Journal of General Microbiology (1988), 134, 1009-1016.
Printed in Great Britain
1009
Role of Lipopolysaccharide and Complement in Susceptibility of
Escherichia coli and Salmonella typhimurium to Non-immune Serum
BY J U A N M . TOMAS,* B L A N C A C I U R A N A , V I C E N T E J . B E N E D ~A N D
ANTONIO JUAREZ
Department of Microbiology, Faculty of Biology, University of Barcelona, Avda Diagonal 645,
08071 Barcelona, Spain
(Received I 1 May 1987; revised 8 December 1987)
The role of lipopolysaccharide (LPS) in the susceptibility of Escherichia coli and Salmonella
typhimurium to non-immune human serum was investigated using serum-sensitive strains of
both enterobacteria. LPS from serum-resistant strains of E. coli and S. typhimurium could
activate and completely remove the serum bactericidal activity, and also showed dosedependent anti-complement activity. These properties were mainly due to the high-molecularmass LPS: the low-molecular-mass LPS from serum-resistant strains of E. coli and S.
typhimuriumhad only a slight effect on the serum bactericidal activity, and showed only low anticomplement activity, even at high concentration. The results suggest that LPS composition,
especially the 0-antigen polysaccharide chains, contributes to the susceptibility of E. coli and S.
typhimurium strains to complement-mediated serum bactericidal activity.
INTRODUCTION
The bactericidal effect of non-immune serum plays an important role in host defence against
bacterial infection. This phenomenon has been widely noted and studied since the late 1800s
(Buchner, 1889) and has been shown to be complement mediated (Roantree & Pappas, 1960).
Although serum resistance of Gram-negative bacteria probably has a multifactorial basis in vim,
the outer membrane clearly is involved as the most peripheral component of the bacterial cell
envelope. Several studies have implicated outer membrane components such as lipopolysaccharide (LPS) (Glynn & Ward, 1970, Munn et al., 1982; Nelson & Roantree, 1967), capsular
polysaccharide (Glynn & Howard, 1970; Sutton et al., 1982) and outer membrane proteins
(Hildebrant et al., 1978; Moll et al., 1980; Taylor & Parton, 1977) in resistance of bacterial
strains to the bactericidal activity of serum.
Complement activation by Gram-negative bacteria can occur via the classical or the
alternative pathway. The classical pathway can be activated directly by the interaction of
antibody with bacterial surface antigens such as the lipid A moiety of LPS (Morrison & Kline,
1977). The alternative pathway may be activated by bacterial surface polysaccharides
independent of antibody (Morrison & Kline, 1977).
In the present study we have investigated the mechanism of complement activation by the
LPS of Escherichia coli and Salmonella typhimurium. We have also examined the role of highmolecular-mass LPS (HMM-LPS) (0-antigen enriched) and low-molecular-mass (LMM-LPS)
(core and lipid A enriched) in serum susceptibility.
METHODS
Bacterial strains and media. The E. coli and S. typhimurium strains used are listed in Table 1. S. typhimurium
strains SA2380 and SA1377 are isogenic mutants of strain Su453. The basal medium for bacterial growth was
Luria broth (LB) (Miller, 1972) or LB with 1.5% (w/v) agar (LB-agar).
Abbreviations: HMM-LPS, high-molecular-mass lipopolysaccharide; LMM-LPS, low-molecular-mass
lipopolysaccharide ; KDO, 2-keto-3-deoxyoctulosonicacid ; NHS, non-immune human serum.
0001-4179 0 1988 SGM
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J. M.
TOMAS
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Table 1. Escherichia coli and Salmonella typhimurium strains
Strain
E. coli 0 5 5 : B5
E. coli PL-2
E. coli Y10
S . typhimurium Su453
S . typhimurium SA2380
S . typhimurium Sa1377
Relevant characteristics*
0-antigen+ ; serum-resistant
galE; 0-antigen-; serum-sensitive
rfbD ; 0-antigen- ; serum-sensitive
0-antigen+ ; serum-resistant
galE; 0-antigen- ; serum-sensitive
rfaC; 0-antigen- ; serum-sensitive
Origin
B. Bachmann, New Haven, USA
1
K. Sanderson, Calgary, Canada
* 0-antigen+/-, presence/absence of HMM-LPS.
Bacterial survival infresh non-immune serum. The survival of exponential-phase bacteria in non-immune human
serum (NHS) was measured as previously described (Tomis et al., 1986). Controls (bacteria in phosphate-buffered
saline : PBS, containing 0.1 5 M-sodium chloride and 0.1 5 M-sodium phosphate, pH 7.2) showed no significant
changes in viable counts over the incubation period. We used heat-inactivated NHS (56 "C for 30 min) as an
additional control. Serum was usually used on the day of collection or stored at - 80 "C.
LPS isolation and subfractionation.LPS from E. coli 055 :B5 or S. typhimuriumSu453 was purified by the method
of Westphal & Jann (1965) or purchased from Sigma. LPS from serum-sensitive strains of E. coli and S .
typhimurium was purified according to Westphal & Jann (1965) with the modification of Osborn (1966).
Lyophilized LPS was solubilized at a final concentration of 7.5 mg ml-' in buffer containing 3% (w/v) sodium
deoxycholate, 0.2 M-NaC1, 5 mM-EDTA, 20 mM-Tris/HCl (pH 8-3), and was applied at rmm temperature to a
column of Sephacryl S-300 (Pharmacia) equilibrated in buffer containing 0.25 % sodium deoxycholate, 0.2 MNaCl, 5 mM-EDTA, 10 mM-Tris/HCl (pH 8.0). Fractions (2.5 ml) were collected at a flow rate of 12 ml h-' and
were analysed directly by SDS-PAGE. Before chemical analyses and serum inhibition studies, fractions were
dialysed extensively against distilled water, first at room temperature and then at 4 "C.
Electrophoretic techniques. SDS-PAGE was done according to the procedure of Laemmli (1970) as modified by
Ames et al. (1974). Samples were mixed 1 :1 with sample buffer (containing 4%, w/v, SDS) and boiled for 5 min,
and 10 pl portions were applied to the gel. LPS bands were detected by the silver stain method of Tsai & Frasch
(1982).
Analytical procedures. Organic phosphate was measured by the method of Ames 8z Dubin (1960) using
NaH2P0,. 2H20as standard, and total carbohydrates by the phenol reaction procedure (Hanson & Phillips, 1981)
with glucose as standard. 2-Keto-3-deoxyoctulosonicacid (KDO) was measured by the thiobarbituric acid method
after hydrolysis of samples in 0.05 M-sulphuric acid for 30 min (Karkhanis et al., 1978).
Inhibition of serum bactericidal activity by complete and fractionated LPS. LPS was suspended in PBS (pH 7.2) to a
final concentration of 1-5 mg ml-I and briefly sonicated at 4 "C until the solution cleared. LPS solution in the
concentration range 0.01-0.2 mg ml-' was added to 0.5 ml serum in a tube. The volume was adjusted to 0.9 ml with
PBS, and the solution was incubated at 37 "C with shaking (200 r.p.m.) for 30 min. Then, 0.1 ml of bacterial
suspension (5 x lo7 c.f.u.) in the exponential phase was added to the tube and incubated at 37 "C for an additional
1 h before dilution plating. Similar experiments were done with LPS fractions lyophilized and suspended in PBS
and added at a final concentration of 0.05 mg ml-l to 0.5 ml serum. Controls without LPS or LPS fractions showed
no inhibition of serum bactericidal activity.
Measurement of the anti-complement activity of LPS from E. coli and S . typhimurium. The anti-complement
activity of LPS or LPS fractions was measured according to Shafer et al. (1984) with slight modifications. Serum
(0.1 ml) was mixed with LPS (0.01-0-2 mg ml-l) or LPS fractions (0.05 mg ml-l) suspended in PBS, or with PBS
alone, to a final volume of 0.2 ml, and incubated with shaking at 37 "C for 30 min. Antibody-sensitized sheep
erythrocytes in 0.2 ml PBS were added to a fourfold dilution of treated NHS and incubated for an additional 30
min at 37 "C. Ice-cold saline (3 ml) was added to the mixture, the cells were pelleted by centrifugation, and the
absorbance of the supernatant was measured at 412 nm. The positive control was sensitized sheep erythrocytes
plus serum without added LPS or LPS fraction, and the negative control was LPS or LPS fraction without added
serum.
Radiolabelled LPS. Bacteria were grown with the addition of ~-[2-~H]glucose
(5 mCi l-l, 185 MBq 1 - I ;
Amersham) and LPS was extracted and fractionated as described above.
Liposomepreparation. Liposomes were prepared according to Porat et al. (1987) and mixed with 3H-labelledLPS
or 3H-labelledLPS fractions (0-5 mg) in a total volume of 0.2 ml. The liposome suspension was agitated vigorously
for 5 min, washed twice with PBS and resuspended in 1 ml PBS. The suspension (0.2 ml) was then incubated at
37 "C for 30 min with one of the following: 20% (v/v) NHS, 20% heat-inactivated NHS, or PBS alone, in a final
volume of 1 ml. Each mixture was chromatographed on Sephadex G-25 and 1 ml fractions were collected and
assayed for radioactivity.
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LPS and serum susceptibility of enterobacteria
Table 2. Inhibition of serum bactericidal activity against E. coli and S . typhimurium strains by
homologous and heterologous LPS
The LPS concentration used was 0.1 mg ml-l. The results are the means of three independent
experiments. NHS pretreated with LPS from S . typhimurium Su453 (serum-resistant) was unable to kill
E. coli strain PL-2 (serum-sensitive), and NHS pretreated with LPS from E. coli 0 5 5 :B5 (serumresistant) was unable to kill s. typhimurium strain SA2380 (serum-sensitive).
Percentage survival of strains after 60 min incubation in:
r
Strain
E. coli
PL-2
Y 10
S . typhimurium
SA2380
SA1377
~~
NHS plus LPS from strains
Control
NHS
<1
<I
<1
<1
I
A
Serum-sensitive
&
PL-2
5
4
SA2380
4
7
Y-10
4
6
SA1377
8
5
7
7
Serum-resistant
0 5 5 : B5
108
107
su453
109
111
RESULTS
Inhibition of serum bactericidal activity by E. coli and S . typhimurium LPS
Purified LPS from E. coli 055:B5 at various concentrations was able to inhibit the
bactericidal activity of NHS against E. coli serum-sensitive strain PL-2. The survival of PL-2
cells in NHS after 60 min incubation at 37 "C was 0, 39, 86, 108, 109, 112% at LPS
concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.4 mg ml-l, respectively. Purified LPS from S .
typhimurium Su453 at various concentrations was also able to inhibit the bactericidal activity of
NHS against S . typhimurium serum-sensitive strain SA2380. The survival of SA2380 cells in
NHS after 60 min incubation at 37 "C was 0, 41, 87, 109, 111, 115% at LPS concentrations of
0, 0.025, 0.05, 0.1, 0.2, 0.4 mg ml-l, respectively.
Table 2 shows the percentage survival of serum-sensitive strains of E. coli and S . typhimurium
in NHS treated with LPS (0.1 mg ml-l) from serum-sensitive and serum-resistant strains of both
enterobacteria. Serum-sensitive strains of E. coli (PL-2 and Y 10) and S . typhimuriurn (SA2380
and SA1377) showed a survival in control NHS of less than 1% after 60 min incubation, whereas
these strains survived well in NHS pre-incubated with LPS obtained from serum-resistant
strains of E. coli and S . typhimurium.LPS from serum-sensitive strains only slightly inhibited the
bactericidal activity of NHS (Table 2).
Subfractionation of LPS
LPS from the serum-resistant strains E. coli 055:B5 and S . typhimurium Su453 were
fractionated as described in Methods. The presence of LPS in column fractions was monitored
by determination of organic phosphate, KDO and total hexose, as well as by SDS-PAGE (Fig. 1
for E. coli 0 5 5 :B5LPS; Fig. 2 for S . typhimurium Su453 LPS).
Purified LPS from E. coli 055 :B5 showed a main peak of total hexose centred in fraction 30.
This fraction also coincided with a peak of KDO (Fig. 1a) and was HMM-LPS (Fig. 1b). In
these peak fractions (28-32) the apparent ratio of hexose to KDO was approximately 25-30 :1.
This value decreased in the following fractions until the main peak of KDO (Fig. 1a), centred in
fraction 42, where the apparent ratio of hexose to KDO was approximately 1-2 :1, a feature
consistent with LPS fractionation on the basis of a decreasing number of repeating units in the
0-antigen side chains. Fractions 38-48 contained LMM-LPS (Fig. 1b).
A similar profile was obtained by fractionation of the S . typhimurium Su453 LPS. The main
peak of total hexose was centred in fraction 28 (Fig. 2a) and coincided with a peak of KDO and
HMM-LPS (Fig. 2b). In these peak fractions (26-30) the apparent ratio of hexose to KDO was
approximately 25-35 : 1. As in the E. coli LPS subfractionation, the hexose :KDO ratio
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J . M.
TOMAS
AND OTHERS
60
1200
50
40
8
-n
3
I
400
H
e4
10
20
30
-25
35
40
50
45
54
Fraction no.
Pure
LPS
24
26
28
30
32
34
36
38
40
42
44
46
48
Fig. 1. (a) Subfractionationof LPS from E. coli 055 :B5 (serum-resistant)by column chromatography.
LPS was applied to a column of Sephacryl S-300 and eluted with 0.25% buffered deoxycholate as
described in Methods. Eluted fractions were analysed for KDO (e-0) and total hexose (O---O)
after extensive dialysis, and by SDS-PAGE in conjunction with silver staining for carbohydrate (see b).
V,, void volume. (b) Analysis of subfractionated LPS from E. coli 0 5 5 :B5 by SDS-PAGE. LPS
fractions from the gel filtration (a) were analysed and stained with silver. The leftmost lane contains
purified LPS from E. coli 0 5 5 :B5;the other lanes are labelled by fraction number (see a).
decreased in the following fractions until the main peak of KDO (Fig. 2a) centred in fraction 42,
where the apparent ratio was approximately 1-2 : 1 . Fractions 38-50 were LMM-LPS (Fig. 2 b).
Inhibition of serum bactericidal activity by E. coli and S . typhimurium LPS fractions
Serum-sensitive strains of E. coli (PL-2) and S . typhimurium (SA2380) showed a high
percentage survival (> 50%) in NHS treated with LPS fractions having a high hexose :KDO
ratio (HMMILPS), such as fractions 28-32 from the E. coli 0 5 5 :B5 subfractionation, or
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LPS and serum susceptibility of enterobacteria
1013
1200
60
50
-n
Pure
-
n
800
22
40
24
26
28
30
32
34 36
38
40
42
44
46
$
48 50
LPS
rig. 2. (a) Subfractionation of LPS from S . typhimurium Su453 (serum-resistant) by column
chromatography. Details as for Fig. 1 (a). (b) Analysis of subfractionated LPS from S . typhimurium
Su453 by SDS-PAGE. The leftmost lane contains purified LPS from S . typhimurium Su453. Other
details as for Fig. I@).
fractions 26-30 from the S . typhimurium Su453 LPS subfractionation. When the hexose :KDO
ratio decreased in the LPS fractions, there was also a decrease in the survival of serum-sensitive
strains in the NHS pretreated with these LPS fractions.
When NHS was treated with LPS fractions of low hexose :KDO ratio (LMM-LPS) (fractions
38-50 in E. coli 0 5 5 :B5 LPS or S . typhimurium Su453 LPS), the survival of serum-sensitive
strains of both bacteria was always below 10%.
Measurement of the anti-complement activity of E. coli and S . typhimurium LPS
The anti-complement activity of LPS from E . coli 0 5 5 :B5 and S . typhimurium Su453 was
measured to determine whether the inhibitory effect of the LPS on serum bactericidal activity
was due to its ability to activate and deplete complement. The anti-complement activity of the
unfractionated LPS from both strains was dose-dependent, as was the anti-complement activity
of the HMM-LPS fractions (Fig. 3). The LMM-LPS fractions of both bacteria had low anticomplement activity even at high concentration (0.2 mg ml-l) (Fig. 3).
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J. M.
TOMAS
AND OTHERS
1I000
750
500
250
'3
1250
cd
.-
3
4
m
I
25
50
1000
I
I
I
100
200
Concn of LPS (pg ml-')
30
40
50 60 70
Tube no.
80
90
100
Fig. 4
Fig. 3
Fig. 3. Inhibition of complement-mediated haemolysis of sensitized sheep erythrocytes after 30 min
incubation in non-immune rabbit serum with LPS fractions from E. coli 055 :B5 and S. typhimurium
Su453. 0 ,HMM-LPS from E. coli 055 :B5; 0 ,LMM-LPS from E. coli 055 :B5; 0 ,HMM-LPS from
S . typhirnurium Su453; m, LMM-LPS from S . typhimurium Su453.
Fig. 4. Distribution of 3H after incubation of liposomes containing 3H-labelled HMM-LPS (a) or
LMM-LPS (b)(both from S . typhimurium Su453) with NHS (0-0) or with heat-inactivated NHS
(.---a) for 30 min at 37 "C.Only a single peak, representing unlysed liposomes, is seen with heatinactivated NHS (a,b) and with liposomes containing 3H-labelled HMM-LPS incubated with NHS (a).
Two peaks are seen with liposomes containing 3H-labelled LMM-LPS (b) after incubation with NHS,
the second peak representing 3H released from lysed liposomes.
Lysis of liposomes containing LPS fractions
Pooled fractions 26-30 (HMM-LPS) and 40-46 (LMM-LPS) from S . typhimurium Su453
3H-labelled LPS were incorporated separately into liposomes, and treated with NHS or heatinactivated NHS. HMM-LPS protected liposomes from lysis by NHS (Fig. 4 a ) : there was no
release of 3H from liposomes containing this LPS fraction. In contrast, liposomes containing
similar concentrations of LMM-LPS were lysed and 3H was released (Fig. 46). Similar
experiments were done with pooled HMM-LPS and LMM-LPS fractions of E . coli 0 5 5 :B5
3H-labelled LPS incorporated into liposomes. As for the S . typhimurium LPS, HMM-LPS, but
not LMM-LPS, protected the liposomes from lysis by NHS (data not shown).
DISCUSSION
The bactericidal effects of immune or non-immune serum are mediated by activated
components of the classical or alternative complement pathway (Pangburn, 1983 ;Taylor, 1983).
Activation of either can lead to membrane damage culminating in cell death (Taylor, 1983).
E. coli (Taylor, 1975) or Salmonella spp. (Joinier et al., 1982) are known to activate both
complement pathways.
Sensitivity of a number of Gram-negative bacteria to the bactericidal activity of non-immune
or immune sera has been attributed to their LPS composition (Munn etal., 1982; Rice & Kasper,
1977; Schiller et al., 1984; Shafer et al., 1984). We found that LPS from serum-resistant strains of
E. coli and S. typhimurium inhibited serum bactericidal activity against serum-sensitive strains
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LPS and serum susceptibility of enterobacteria
1015
from both enterobacteria, whereas LPS from serum-sensitive strains of E . coli and S.
typhimurium was only slightly inhibitory even at high concentrations. Furthermore, we found a
correlation between the anti-complement activity of LPS, measured as the percentage inhibition
of haemolysis with sensitized erythrocytes, and its inhibitory effect on serum bactericidal
activity .
As described previously (MacIntyre et al., 1986; Peterson & McGroarty, 1985), the LPS
fractions with HMM-LPS contained a high number of 0-antigen side-chain repeating units.
This feature was consistent with the apparent ratio of hexose to KDO in the subfractionation of
LPS from E. coli 055 :B5 and S . typhimurium Su453. In the E. coli 055 :B5 LPS subfractionation
we also found, as described by Peterson & McGroarty (1989, LPS bands of very high molecular
mass, which we also assume represented very long polymers of 0-antigen not covalently
attached to lipid A (capsular polysaccharide) as described by some authors (Goldmann et al.,
1984). These carbohydrate bands of very high molecular mass were not observed in S .
typhimurium LPS subfractionation, as was also described by Peterson & McGroarty (1985) for S .
typhimurium and S . minnesota.
The E. coli and S . typhimurium LPS fractions containing HMM-LPS could activate and
remove the serum bactericidal reaction, and could also reduce the percentage of haemolysis in a
mixture with sensitized sheep erythrocytes, indicating a correlation between the effect on serum
bactericidal activity and the anti-complement activity of these LPS fractions. Also, 3H-labelled
HMM-LPS from E. coli or S . typhimurium prevented lysis of liposomes containing these LPS
fractions when mixed with NHS.
E. coli and S . typhimurium LPS fractions containing LMM-LPS with an apparent
hexose :KDO ratio of 1-2 : 1 (i.e. a small number of 0-antigen side chains per lipid A-KDO
molecule) were unable to significantly activate and remove the serum bactericidal reaction, or to
inhibit the haemolysis of sensitized sheep erythrocytes. Furthermore, 3H-labelled LMM-LPS
fractions from E. coli or S . typhimurium were unable to prevent lysis of liposomes containing
these LPS fractions when treated with NHS.
All these facts suggest that the 0-antigen side-chains are the main part of the LPS molecules
from E. coli and S . typhimurium involved in serum resistance by virtue of their anti-complement
activity. This point seems to correlate with the results of other laboratories indicating that C3b
(a component of the complement system) preferentially attaches to the longest 0-antigen chains
in LPS molecules (Goldmann et al., 1984; Liang-Takasaki et al., 1983).
B. Ciurana was supported by a fellowship from Fondo de Investigaciones Sanitarias.
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