl-T EPS at various stages of purification starting with a 200
ml culture of each strain grown in defined medium for 48h at 37 C.
The data reveal yields of between 24 and 80% throughout the
purification procedures. The final yield for KPl-0 ECPS was 22.5%
while the final yield for the KPl-T ECPS was 7.4%. Much of the
KPl-T ECPS was lost during purification on DEAE Sephacel, which was
also shown above not to further purify the ECPS from either the
KPl-0, KPl-T or the KP2 2-70 strain. Indeed, the eluted fractions
from ion exchange appear to be less pure than any of the fractions
before or after this purification step. These purification steps
apparently have not purified the KP2 ECPS beyond the level of
purification achieved by ethanol fractionation for the KPl ECPS.
Ethanol fractionation resulted in a product that is 43 to 52% ECPS
by weight, while no other subsequent fractions from the
purification schema, except for the LMW fraction from gel
109
Table 22. Percent Yield Obtained from ECPS Pur i f i ca t ion of KPl-0
Stage Preparation ygECPS/mg dry wt^ Total ECPS Yield(%)'
I 48h Dialyzed 79.3+10.2 108.2+2.0 100
Supernatants
II Ethanol 522.2+24.6 62.7+3.0 58
Fractionation
III DEAE 185.4^13.6 30.4+^2.2 28
Chromatography
IV BGA-150m
Chromatography HMW 287.0+38.3 2.4+0.3
IV BGA-150m 23
Chromatography LMW 471.8+96.4 21.9+4.5
The quantity of ECPS in the samples were determined from uronic acid measurements performed by the procedure of Blumencrantz et al. (8).
Yields were determined as the percent of ECPS at each stage of purification compared to the amount in the 48h dialyzed supernatant.
no
filtration, were of equal purity. The KPl LMW fraction retained
the highest purity of all the KPl fractions in that it contained
lower quantities of ELPS (7 yg per 100 yg ECPS) than the ethanol
extracted fraction (12 yg ELPS per 100 yg ECPS).
Due to the rather poor level of purity obtained for the ECPS
of the KPl strains by the procedures above, a different method was
utilized to purify the KPl ECPS from the KPl-0 strain. Ethanol
fractionated KPl-0 EPS was resuspended in DH^O and subjected to
electrodialysis (ED) at 2000 V. All of the DH^O in the cathode and
anode chambers were collected, lyophilized and referred to as
fraction I (Fr I). The electrodialyzed EPS was then fractionated
with cetavlon. The cetavlon precipitate, labelled Fraction II (Fr
II), and the cetavlon supernatant, labelled fraction III (Fr III)
were both washed with ethanol (3 times) and the product was tested
for its content of ECPS, ELPS and protein. The results of these
procedures are presented in Table 23. With 150 mg of starting
material, 130 mg total product were obtained gravimetrically after
these purification steps. Fr I was shown to contain no detectable
hexose or uronic acid and comprised at least 19 mg of material.
Therefore nearly 13% of the starting material was dialyzed free of
the EPS during ED. Fr II was found to comprise about 71% of the
weight of the starting material, and 77% of Fr II was found
chemically to be ECPS while 3% was ELPS, 3.5% was protein and 16%
was unaccounted for by these methods. Fr III, on the other hand,
comprised only 3% of the starting material. It was determined
Ill
Table 23. Pur i f i ca t ion of KPl-0 (EtOH) EPS by ED, Cetavlon and Gel F i l t r a t i o n
Sample
KPl-0 (EtOH)^
Fr 11^
Fr I I I ^
Fr I^
Fr I I , S-2B^
Fr I I , S-2B,ED
KPl-0 (EtOH)ED
mg dry wt
150.0
106.0
4.5
19.0
58.0
9 ^p
^ NP
ECPS
522.17+24.64
766.77+14.36
494.40+.56.15
ND" '
620.14j: 3.13
732.10+35.42
802.47^18.91
ELPS^
62.66+2.96
29.70+0.86^
212.73+2.96
ND
3 . 0 5 + 1 . 3 1
6 . 1 5 + 0 . 6 6
54.37+0.87
Protein
39.0+1.5
34.8+.7.6
NP^
ND
9 . 2 + 2 . 0
1 9 . 6 + 2 . 7
NP
Units are in yg per mg dry weight.
^Ethanol extracted EPS.
^Fr I I ; the cetavlon prec ip i ta te from KPl-0 (EtOH) EPS washed x3 wi th EtOH.
^Fr I I I ; the supernatant from the cetavlon step washed x 3 with EtOH.
^Fr I ; the dialysable material col lected during ED (2000 molecular weight c u t o f f ) .
^Fr I I , S-2B; Fr I placed on Sepharose - 2B and the LMW component co l lec ted .
^Fr I I , S-2B, ED; Material in footnote d re-electrodialyzed.
•^KPl-O (EtOH) ED; KPl-0 (EtOH) EPS subjected to ED (separate experiment).
^NP; not performed.
"^ND; none detected.
112
chemically that 49.5% of Fr III consisted of ECPS while 21.3% was
ELPS, which left 30% of Fr III unaccounted for by these methods. A
portion of Fr II was then placed on a S-2B gel filtration column
and an elution profile was obtained as seen in Figure 15. This
figure illustrates that now the vast majority of the hexose
containing material elutes at a well defined LMW peak. Tube
fractions 25-45 were collected, dialyzed and lyophilized and
retested for ECPS, ELPS and protein content as seen under the label
Fr II, S-2B in Table 23. Nearly 90% of the ELPS has been removed
from this fraction, when comparing it to the Fr II starting
material, as well as 75% of the protein. However, the ECPS per mg
dry weight was nearly 15% less than that of Fr II. The Fr II, S-2B
material was then electrodialyzed to remove the salts acquired from
gel filtration. This final preparation, labeled Fr II, S-2B, ED in
Table 23, is now comparable to Fr II in its ECPS content (73%
compared to 77% respectively) and still shows comparatively low
levels of ELPS and protein. Finally, in a separate experiment
KPl-0 (EtOH) EPS was subjected to ED and then tested for ECPS and
ELPS content as shown in Table 23 under the label KPl-0 (EtOH) ED.
The ED step shows a 28% increase in the quantity of ECPS per mg dry
wt with comparable levels of ELPS, when compared to the KPl-0
(EtOH) EPS. Table 24 shows the amounts of ELPS and ECPS that were
contained in 100 yg ECPS from all of these purification steps.
Greater than 90% of the ELPS and between 65 and 80% of the protein
were removed from the KPl-0 (EtOH) EPS by these methods as seen in
113
114
80 '
70
- 6 0 ^ E ^ 50 r =k
U 4 0 o ^ 30 X
2 0
10
—
-
-
A A
10 20 30 40 50 60
FRACTION NUMBER
115
Table 24 Purification of KPl-0 (EtOH) EPS bv Fn cetavlon and S-2B: Percent of^LPs! Protean'''
and Other Materials
^^m}± ELPS/ECPS^ Protein/Frpc;a n^u R ^^o^^^ri/tCPS Other material/ECPS^
KPl-0 (EtOH)^ 12.00 7.47
F^ n ^ 3.87 72.04
4-54 22.01
' ' ' ' ' " ^ 4 3 . 0 3 NPJ- .• d
^' I ' ND " Mnl
Fr I I , S-2B^ 0 . 1 6
Fr I I , S -2B , ED^ 0 . 8 4
NP
ND' i p J
T-48 5 9 . 2 8
2 - 6 8 3 3 . 0 8
a A^un t of ELPS, protein or other materials in pg per 100 .g ECPS.
'•^As In Table 23.
116
this table. Extracting the EPS with cetavlon alone without prior
ED gave a similar elution profile as was seen for the EtOH
extracted fractions and a similar level of purity (559.15 and
607.49 yg ECPS per mg dry weight and 20.89 and 52.05 yg ELPS per mg
dry weight for KPl-0 and KPl-T samples respectively).
Fr III (3.5 mg) was applied to a BioGel P-300 gel filtration
column (P-300), equilibrated with 0.1 M ammonium acetate, pH 8.1,
containing 0.1% SDS, after boiling for 5 min in the column buffer.
The elution profile in Figure 16 shows the hexose containing
fractions obtained. The Limulus Amoebocyte Lysate assay indicated
that the majority of the ELPS was located under the peak that
eluted at tube fractions 26-34. This latter pool was collected,
washed 3 times with EtOH and tested for its ECPS and ELPS content.
It was found that this pool was enriched with ELPS (45 yg of ELPS
per 100 yg of ECPS), more so than all other samples encountered.
The KP2-0 EPS was also subjected to the above purification
procedures (EtOH ED CET S-2B). No substantial difference was
seen in the quantitites of ECPS or ELPS between the KP2-0 (EtOH)
EPS and the EPS obtained by the above methods. However, the Fr III
material obtained from the supernatant after cetavlon extraction
was enriched with ELPS (38.1 yg ELPS per 100 y5 ECPS) compared to
the Fr II precipitate from cetavlon treatment (4.21 yg ELPS per 100
yg ECPS). Table 25 summarizes the data obtained from these studies
with the KP2-0 EPS. Figure 17 shows the elution profile of these
materials on S-2B. KP2 2-70 (A) EPS was also subjected to ED and
117
118
50
4 0 -
•g 3 0
C3>
3 U l CO
o X Ul X
20r
0 -
20 30 40 50 60
FRACTION NUMBER
119
120
FRACTION NUMBER
121
Table 25. Pur i f i ca t ion of KP2-0 EPS by ED and Cetavlon
Sample ELPS/ECPS^
KP2-0 (EtOH)^ 3.25+0.01
KP2-0 (EtOH)ED^ 3.20+0.27
KP2-0 (EtOH)ED, CET PPT^ 4.21+0.18
KP2-0 (EtOH)ED, CET SUPE^ 38.05+3.59
^Amourt of ELPS in yg found per 100 yg of ECPS (N=2).
^KP2-C (EtOH); Ethanol extracted KP2-0 supernatants from growth (48h) at 37 C in defined medium.
^KP2-0 (EtOH)ED; Electrodialysis of material in footnote b.
CET PPT; Ethanol washed precipitate from cetavlon extraction of material in footnote c.
^CET SUPE; Ethanol washed supernatant as in footnote d.
122
then fractionated with cetavlon, and both the cetavlon precipitate
(Fr II) and the cetavlon supernatant (Fr III) were ethanol
extracted. Table 26 shows the KP2 2-70 (A) EPS data utilizing
these purification methods. Figure 18 shows the elution profile
obtained for the Fr II material applied to the S-2B gel filtration
column. It can be seen in Figure 17 that these purification
procedures have produced a better separation of the two major
fractions of KP2-0 EPS (compare with Figures 11 and 22), but did
not dissociate the HMW form. Figure 18 shows that cetavlon
extraction did not further dissociate the HMW form of KP2 2-70 EPS
over that of ED alone, but, rather, tended to increase the
concentration of the HMW form with a decrease in the proportion of
lower molecular weight peaks. It can be seen in Figure 18 that
whenever ED was a part of the purification procedure, a more
prominent LMW fraction (tube fractions 22 to 44) can be seen,
regardless of cetavlon use in the purification protocol. Cetavlon
extraction alone does not reduce the proportion of HMW EPS to LMW
EPS, when compared to the profile for KP2 2-70(A) EPS, but the LMW
regions are markedly different from one another. Since cetavlon
extracts only acidic polysaccharides, the LMW peaks observed in
Figure 18 for the profile of KP2 2-70 (A) EPS, after tube fraction
36, are most likely composed of neutral polysaccharides that are
removed during cetavlon extraction. It is therefore likely that
only ED can effect a dissociation of HMW EPS, and that cetavlon
extraction serves to remove neutral polysaccharides, such as LPS,
from the EPS, which was found to be the case shown in Table 26.
123
Table 26. Purification of KP2 2-70 EPS by ED and Cetavlon
Sample^
KP2 2-70(A)
KP2 2-70(A), ED
KP2 2-70(A), ED CET PPT
KP2 2-70(A) CET PPT
KP2 2-70(A), ED, CET SUPE
KP2 2-70(A), CET SUPE
^KP2 2-70(A) EPS was subjected to ED (KP2 2-70(A), ED) at 2000 V and cetavlon extracted to give both the cetavlon precipitate (KP2 2-70(A), ED, CET PPT) and ethanol extracted material from the cetavlon supernatants (KP2 2-70 (A), ED, CET SUPE). KP2 2-70(A) EPS was also cetavlon extracted without ED (KP2 2-70, CET PPT) and ethanol extracted materials from the cetavlon supernatant were obtained (KP2 2-70(A), CET SUPE).
Amount of ELPS present in yg per 100 yg of ECPS.
^Amount of protein present in yg per 100 yg of ECPS.
^No ECPS detected.
^Not determined.
ELPS/ECPS^
3.67+0.19
3.26+0.23
3.42+0.29
2.89+0.12
46.15+0.11
d
Protein/ECPS^
2.51+0.09
0.04^0.03
0.07+0.04
ND^
2.38+1.50
d
124
125
6 0 ff
50
40
C7>
3 Ul 3 0 cn O X Ul X
20
10 -
• J — • -
FRACTION NUMBER
126
Effect of Purified Extracellular Products
from K. pneumoniae on Virulence
in a Mouse Model
The majority of the studies in this section were performed
with the moderately virulent KPl-T strain to test whether the
virulence of this strain could be enhanced by co-injection with
EPS at various stages of purity. These studies were performed
first by utilizing KPl EPS, as the material co-administered to mice
by IP injections, along with serial ten-fold dilutions of KPl-T.
At the dosages administered it was found that only the KPl-T (A)
and the LMW fractions of EPS from KPl-0 or KPl-T did not
significantly enhance the virulence of KPl-T over control values.
This was true for the LMW EPS fractions even at the high doses
(108-149 yg/mouse) administered. Table 27 displays the Alog-jQ LD^Q
per milligram of ECPS administered to these mice [A(log.|Q LDgQ)/mg
ECPS]. A wide range of differences are seen to exist among the
values presented in this table (from -1.00 to -40.00), and these
differences seem to correlate inversely with the degree of
purification. For example the A(log^Q LD^QJ/mg ECPS for the acid
or neutral ECPS from KPl-0 (-35.7 to -40.0) had at least three
times the virulence enhancing capability as did the same EPS
further purified by gel filtration (-3.89 to -11.78). These
differences reach significance at the levels shown in Table 28.
The KPl-0 HMW (N) EPS virulence enhancement values were also
significantly greater (p<0.01) than the KPl-0 LMW EPS values. Thus
127
Table 27. E f fec t of KPl EPS on KPl-T Vi ru lence in the Mouse Model
FCPS Adog^Q LD5Q)/mg ECPS a
KPl-0 (N)'^ -35.73 + 10.2
KPl-0 (A)^ -40.00 + 13.3
KPl-T (N)^ -9.12 + 0.99
KPl-T (A)^ -7.38 + 4.57
KPl-0 HMW (N)^ -11.78 + 0.52
KPl-0 LMW (N)^ -3.89 + 1.87
KPl-T HMW (N)^ -9.08 + 3.51
KPl-T LMW (N)^ -1.00 + 0.30
^A(log,Q LDr^)/mg ECPS; Change in the log.Q LD^Q compared to contrAV t r i a l s per mg ECPS co- in jected with tne KPl-T s t ra in ,
^Neutral EPS f rac t i on from DEAE-Sephacel.
^Acid EPS f rac t i on from DEAE-Sephacel.
*^High molecular weight f rac t ion from neutral EPS applied to Sepharose 2B gel f i l t r a t i o n .
^Low molecular weight f rac t ion as in footnote d.
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128
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129
as purification proceeded for the KPl-0 EPS, virulence enhancement
significantly decreased.
There were no significant differences seen between the KPl-T
(A), KPl-T (N) and the KPl-T HMW EPS in their virulence enhancing
properties for the KPl-T strain. However, the KPl-T (N) and the
KPl-T HMW (N) ECPS were significantly greater virulence enhancers
for KPl-T than was the KPl-T LMW EPS (p values are shown in Table
28). The A(log^Q ^^^0^/^^ ^^P^ values for KPl-T (N), KPl-T (A) and
KPl-T HMW are all quite similar (-9.12, -7.38, and -9.08,
respectively) whereas the value for KPl-T LMW is -1.00. Thus for
both the KPl-0 and the KPl-T EPS, the LMW fraction showed
significantly less virulence enhancement for KPl-T in the mouse
model than the HMW fractions from either strain.
When comparing the KPl-0 EPS to the KPl-T EPS at various
stages of purity as to their virulence enhancing capabilities, it
was found that the KPl-0 (N) and the KPl-0 (A) EPS were
significantly more virulence enhancing than the same fractions of
KPl-T EPS, but the HMW (N) EPS fractions from either strain did not
differ significantly in this parameter (see Table 28). The LMW EPS
from KPl-0 (N) did, however, differ significantly (p<0.05) from the
KPl-T LMW EPS, even though both values were small compared to the
other values obtained for KPl EPS in Table 27. With the exception
of KPl-0 (N) and KPl-0 (A) EPS on one extreme, and KPl-0 LMW and
KPl-T LMW EPS on the other, the remaining four fractions of KPl EPS
show very similar A(log^Q LD^Q)/mg ECPS values, the mean of these
values being -10.58 ± 3.60.
130
Cetavlon extracted EPS from KPl-0 cultures [KPl-0 (CET) EPS]
was also tested in the mouse virulence assay, with both the KPl-T
and the KP2-0 s t r a i n , to see i f a d i f fe ren t pu r i f i ca t i on procedure
could a f fec t the virulence enhancement properties of KPl-0 EPS.
At 200 yg/mouse, the KPl-0 (CET) EPS was shown to enhance the
virulence of both the KPl-T and the KP2-0 s t ra in s i gn i f i can t l y over
control values. The A(log^Q ^^Q^^^ ECPS for KPl-0 (CET) EPS in
the virulence studies with the KPl-T s t ra in (-8.40 + 0.92) is not
s i g n i f i c a n t l y d i f f e ren t from the mean value (-10.58 ± 3.60)
obtained in the ea r l i e r studies in th is sect ion. Therefore the EPS
of KPl-0 retains i t s virulence enhancing properties whether an
ethanol-based or a cetavlon-based extract ion is u t i l i z e d .
The next series of studies were performed in order to
determine the a b i l i t y of the KP2 EPS to enhance the virulence of a
KPl organism. KP2 EPS, at various stages of p u r i f i c a t i o n , from
e i ther the v i ru len t KP2 2-70 or the moderately v i ru len t KP2-0
s t r a i n , were co-administered IP with the KPl-T s t ra in into mice,
and lOrr. values were obtained. Appendix 15 displays the dosages of
the various EPS f rac t ions along with the log-jg LDrg data and the
A(logiQ LDCQ) wi th respect to contro ls . S ta t i s t i ca l analysis of
these values showed that (1) administrat ion of KP2 2-70 ethanol
extracted [KP2 2-70 (EtOH)] EPS at 336.2 yg ECPS/mouse enhanced the
virulence of KPl-T s i gn i f i can t l y over control values (p<0.005); (2)
administ rat ion of KP2 2-70 (A) EPS into mice at a dosage of 454.1
yg ECPS/mouse, but not at 204.7 or 101.5 yg ECPS/mouse, enhanced
the virulence of KPl-T s i gn i f i can t l y over control values; and (3)
131
administration of KP2-0 (A) EPS at 428.6 yg ECPS/mouse did not
significantly enhance the virulence of KPl-T in the mouse model.
Table 29 shows the A(log^Q '-D5Q)/mg ECPS for each of the ECPS
fractions utilized in these studies. The KP2 2-70 (EtOH) fraction
is seen here to have produced the greatest increment of virulence
enhancement for the KPl-T strain in the mouse model (-5.38 +0.38),
followed by the KP2 2-70 (A) EPS at the higher doses (-3.56 + 0.48
and -4.21 + 1.18). The KP2-0 (A) EPS produced a relatively small
increment in virulence enhancement for KPl-T (-2.03 + 2.77) as well
as did the lowest dosage of KP2 2-70 EPS (-1.28 + 2.35). None of
these differences in virulence enhancement among the KP2 EPS
fractions were significantly different, however. Table 29 also
shows the A(log^Q '-' SO /' ^ ECPS obtained for the six KP2 2-70 (A)
EPS trials at three different doses. Four of the six values used
to calculate this mean also approximated the mean closely, whereas
two of the data points (one in the highest and one in the lowest
dosage) were unrelated to the mean obtained. Similarly, the two
data points used to obtain the mean A(logiQ LDrQ)/mg ECPS value for
KP2-0 (A) EPS were dissimilar. A high degree of variability was
thus seen in these experiments, as well as in the experiments
utilizing KPl EPS to enhance the KPl-T strain in the mouse model.
When comparing the ffect of KPl EPS to the effect of KP2 EPS
on the virulence of KPl-T in the mouse model, the following trends
were evident: (1) the HMW fractions or the fractions from
ion-exchange for the KPl EPS are between 2 and 10 times more
virulence enhancing for the KPl-T strain than are the KP2 EPS
132
Table 29. Effect of KP2 EPS on KPl-T Virulence in the Mouse Model
hPb
KP2 2-70 (EtOH)^
KP2 2-70 (A)^
KP2 2-70 (A)
KP2 2-70 (A)
KP2-0 (A)^
^As in Table 27.
yg ECPS/mouse
336.2
101.5
204.7
454.1
428.6
Adog^Q LD3Q)/mg ECPS
-5.38 ± 0.38
-1.28 +2 .35^
-3.56 + 0.48^
-4.21 + 1.18^
-2.03 + 2.77
Ethanol extracted EPS from 48h cu l tura l supernatants.
^Acid EPS f rac t ion from DEAE-Sephacel
^The mean and standard deviat ion of a l l the KP2 2-70 (A) EPS t r i a l s (N=6) was -3.02 ± 1.83.
133
fractions; and (2) the LMW fractions of KPl EPS are equal in
potency to the KP2 EPS in enhancing the virulence of KPl-T. Table
28 shows the p values for the comparison of the means of the
A(log^Q '- So'/' S ECPS for KPl and KP2 EPS. The KPl-0 (N), KPl-0
(A) and the KPl-0 HMW EPS are all significantly greater virulence
enhancers than any one of the KP2 EPS fractions, while the KPl-T
(N) EPS fraction is significantly more potent as a virulence
enhancer of KPl-T than two of the three KP2 EPS fractions [KP2 2-70
(A) and KP2-0 (A), but not KP2 2-70 (EtOH) EPS]. The KPl-T HMW
EPS was a significantly greater virulence enhancer than the KP2
2-70 (A) EPS (p<0.02) but did not differ significantly from the
KP2-0 (A) or the KP2 2-70 (EtOH) EPS. There were no significant
differences seen between the KPl-T (A) or the KPl-0 LMW EPS when
compared to any of the KP2 EPS fractions in virulence enhancement
of KPl-T. Finally, the A(log^Q ^^SO^^^^ ^^^^ ^ ° ^ ^^^ ^^^''^ "" ^
EPS was significantly lower (p<0.005) than the value obtained for
the KP2 2-70 (EtOH) EPS, but not different significantly from the
other KP2 EPS fractions.
The reverse experiment was then performed, wherein a KP2
strain was utilized in standard mouse virulence assays, with the
additional co-administration of EPS from KPl or KP2, to see if EPS
from either serotype could enhance the virulence of the KP2 strain.
The moderately virulent KP2-0 strain was chosen for these trials,
and either KPl-0 (N) EPS at 40.3 or 200.0 yg ECPS per mouse were
co-administered. The A(log^Q ldc^Q)/mg ECPS are given in Table 30.
These data reveal that the virulence of the KP2-0 strain was
134
Table 30. E f fec t of KPl or KP2 EPS on the V i ru lence o f KP2-0 in the Mouse Model
EPS Adog^Q LDgQJ/mg ECPS^
KP2 2-70 (EtOH)^ -1.70 + 0 . 4 6
KPl-0 (N)^ - 3 . 9 7 + 0 . 0 0 (p<0.005)^
KPl-0 (CET)^ -5.08 + 0.88
^As i n Table 27.
^KP2 2-70 (EtOH); Ethanol ex t rac ted EPS from 48h supernatants of KP2 2-70 grown in def ined medium a t 37 C.
^KPl-0 (N) ; Neutral EPS f r a c t i o n from DEAE sephacel.
*^KPl-0 (CET); Cetavlon ex t rac ted EPS
^ S t a t i s t i c a l ana lys is o f the means comparing the A(log•,f^ LDt-p,)/mg ECPS of KP2 2-70 (EtOH) EPS to t ha t o f KPl-0 (N) EPS.""
135
enhanced in the mouse model by co-injection of EPS from either the
KPl-0 or the KP2 2-70 strain. When cetavlon extracted KPl-0 EPS
was co-injected with the KP2-0 strain, a A(logiQ LDgQ)/mg ECPS
value of -5.08 j 0.88 (N=2) was obtained, which was not
significantly different than the value obtained for KPl-0 (N) EPS.
Both values, however, were significantly greater than that obtained
for the KP2 2-70 EPS with the KP2-0 strain. In general, however,
the extent of virulence enhancement was relatively small compared
to that manifested by the KPl-T strain with these same EPS
fractions. The KP2 2-70 (EtOH) EPS enhanced the virulence of KPl-T
with approximately three times the magnitude of that which it
enhanced the KP2-0 strain (-5.38 compared to -1.70, respectively)
per mg of ECPS. This difference is significant at the p<0.001
level. The same phenomenon was observed for the effect of KPl (N)
EPS on KP2-0 and KPl-T virulence, namely that the KPl-0 (N) EPS was
significantly more virulence enhancing (p<0.01) for KPl-T than for
KP2-0 (-35.7 compared to -3.97, respectively) per mg of ECPS.
Finally, when comparing the effect of KPl EPS versus the KP2 EPS in
the virulence enhancement of KP2-0, it was seen that the KPl-0 (N)
EPS was significantly more enhancing (p<0.005) than the KP2 2-70
(EtOH) EPS. It has thus been determined that the KPl EPS was
significantly more virulence enhancing for both serotype 1 and
serotype 2 K.. pneumoniae than the KP2 EPS in the mouse model.
Furthermore, the virulence of the KPl-T strain was affected
significantly more so by the co-administration of either KPl or KP2
EPS than was the KP2-0 strain.
136
The next set of experiments were performed to determine
whether the virulence enhancement properties of EPS could be
influenced by electrodialysis (ED) of the EPS as a purification
step before utilization in the mouse model. Both the KP2 2-70
(EtOH) and the KP2-0 (EtOH) EPS were electrodialyzed at lOOOV until
no further marked increases in mA were observed over a 30 minute
period. Both of these types of KP2 EPS were then injected
separately at various dosages with the KPl-T strain into mice.
Table 31 summarizes the results for KP2 2-70 (EtOH) EPS in terms of
the A(logiQ LDcgj/mg ECPS. It can be seen from this table that the
EPS before ED enhanced the virulence of KPl-T to a significantly
greater extent (p<0.005) than did the EPS after ED (-5.38 compared
to -3.01, respectively). Table 32 summarizes these results in
terms of the A(logiQ LDcQ)/mg ECPS. In contrast to the effect of
^Samples were prepared from ED of the ethanol f ract ionated prec ip i ta ted from 48h supernatant f l u i d of KPl-0 cultures followed by cetavlon f rac t i ona t ion . The Fr I I sample is the cetavlon p rec ip i t a te . The Fr I I S-2B (ED) sample is material obtained from placing the Fr I I sample on S-2B, co l lec t ing the LMW f rac t ion and e lect rod ia lyz ing at 2000V. The Fr I I I sample is the ethanol extracted material obtained from the supernatants a f te r cetavlon ex t rac t ion .
As in Table 27: footnote a.
^As in Table 34: footnote b.
No ECPS was detected in th is f rac t ion as determined by the uronic acid assay of Blumencrantz et a l . (7 ) .
146 Structural Studies on the EPS Produced
by K. pneumoniae
The primary focus of these studies was on the effects of ED on
the EPS from the KP2 strains. Ethanol extracted KP2 2-70 [KP2 2-70
(EtOH)] or KP2-0 [KP2-0 (EtOH)] EPS from 48h supernatants were
resuspended in column buffer (0.01 M Tris, pH 12) and applied to
either BGA-150 m or S-2B gel filtration columns equilibrated with
the same buffer. A portion of these samples were subjected to ED
for various time periods before application to the column. Figure
19 shows the effect of ED on the elution profile from BGA-150 m for
the KP2 2-70 (EtOH) EPS at 4 mg dry weight per ml. Before ED there
were two prominent hexose containing peaks, one eluting at the void
volume of the column (HMW) and one retained by the column (LMW)
with a peak of hexose activity at fraction 33-36. After subjecting
the KP2 2-70 (EtOH) EPS to ED at 400V (intermediate ED), an elution
profile on BGA-150m was obtained, denoted by the profile seen in
Figure 19. In this profile at least two thirds of the HMW fraction
from the profile before ED is now absent, and the LMW component has
increased in magnitude. The 400V ED product was then subjected to
ED at lOOOV and the resulting material was placed on BGA 150m. The
elution profile for this final ED product of KP2 2-70 (EtOH) EPS
can be seen in Figure 19. The HMW component in this final ED
profile is now virtually absent and the LMW peak is apparently more
refined (higher concentrations of hexose containing material in a
fewer number of fractions). It was found that approximately 10% of
the hexose activity was lost during ED and the majority of this
hexose activity was recovered in the fluids collected outside of
147
148
I I 1 1 1 1 — O «rt O lO O uo r o CM CM — —
( I O J / D T / ) 9S0X9H
149
the ED dialysis bag (in the cathode and anode fluids). Subsequent
studies were performed utilizing dialysis tubing with a 1000 or
2000 molecular weight cutoff rather than a 10,000 molecular weight
cutoff, so as to retain all of the hexose activity within the
dialysis tubing.
The KP2 2-70 (EtOH) EPS at 4 mg/ml was again subjected to ED,
this time at 400V, lOOOV and then at 2000V and subjected to RID in
agarose impregnated with antiserum prepared in rabbits against the
KP2 2-70 organism. Figure 20 shows the precipitin zones formed from
these antigen-antibody interactions in agarose at various stages of
ED. Well a in Figure 20 shows the KP2 2-70 (EtOH) EPS before ED.
In this figure a number of diffuse precipitin zones can be seen
within a rather broad and hazy background. When this material was
subjected to ED at 400V the RID profile seen in well b in Figure 20
was obtained. At this stage one predominant precipitin zone was
observed, with a diameter far less than that in well a. Apparently
a small portion of the EPS was also precipitating with antibody at
the periphery of the sample well. It was not certain, however,
whether this precipitate was due to antibody- antigen interactions.
Figure 20, well c, shows the RID profile for KP2 2-70 (EtOH) EPS
after ED at lOOOV, and is essentially the same profile as that seen
in well b. To be sure that ED was complete, the EPS was subjected
to 2000V, and the RID profile seen in Figure 20, well d, was
obtained. In this figure it can be seen that the zone diameter of
the precipitin ring has apparently increased over that of well c,
even though the concentration of ECPS placed in this well was
150
151
i^isissssas^
152
approximately the same as that in the other RID profiles.
From the data obtained from gel filtration and RID studies on
electrodialyzed products of KP2 2-70 (EtOH) EPS, it can be
concluded that ED affects the EPS in two different fashions: 1) HMW
components have apparently dissociated and have given rise to LMW
components and, 2) The LMW components have become more homogeneous
in molecular size as ED proceeded. To substantiate these
conclusions the KP2 2-70 LMW EPS from the BGA-150m profile of KP2
2-70 (EtOH) EPS electrodialyzed at lOOOV (Figure 19) was pooled,
collected, dialyzed and lyophilized to dryness. A comparatively
small sample (2mg dry weight) of the LMW EPS was reapplied to
BGA-150m before and after another round of ED at 1000 V. The
elution profile for the KP2 2-70 (EtOH) LMW EPS before and after ED
can be seen in Figure 21 . Before ED it can be seen that the
elution profile contains a predominant HMW peak that eluted at the
void volume (fractions 16-18), even though no HMW material was
carried over from the initial column run. Also at this low
concentration of EPS one can now see that the LMW fraction
apparently consisted of a number of distinct lower molecular weight
species that, with larger sample volumes, seemed to coalesce into
one broad fraction. These individual LMW fractions were somewhat
symnetrical and occured in the profile at a rather distinct
periodicity (peaks occured for every 60 ml of eluant on the
average). After ED an elution profile was obtained as seen in
Figure 21. The HMW component was again seen to diminish, while the
LMW components seem to have become somewhat less broad and more
refined toward the center of the LMW region. Moreover a new peak
153
154
8
o» 6 U l CO
o X Ul X i
30 40
FRACTION NUMBER
155
was seen near fraction 70.
The ethanol extracted 48h supernatant from the KP2-0 strains
[KP2-0 (EtOH) EPS] was then subjected to ED at up to 2000V to
compare the elution profiles of this ECPS on gel filtration before
and after ED. Figure 22 depicts the elution profile of the KP2-0
(EtOH) EPS before and after ED at 2000V on BGA-150m (6 mg dry
weight applied). Before ED the hexose and uronic acid activity was
limited largely to the void volume of the column, with some
evidence of a second fraction manifested as a shoulder of hexose or
uronic acid activity to the right of the void volume peak at around
fraction 24, and a third small fraction that peaked at fraction 42.
After ED a slight shift to the right is noted, with more hexose
activity found in later fractions (fractions 27-60) and a small,
though not well-defined peak at fraction 57. To demonstrate the
existence of two juxtaposed peaks of activity at or near the void
volume, a much smaller sample of KP2-0 (EtOH) EPS was applied to
the same column and the elution profile can be seen in Figure 23.
In Figure 23' the peak to the right of the void volume fraction was
somewhat more evident in the profiles before and after ED but in
contrast to the EPS from KP2 2-70, the HMW fraction was not
drastically diminished in magnitude after extensive ED.
The ability of ED to dissociate higher molecular weight KP2
2-70 EPS to that of lower molecular weight components argues for
the presence of electrophilic interactions between polysaccharide
strands. During ED it was found that as the mA increased across
the terminals, there was a concommitant increase in pH at the
cathode and a decrease in pH at the anode. The surge in mA during
156
157
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158
159
CP
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FRACTION NUMBER
160
ED tended to produce high temperatures, so it was necessary to
change the DH^O in all chambers when a current of approximately 25
mA was reached. The column labeled "Wash" in Table 37 refers to
these DH^O changes as ED proceeded, the washes being numbered
progressively. The pH of the DH2O was measured, as well as was the
pH in the cathode and the anode, once this 25 mA current was
achieved, for each wash. The change in the pH in the cathode or
the anode was calculated by subtracting the pH of DHpO from the
resulting pH in either chamber. The ratio of the change in the pH
in the cathode to the change in pH in the anode (ApH cathode/ApH
anode) was then determined, and these results are listed in Table
37. From this table it can be seen that the highest ratio between
the pH changes at the cathode and the anode occured within the
first three washes, where over 2 pH unit changes have occurred in
the cathode for ewery one pH unit change in the anode. Washes 4 to
10 produced a ratio of 1.76, and washes 11-19 produced a 1.60 pH
change ratio, which were both significantly less in magnitude than
the ratio obtained in the first three washes, but were not
significantly different from each other. Therefore the very early
stages of KP2 2-70 (EtOH) EPS electrodialysis showed a greater
surge of cations to the cathode than the latter stages of ED in
relation to the surge of anions to the anode. In a total of 19
washes the overall ApH cathode/ApH anode value was determined to be
1.75.
During ED of KP2 2-70 (EtOH) EPS, the DH2O from both the anode
and the cathode chambers were pooled separately, lyophilized and
resuspended in concentrated volumes to test for certain divalent
161
Table 37. The pH Differential of the Anode and Cathode Chambers During Electrodialysis
ApH Cathode/ApH Anode^
2.16 + 0.15
1.76 + 0.06
1.60 +. 0.13
1.75 +0.22 (N=19)
The number of changes of DHpO in the electrodialysis chambers as electrodialysis proceeded.
ApH Cathode/ApH Anode; the ratio of the pH changes in the cathode chamber and the anode chamber with respect to the pH of dH^O (pH = 6.10). ^
Wash^
1 - 3
4 - 10
11 - 19
Total
162
cations [calcium (Ca"*" ) and magnesium (Mg"^^)] in the cathode wash
and phosphate (P0^~^), which is the major anion in the defined
medium, in the anode wash. Table 38 shows the results of the
quantitation of these ions in the first three washes during ED. It
was found that the major divalent cation dissociated from the KP2
2-70 EPS during the first 3 washes of ED was Mg" ^ (1648.8 yM),
+2 while 829.0 yM of Ca was determined from the same cathode sample. _3
No PO. was found in the cathode sample but 779.3 yM was found in
the corresponding anode sample after 3 ED washes. Comparatively
+2 +2 small amounts of Mg and Ca were found in the anode also (22 and
51 yM, respectively). These data indicate that approximately 3.2
moles of these divalent cations are retrieved at the cathode for _3
ewery 1 mole of PO^ retrieved at the anode.
+2 +2 Table 39 shows the quantities of Mg and Ca found in yg per
mg of ECPS for both the KP2 2-70 (EtOH) and the KP2-0 (EtOH) EPS
before and after ED at 400V. In this table it can be seen that the
vast majority of Mg is lost from either EPS fraction after ED at
+2 400V, while approximately two-thirds of the Ca is lost during the
_3 same interval. Table 40 shows the effect of ED on the PO^
concentration for both KP2 2-70 (EtOH) and KP2-0 (EtOH) EPS.
Nearly 50% of the PO." was found to be extractable from both KP2
EPS fractions after ED at 400V. Further ED at lOOOV removed 18%
more PO.'^ from the KP2 2-70 (EtOH) EPS and 32% more PO^'^ from the
KP2-0 (EtOH) EPS. Figure 24 shows a histogram of the effect of ED
on the concentration of PO "' [P04"^] in mM, both in the sample
(open bars) and in the anode (stippled bars) for KP2-70 (EtOH) EPS
at various intervals of ED. The cross-hatched bars reflect the
163
"°'^^ 22.0+0.3 51.0+18.4 779.3+2.80
Cathode 1648.8+14.7 829.0+7.1 ND"
ND; none detected.
164
Table 39. Effect of ED on the Quantity of Divalent Cations Found in KP2 EPS
EPS
KP2-70 (EtOH) Before ED
After ED^
KP2-0 (EtOH) Before ED
After ED^
yg Mg'*" /mg ECPS
8.44+1.07 (N=4)^
0.10+0.05 (N=3)
8.50+2.76 (N=4)
0.54+0.37 (N=4)
yg Ca'^^/mq ECPS
1.70+0.15 (N=2)
0.62+0.09 (N=3)
1.14+0.05 (N=2)
0.38+0.00 (N=2)
ED proceeded at 400V until no marked surge in mA was noted over a 30 min period.
'N equals the number of determinations made from two separate preparations.
165
Table 40. Effect of ED on the Quantity of Phosphate Found in the KP2 EPS
EPi yg P04"^/mq ECPS (N=2)^
KP2 2-70 (EtOH) Before ED 36.81 + 1.31
400V ED^ 17.83 + 2.41
lOOOV ED^ 11.05 + 1.64
KP2-0 (EtOH) Before ED 2 9 . 2 8 ^ 2 . 8 2
400V ED 15.69 +_ 0.58
lOOOV ED 6.24 + 0.13
N equals the number of phosphate determinations made on the same sample.
400V ED; Electrodia lys is at 400V un t i l no marked surge in mA was noted over a 30 min period.
lOOOV ED; Electrodia lys is at lOOOV as in footnote b.
166
167
( V ) (l"^/^iJLi)Sd03 i n ^ fO CVJ —
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168
concentration of ECPS in mg/ml. The [PO^"^] in the sample before
ED (position A) was calculated to be 55.5 ymoles, but after ED at
400V (position B) the [PO^"^] in the sample diminished to 30.9
ymoles, while the [PO^"^] recovered in the anode equalled 19.9
ymoles (92% recovery of [PO^"^] from position A to position B).
After ED at lOOOV (position C), 15.4 ymoles of [PO^"^] remained in
the sample, while 9.1 ymoles were recovered in the anode (a
recovery of 79% from position B to position C). Finally, the ECPS
concentration in the sample remained essentially the same
throughout ED as seen in this figure. The results from this study
and the above data show that the EPS from two strains of KP2
contained appreciable amounts of both cations and anions even after
extensive dialysis, and the majority of the cations and anions
measured were extractable by the ED procedures, as shown both in
the concentrations of ions retained by the sample and the ions
recovered at the ED terminals.
The major difference between the two KP2 EPS was that the KP2
2-70 EPS was seen to readily dissociate into a LMW component after
ED while the majority of the KP2-0 EPS stayed in the HMW form even
after extensive ED. A study was then undertaken to see whether the
KP2-0 EPS could be dissociated into a lower molecular weight form
by the addition of sodium dodecyl sulfate (SDS). The KP2 (EtOH)
EPS was first electrodialyzed at 2000V to rid of all possible
hydrophilic associations of EPS due to inorganic cations and
anions. The electrodialyzed EPS was then subjected to boiling in
2% SDS for 5 min to disrupt noncovalent, hydrophobic interactions.
169
and applied to a Sepharose 2B column equilibrated with 0.01 M Tris,
pH 12, and 0.1% SDS. The elution profile obtained is displayed in
Figure 25, and fractions were monitored for both hexose and uronic
acid activity. Both the hexose and uronic acid profiles exhibited
two major fractions on S-2B; the HMW void volume fraction and a
second fraction immediately to the right of the HMW fraction, as
was seen in the profiles for KP2-0 EPS after ED alone (Figure 22).
A third and a fourth minor fraction can also be seen in this
profile and correspond proportionally to the same minor fractions
seen after ED alone (Figure 22). Although the two major fractions
produced on gel filtration after ED in the presence of SDS seem to
have been more clearly separated from one another than in the
profile obtained from ED alone, the differences are minor at best.
Thus, with the methods used in these studies to (1) remove all
dialysable ions that may contribute to the aggregation of the KP2-0
EPS and (2) to disrupt any noncovalent, hydrophobic interactions
between KP2-0 EPS polymers, it was found that no major shift to a
LMW form of ECPS could be achieved, although a slight shift to a
still rather high molecular weight fraction was evident.
The EPS from KPl-0 48h ethanol extracted supernatants was also
subjected to ED at 2000V to determine the effect of ED on the S-2B
elution profile of this material, which is shown in Figure 26.
Again, a relatively small quantity (1 mg total ECPS) was placed on
the column to avoid the coalescence of adjacent peaks (see Figure 7
for comparison). In both the profile before ED and after ED, a
number of symmetrical and rather evenly spaced peaks can be seen.
170
171
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3 Ul 8 30H X UJ X
20 30 40
FRACTION NUMBER
172
173
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174
which do not necessarily correspond to the fractions seen in
earlier profiles (see Figure 7). Both profiles in Figure 26 have
retained a similar quantity of the HMW component of KPl-0 EPS,
though there appears to be a shift to the right of 4 fractions for
the ED profile. Most of the other fractions coincide as to
fraction number when comparing the two profiles in the LMW region,
except for the materials eluting at the far right of the profile
(between tube fractions 55 and 72). There were, however,
quantitative differences between many of the coincidental fractions
in the two profiles. For example, the fractions which eluted at
tube fractions 23, 27 and 34 for the ED profile were of greater
magnitude with regard to hexose than were the corresponding
fractions seen in the profile before ED, whereas the peaks of
hexose at tube fractions 38 and 43 were much larger in the profile
before ED than in the corresponding fractions after ED. There was
then an apparent shift of hexose from later to earlier fractions in
the LMW region as a result of ED, as well as an apparent shift of
the HMW fraction to a higher elution volume. There were also two
prominent peaks of hexose in the material after ED at tube
fractions 53 and 65 that were in contrast to the material before
ED.
Two different methods were utilized to ascertain the
contribution of hydrophobic interactions in the KPl-0 EPS, both of
which were based on the assumption that lipid groups are covalently
linked to either the ECPS or to the contaminating ELPS and that
these hydrophobic groups may tend toward micellar formation in
175
aqueous solutions (see Results, Section G). The first of these
methods was hydrolysis of KPl-0 EPS with 60% hydrofluoric acid (HF)
at 0 C, which has been shown to liberate acylglycerols and
diacylglycerols from the capsular polysaccharides of the Group B
meningococci, but leave the glycosidic linkages intact (37).
Figure 27 shows the elution profile on S-2B of KPl-0 EPS obtained
before and after HF .treatment, utilizing the EPS obtained from
dialyzed 48h supernatants. The profile after HF treatment is seen
here to be a more homogenous preparation of LMW EPS, with much of
the higher molecular weight hexose activity absent and a more
refined singular LMW fraction. The second method used to define
these putative hydrophobic interactions was that of treatment of
the KPl-0 EPS with 0.5 M NaOH (saponification). Figure 28 shows
the effect of saponification of KPl-0 (N) ECPS on the elution
profile obtained on S-2B. The profile obtained before
saponification contained a number of prominent peaks. After
saponification the vast majority of the hexose activity was found
to elute between tube fractions 32 and 50. Thus with HF treatment,
or with NaOH treatment of KPl-0 EPS, the fractions in the HMW
region were not as prominent and the LMW fraction apparently became
more homogeneous. It was also found that ED of the KPl-0 EPS,
followed by cetavlon fractionation, produces essentially the same
profile, with the LMW peak predominant (Figure 15). However, there
is a difference in the elution volume between the LMW fractions
from HF and NaOH treatment on the one hand and the LMW fraction in
Figure 15.
176
177
130
FRACTION NUMBER
178
179
FRACTION NUMBER
180
Further studies on the saponif icat ion of KPl-0 EPS, as well as
on various f ract ions of KPl and KP2 EPS from other sources,
revealed that f a t t y acids (FA) were being released from a l l of
these f rac t i ons , even a f te r the EPS was pre-extracted with organic
solvents to remove any noncovalently attached l i p i d s . The presence
of covalently l inked f a t t y acids was demonstrated by the i r release
from the EPS treated with 0.5 M NaOH, and the i r conversion to
v o l a t i l e methyl esters to be detected in gas l i qu id chromatography
(GLC). A known concentration of bacterial methyl ester standard
was run along with j<. pneumoniae EPS to quanti tate the to ta l amount
of f a t t y acid methyl ester (FAME) released by a known quantity of