Surgical Infectious Disease Laboratory,
University of Virginia, Charlottesville,
Virginia1;
Department of Surgery,
University of Alabama, Birmingham, Alabama2; and
Department of Surgery, University of South Florida, Tampa,
Florida3
Received 30 December 1997/Returned for modification 5 March
1998/Accepted 22 May 1998
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INTRODUCTION |
Escherichia coli remains
the most common gram-negative bacterial species isolated from
infections in hospitalized patients (17), and despite
significant advances in antimicrobial therapy and critical care
technology, mortality from sepsis caused by gram-negative organisms
remains 40% or higher (26, 44, 58, 63). Pathogenic E. coli organisms make a variety of virulence factors, and among them
are the group of toxic proteins called RTX (repeats in toxin)
cytolysins. Alpha-hemolysin (Hly) is the prototype RTX cytolysin
(31). At least 40% of pathogenic E. coli
organisms produce it, while most fecal isolates do not (6, 11, 27,
29, 49, 50, 51). Production of Hly is associated with
enhanced E. coli virulence (35, 59, 60). It
remains unclear (i) what effector mechanism(s) mediates its lethality, (ii) whether Hly fatty acid acylation is critical to Hly's
clinically relevant functions (e.g., causing cell lysis, eliciting
interleukin-1 (IL-1), and mediating death), and (iii) whether
Hly-associated lipopolysaccharide (LPS) is responsible for the
enhanced virulence of hemolytic E. coli (LPS is believed to
be bound to Hly [9, 10, 55]). To study these
issues, three transformed E. coli strains (WAF108, WAF270,
and WAH540), differing only in their ability to produce and secrete
functionally active Hly, as defined by its ability to lyse
erythrocytes in vitro, were used. WAF108 produces no Hly
(Hlynull), WAF270 produces hemolytic, fully acylated
Hly (Hlyactive), and WAH540, a newly constructed
strain, produces full-length, nonacylated, functionally inactive
Hly (Hlyinactive) (its genetic construct encodes
the same peptide sequence as that of the Hly of WAF270 [8,
61]).
We have previously shown that live Hly-producing E. coli
WAF270 is lethal at 108 CFU intraperitoneally (i.p.) and
elicits a distinct IL-1
spike 4 to 6 h after infection while
nonhemolytic E. coli WAF108 does not elicit this IL-1
spike and is nonlethal at the same inoculum dose (35). Tumor
necrosis factor (TNF) blockade with antibodies to TNF- and TNF-
failed to abrogate the lethality of WAF270 in this study
(35). Although Hly is known to cause IL-1 release from monocytes in vitro (5), the relationships between
E. coli, Hly, the acylation state of Hly, host IL-1
release, and mortality are unclear.
Despite the characterization of many LPS-mediated responses
(19, 22, 24, 30, 40, 43, 45, 52, 64), the contribution of
the proinflammatory cytokines to the toxicity and host clearance of
live gram-negative bacterial infections remains ill defined. Efforts at
using anti-LPS monoclonal antibody (MAb), anti-TNF MAb, and IL-1
receptor antagonists to treat sepsis caused by gram-negative organisms
in human and large-animal studies have been discouraging (26, 44,
58, 63). Alternatively, IL-1R1 knockout (KO) mice were partially
resistant to lethal E. coli peritonitis (1). In
the present study, we hypothesized that Hly-provoked IL-1 signaling with or without TNF signaling was responsible for the enhanced lethality of Hly-producing E. coli. We also proposed
that the nonacylated Hly of WAH540 would not activate monocytes,
elicit IL-1, or enhance the lethality of E. coli as the
acylated Hly of WAF270 would, implying that both the hemolytic and
the nonhemolytic activities of Hly depend on its fatty acid
acylation via expression of the hlyC locus.
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MATERIALS AND METHODS |
Bacteria.
The human fecal isolate E. coli J198
(O22 ColV
Hly) (59) and the
laboratory E. coli strains WAM783 (DH1 transformed with pSF4000
BamHI [8, 60]), WAF108 (O22
Hlynull, ampicillin resistant [Ampr],
chloramphenicol resistant [Cmr]) (57), WAF270
(O22 Hlyactive Cmr) (35), and
WAH540 (O22 Hlyinactive Cmr) were used in
the following studies.
Bacteria were grown in tryptic soy broth at 37°C for 6 or 9 h
unless otherwise stated. Ampicillin (100 µg/ml) was added for growing
WAF108, and chloramphenicol (20 µg/ml) was added for growing WAM783,
WAF108, WAF270, and WAH540. The bacteria were washed, and
concentrations were estimated by measuring absorbances at 600 nm
(A600) and confirmed by plating on tryptic soy
agar with 5% sheep blood overnight (to verify the presence or absence
of hemolysis).
WAF108, WAF270, and WAH540, all transformants of J198, are genetically
identical except for the presence of slightly modified constructs of
the plasmid pSF4000 containing the Hly determinant, hlyCABD (59). The hlyA locus encodes
the secreted Hly protein (23, 33), while hlyC
encodes posttranslational modifications (covalent fatty acid acylation
of lysine residues) activating the protein (28, 32, 37, 54).
The hlyB and -D loci encode transport genes
facilitating translocation of Hly (7, 41). WAF108
contains the plasmid pSF4000:Tn1 and produces no Hly
(Hlynull) (59). The transposon
Tn1 is inserted into the hlyA locus. WAF270
contains the complete pSF4000, enabling production and secretion of
fully modified Hly (61). WAH540 contains a pSF4000 plasmid in which the only BamHI fragment is deleted
(8). This fragment is approximately 2.9 kb in length and
includes the first 483 bp of the hlyC locus (633 bp total).
This deletion has no effect on the expression of downstream coding
segments, since the complete hlyA locus is expressed. WAH540
was constructed by electroporating pSF4000BamHI
(isolated from E. coli WAM783 [8] with a
plasmid kit [Qiagen, Inc., Chatsworth, Calif.] according to the
instructions of the manufacturer) into E. coli J198 at a
field strength of 14 kV/cm and a pulse length of 5 ms, as previously described (13, 21). This enabled the production and
secretion of full-length, nonacylated Hly
(Hlyinactive) by new transformants. Transformants
displaying restriction patterns in agreement with the
predicted restriction map for pSF4000BamHI (8, 60, 61) were assayed for expression of the Hly gene and hemolytic activity. A single transformant which expressed the
highest level of Hlyinactive among the transformants
was isolated and denoted WAH540. Linearized (digested) plasmid from
WAH540 showed a single band of approximately 12.3 kb, matching the
linearized stock plasmid from WAM783. The restriction pattern of the
putative E. coli WAH540 matched the predicted restriction
map of pSF4000BamHI (8, 60, 61).
Hemolysin gene expression and hemolytic assay.
Culture
supernatants of WAF108, WAF270, and WAH540 were clarified with
0.2-µm-pore-size filters and partially purified by ultrafiltration
through Centricon 30 centrifugation filters (Amicon, Beverly, Mass.).
Bacterial products (Hly and others) were partially purified from
9-h cultures containing 6 × 1010 CFU in 25 cm3 of medium into ending volumes of approximately 500 µl. Partially purified supernatant preparations from equivalently
sized cultures were separated by sodium dodecyl sulfate-7.5%
polyacrylamide gel electrophoresis. After protein transfer,
immunoprobing was performed with the murine anti-Hly MAbs B7 and G8
(gifts of R. A. Welch) as previously described (12,
42). Supernatants from 9-h bacterial cultures were filtered and
diluted with 10 mM CaCl2-150 mM NaCl in a microtiter
plate. Sheep erythrocytes (RBC) were washed and resuspended to 2%
(wt/vol) in CaCl2-saline, and 100 µl was added to each
well. Following a 1-h incubation at 37°C, unlysed cells were pelleted
and the percent lysis was calculated by measuring the
A415 of the supernatant, as previously described
(60).
LPS O22 purification and characterization.
Whole bacteria
were lyophilized and crushed into powder. By a modified phenol-water
extraction method (2), LPS was extracted from each strain.
Each purified LPS was lyophilized, and its purity was confirmed by
sodium dodecyl sulfate-14% polyacrylamide gel electrophoresis,
spectral analysis (A245-290), and agarose gel
electrophoresis. The LPS serotype was confirmed by immunoblot analysis
with rabbit anti-LPS O22 antiserum (E. coli Reference Center, Pennsylvania State University) as previously described (12, 47). Endotoxin was quantified by a chromogenic
Limulus amebocyte lysate (LAL) (BioWhittaker, Inc.,
Walkersville, Md.) assay according to the instructions of the
manufacturer. Whole bacteria (10, 102, 103, or
104 CFU) and culture supernatants from cultures diluted to
contain equal amounts of bacteria (as determined by overnight plating) were assayed for total LPS content. Aliquots of these samples were also
compared by immunoprobing with rabbit anti-LPS O22 antiserum and by
densitometry (12, 34, 47). Endotoxin bioactivities were
compared by measuring splenocyte proliferation and TNF- release.
Naive BALB/c murine splenocytes (2 × 105) were
stimulated for 24 h with 5 µg of purified LPS O22/ml,
derived from WAF108, WAF270, and WAH540 in RPMI-10% fetal calf
serum (FCS). The cell cultures were then pulsed with
[3H]thymidine (56), and the cells were
harvested and counted for scintillation. Similarly, human
peripheral blood mononuclear cells (PBMC) (2 × 105)
were stimulated for 6 to 24 h with 1 to 10 ng of LPS O22/ml, and
the supernatants were harvested for TNF- quantitation by enzyme-linked immunosorbent assay (ELISA) with anti-human TNF- MAb
(Pharmingen, San Diego, Calif.) (36).
Bacterium-induced monocyte IL-1 release.
Human PBMC were
separated by sodium diatrizoate polysucrose gradient (Histopaque-1077;
Sigma), washed, and then incubated with viable E. coli
WAF108, WAF270, or WAH540 at bacteria-to-monocyte ratios of 0.04 to
4,000 CFU/monocyte for 90 min at 37°C in RPMI-10% FCS, as
previously described (5). Fresh cell supernatant IL-1 determinations were made by a sandwich ELISA technique with anti-human IL-1 MAbs (Endogen, Cambridge, Mass.) according to the
manufacturer's specifications.
Animals.
Male and female BALB/c, C57BL/6 (Taconic,
Germantown, N.Y., and Hilltop Lab Animals, Inc., Scottsdale, Pa.),
C3H/HeJ (Jackson, Bar Harbor, Maine), and receptor-deficient mice
(described below) were housed in a pathogen-free environment and fed
lab chow and water ad libitum according to National Research Council
standards. All procedures were approved by the University of Virginia
Animal Use Committee.
Mice deficient in the expression of either the type 1 IL-1 receptor
(IL-1R1 KO) or the type 1 TNF receptor (TNFR1 KO) were generated by
gene targeting through homologous recombination in murine embryonic
stem cells, as described previously (8, 38, 39). Transgenic
homozygous IL-1R1 KO animals were subsequently bred through multiple
generations prior to their use in this study. Homozygous IL-1R1 KO and
TNFR1 KO animals were mated to produce doubly heterozygous mutant mice.
Doubly heterozygous mutants were then mated to produce doubly
homozygous mutants, dual (IL-1R1-TNFR1) KOs. All animals used were
genotyped for the IL-1R1 and TNFR1 alleles by PCR (39).
Bacterial dose responses, bacterial kinetics, and serum cytokine
responses.
BALB/c, C57BL/6, C3H/HeJ, and KO mice were given doses
ranging from 107 to 5 × 109 CFU of
E. coli WAF108, WAF270, or WAH540 in 0.15 M NaCl i.p. The
50% lethal dose (LD50) and LD100 for each
transformant (calculated by the Reed-Muench method
[62]) were determined for BALB/c and C57BL/6 mice. To
determine the LD100 of endotoxin alone (independent of
other bacterial products), BALB/c mice were challenged i.p. with doses
of 1 to 60 mg of LPS O22/kg of body weight. In vivo growth-elimination
kinetics were then established by injecting i.p. a sublethal dose
(107 CFU) and a lethal dose (108 CFU for WAF270
or 3 × 109 CFU for WAF108 and WAH540). Diluents of
homogenized lung, liver, spleen, and kidney tissue and both blood and
peritoneal lavage fluid were collected from the animals 4 and 18 h
after injection and plated on blood agar with the respective
antibiotic. When indicated, serum samples for cytokine determinations
were obtained from the mice at 90 min and 5 and 10 h after
bacterial challenge. Serum samples were stored at 
80°C until being
assayed. Serum IL-1 and TNF- levels were determined with ELISA
kits (Genzyme Diagnostics, Cambridge, Mass.) according to the
instructions of the manufacturer.
IL-1 receptor blockade prior to bacterial challenge.
BALB/c
and C57BL/6 mice were treated intravenously with 200 µg of hamster
anti-mouse monoclonal immunoglobulin G (IgG) anti-IL-1R1 antibody
(M147; a gift of J. Sims, Immunex Corp.) (46, 57) or 200 µg of Syrian hamster anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) in 100 µl of phosphate-buffered saline-10% murine serum 2 h prior to bacterial infection. The dose used to block IL-1R1 in vivo was based on the work of Rogers et
al., who used the identical antibody in a similar murine system (46). The mice were then inoculated i.p. with 5 × 108 CFU of WAF108, WAF270, or WAH540.
Statistics.
Parametric data were compared by analysis of
variance (ANOVA) and then post hoc by Tukey's honestly significant
difference tests. Nonparametic data were compared by 
2
analyses followed by Pearson 2 and Fisher exact tests,
with Bonferroni corrections for multiple comparisons. Survival
statistics were also compared by product-limit (Kaplan-Meier) analysis
when applicable.
| |
RESULTS |
Characterization of E. coli WAH540 and
nonacylated Hlyinactive.
All
transformants had nearly equivalent growth kinetics in vitro. WAF270,
but not WAF108 or WAH540, displayed significant hemolytic activity on
blood agar cultures. Quantitative hemolytic assays confirmed WAF270's
hemolytic activity (100 and 10% RBC lysis occurred at 16- and
2,096-fold supernatant dilutions, respectively). WAF108 and WAH540
supernatants caused no RBC lysis. Immunoblots of bacterial culture
supernatants revealed Hly (active or inactive) expression
from WAF270 and WAH540 but none from WAF108, as predicted (Fig.
1). WAH540 generated more
Hlyinactive per CFU of bacteria than WAF270 generated
Hlyactive. The relative amount of LPS O22 expression by
these bacterial transformants correlated with the amount of Hly
expressed. Chromogenic quantitative LAL assays confirmed that
E. coli WAH540 produces more LPS per CFU than WAF270,
which produces more than WAF108: WAH540, WAF270, and WAF108
contained 0.95 (±0.02), 0.76 (±0.01), and 0.58 (±0.02) pg of
LPS/102 CFU, respectively, and released 0.27 (±0.07), 0.08 (±0.003), and 0.02 (±0.0001) pg/ml of diluted supernatant,
respectively (P 
0.002 by ANOVA and post hoc
analyses). Densitometric analysis of LPS immunoblots (targeting the O22
polysaccharide) of whole bacteria and supernatants from
equivalently sized cultures confirmed these relative differences in LPS
expression (Fig. 2). The in vitro
cellular stimulatory effects of the LPS produced by the three
transformants were equivalent, as measured by the proliferative capacity of murine splenocytes ([3H]thymidine
uptake [data not shown]) and TNF- release of human PBMC
(Fig. 3). In vitro exposure of human PBMC
to live WAF270 elicited markedly higher levels of IL-1 secretion
than exposure to live WAF108 or WAH540 (Fig.
4); this is similar to what other hemolytic E. coli strains have been shown to elicit
(5).
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FIG. 1.
Immunoblots of filtered culture supernatants of WAF108,
WAF270, and WAH540 with two MAbs against Hly, B7 and G8. Protein
mass markers appear on the left in each blot.
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FIG. 2.
Semiquantitative LPS immunoblot with rabbit anti-LPS O22
antiserum of 104 CFU of whole E. coli cells
(rows 1, 3, and 5) and supernatants (s) from equivalently
sized E. coli cultures diluted 1:104 (rows 2, 4, and 6). The relative densitometric values (and percentages) are shown
below each blot; 100% corresponds to the blot of whole bacterial or
supernatant samples (WAH540 and WAH540s) with the highest
density, to which the others are compared.
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FIG. 3.
Supernatant TNF- ELISA. Human PBMC (2 × 105, in triplicate) were stimulated for 24 h in
RPMI-10% FCS with 1 or 10 ng of purified LPS O26:B6 or LPS O22/ml,
purified from E. coli WAF108, WAF270, or WAH540, and the
supernatants were assayed for TNF-. Error bars indicate significant
errors of the means. There were no differences in TNF- secretion
between transformants when the cells were stimulated for less time
(i.e., 6 to 12 h).
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FIG. 4.
Human monocyte IL-1 release in response to viable
E. coli mutants after 90 min of exposure in vitro. Note that
WAF270 induced significantly higher levels of IL-1 secretion at all
ratios of bacteria to monocytes. P 0.0003 by ANOVA;
error bars indicate standard errors of the means.
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Animal studies.
BALB/c and C57BL/6 mice responded similarly to
each E. coli transformant (WAF108, WAF270, and WAH540).
E. coli WAF270 was significantly more lethal than
WAF108 and WAH540 when given at equal doses over the range of
5 × 107 to 5 × 109 CFU. The
LD50 and LD100 were estimated at 6.5 (±1) × 107 and 1.1 (±0.5) × 108 CFU for
WAF270, 7 (±1.5) × 108 and 3 (±1.5) × 109 CFU for WAF108, and 8 (±1.5) × 108 and 4 (±1.5) × 109 CFU for WAH540, respectively. A
dose of 108 CFU of WAF270 was 100% lethal, but
108 CFU of WAF108 or WAH540 was nonlethal (0%). At their
respective lethal doses, WAF270 caused death significantly more
rapidly than WAF108 and WAH540 (Fig. 5).
WAF270 was also lethal at significantly lower doses than WAF108
or WAH540 in C3H/HeJ mice: e.g., 5 × 108 CFU of
WAF270 was 100% lethal, while the same dose of WAF108 or WAH540 was
nonlethal (n = 8 to 10/group). Challenge (i.p.) with
purified LPS O22 caused 100% mortality at a dose of 40 mg/kg 48 h
after injection. The amount of LPS contained within an
LD100 inoculum of WAF270, determined as described above by
the LAL assay, was 0.76 µg/108 CFU, representing an
initial LPS load over 103-fold less than the
LD100 of purified LPS O22. Four or 18 h after lethal
or sublethal bacterial challenge, the amount of live bacteria recoverable from the peritoneum, lung, liver, kidney, spleen, and
blood was equivalent for all three E. coli transformants (1 to 10% recovery relative to the initial inoculum, depending on the
organ site).
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FIG. 5.
Times of death after i.p. infection with
LD100 of WAF108, WAF270, or WAH540 for C57BL/6 mice. n,
number per group; P = 0.000001 by Kaplan-Meier
analysis.
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Bacterial challenge with each E. coli transformant elicited
significant TNF- secretion by 90 min after challenge (Table
1) in a manner similar to that caused by
LPS O22 when given alone (data not shown). Serum IL-1 was detectable
by 90 min in mice challenged with WAF108, WAF270, or WAH540. The serum
IL-1 level decreased by 5 h in WAF108- and WAH540-challenged
animals but increased in WAF270-challenged animals (Table 1).
Anti-IL-1R1 MAb did not alter the lethality of E. coli WAF270 (Fig. 6).
Similarly, WAF270 was significantly more lethal than WAF108 and WAH540
in both IL-1R1 and dual-KO mice at the same doses, as was
demonstrated in wild-type mice (Fig.
7). There was no significant difference
between the lethalities of individual bacterial transformants
(WAF108, WAF270, or WAH540) in IL-1R1 KO mice and those in
matched wild-type mice at doses from 7 × 107 to
5 × 108 CFU; there were only slight differences
between lethalities in dual (IL-1R1-TNFR1)-KO mice
challenged with WAF108 or WAH540 and those in matched wild-type mice at
these doses.
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FIG. 6.
Survival curves as a function of time following i.p.
bacterial challenge (5 × 108 CFU) in wild-type
(C57BL/6) mice pretreated with anti-mouse IgG (Sham) (A) or anti-IL-1R1
MAb (B). n, number/group; *, P < 0.000001 and < 0.0001, by 2 analysis and post hoc Fisher exact tests,
respectively. Note that the abscissas are not on a linear scale.
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FIG. 7.
Survival curves as a function of time following i.p.
bacterial challenge (5 × 108 CFU) in wild-type (A),
IL-1R1 KO (B), and dual (IL-1R1-TNFR1)-KO (C) mice with WAF108,
WAF270, or WAH540. n, number per group; *, P < 0.000001 and < 0.0001, by 2 analysis and post hoc
Fisher exact tests, respectively. Note that the abscissas are not on a
linear scale.
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DISCUSSION |
Investigators continue to develop simple models to explain
bacterial pathogenesis, but it is becoming clearer that host-bacterial interactions are extraordinarily complex, so that attempts at targeting
human therapies toward specific effector molecules (e.g., TNF and
IL-1) or bacterial products (e.g., LPS) within the framework of
these paradigms have been discouraging (26, 44, 48, 58, 63).
We postulate that in order for these types of approaches to be
effective the therapies will need to be multitargeted and relatively
specific to the type of pathogen. Hly is another potential target.
It is well characterized biochemically and has a wide range of effects
both in vivo and in vitro, including lysis of erythrocytes,
fibroblasts, leukocytes, and epithelial and endothelial cells (14,
15, 53); cellular ATP depletion (5); promotion of
superoxide anion release (4); and inhibition of macrophage antigen presentation (18). In this study we have increased
our understanding of the pathogenesis of hemolytic E. coli
infections.
We demonstrate that fatty acid acylation of Hly is necessary in
order for the live hemolytic E. coli transformant, WAF270, to elicit IL-1 from monocytes in vitro, to cause hemolysis, and to
enhance its virulence in vivo. The two nonhemolytic E. coli transformants, WAF108 and WAH540, which produce no Hly
(Hlynull) and nonacylated Hly
(Hlyinactive), respectively, required doses more than
10-fold that of WAF270, which produces fully acylated Hly
(Hlyactive), in order to induce mortality. The WAF270
transformant elicited an IL-1 (and an IL-1 [35])
secretion pattern both in vivo and via monocytes in vitro that
was markedly different from that elicited by either WAF108 or
WAH540. The hemolytic transformant elicited high levels of IL-1,
while the nonhemolytic strains elicited only low levels in
patterns similar to those elicited by endotoxin challenge.
Hlyactive (from WAF270) and Hlyinactive
(from WAH540) had identical electrophoretic properties and were recognized by two anti-Hly MAbs which bind to distinct epitopes of
Hly, although Hlyinactive is nonhemolytic and
Hlyactive is very hemolytic. Therefore, we conclude
that fatty acid acylation of Hly via expression of the
hlyC locus is critical to the function of Hly and the
virulence of hemolytic E. coli.
Demonstrating the lethal potential of purified Hly alone has been
difficult due to its instability ex vivo and its association with LPS.
In prior studies, where we compared the lethal potential of
partially purified culture supernatants from WAF108, WAF270, and
WAH540 cultures, WAF270 supernatant appeared to be more lethal than the
others (unpublished results). In the present study we recognized that
concentrates of the culture supernatants contain large amounts of LPS
bound to Hly. The partial purification process itself reduced the
hemolytic activity of the preparations and probably changed the
conformation of Hly (9, 10, 20, 55). Thus, it is
likely that Hlyactive or
Hlyinactive is expressed in conjunction with LPS in the
respective transformant, WAF270 or WAH540.
We have demonstrated that these transformants express antigenically and
functionally identical endotoxins, but they express different
quantities of LPS. It appears that the amount of LPS expressed by a
transformant correlates with the relative differences in
Hlynull, Hlyactive, or
Hlyinactive production by the transformant. In other
words, WAH540 expresses the most Hlyinactive and
consequently the most LPS. This was confirmed by both LAL and
immunoblot assays. Despite the association between Hly and LPS
expression, we demonstrated that the release of Hly-associated LPS
does not seem to be primarily responsible for the enhanced lethality of
Hly-producing E. coli (WAF270), since WAH540 (which expresses more LPS) is actually considerably less virulent than WAF270
and equivalent in virulence to WAF108 (which expresses the least LPS).
The LPS expressed by each transformant has the same stimulatory effect
in vitro, and thus, their endotoxins seem equally potent. Furthermore,
at their respective LD100s, an inoculum of WAF270 contains
markedly less LPS O22 than does an inoculum of WAF108 or WAH540 (0.76 µg versus 5.8 or 9.5 µg, respectively). At equal doses of
108 CFU/mouse in which the initial inoculum of WAF270,
WAF108, or WAH540 contained similar amounts of LPS (0.76, 0.58, and
0.95 µg, respectively), only challenge with WAF270 caused mortality. Additionally, equivalent amounts of each specific transformant were
recoverable from the lung, spleen, liver, kidney, blood, and peritoneum
during the acute phase of a lethal infection, suggesting that the
transformants have similar in vivo growth-elimination kinetics, but
only WAF270 caused early mortality (<8 h). WAF108 and WAH540 caused
late mortality (36 to 72 h), similar to the lethality induced by
challenge with LPS O22 alone (48 h). Finally, endotoxin-tolerant
C3H/HeJ mice responded to WAF270 in a manner similar to that of the
endotoxin-sensitive strains (BALB/c and C57BL/6). Collectively,
these data suggest that LPS is not the primary factor responsible for
hemolytic WAF270-induced lethality at LD100 or less. The
increased lethality of WAF270 appears to be directly related to the
expression of acylated Hly.
Both IL-1 and TNF- have detrimental effects which correlate with the
severity of certain types of infections in animal models. However, they
do not appear to be critical mediators of the lethality of hemolytic
E. coli infection in our model. Because of the
associated IL-1 spike recognized during WAF270 infection
(35), we initially hypothesized that IL-1 signaling
(with or without TNF signaling) was the effector mechanism
mediating the lethality of hemolytic E. coli. In the
present study we show that WAF270 challenge similarly elicited a
sustained level of IL-1 secretion. However, we demonstrate that
neither preventing IL-1 signaling with IL-1R1 MAb blockade in wild-type
mice nor challenging IL-1R1-deficient mice altered the lethality of
hemolytic E. coli (WAF270). The lethality profiles of WAF108
and WAH540 were also similar in wild-type and KO animals. Thus,
mortality from any of the E. coli transformants does not appear to be mediated via signaling through IL-1R1.
Significant TNF- secretion was also demonstrated after
challenge with either WAF108, WAF270, or WAH540 at 90 min,
as would be expected. These elevated serum TNF- levels
decreased by 5 h after challenge. In our model, TNF's role in
Hly-producing E. coli challenges did not appear to
be different than in non-Hly-producing E. coli
challenges, because WAF108, WAF270, and WAH540 all elicited similar TNF- secretory profiles. TNF- and - antibody blockade previously failed to alter WAF270-induced mortality
(35), and now we demonstrate that dually (IL-1R1-TNFR1)
deficient mice were not protected from WAF270-induced mortality.
Collectively, the results of these studies support the notion
that there is no correlation between IL-1 or TNF signaling and
hemolytic E. coli WAF270-induced mortality.
The mechanism by which Hly leads to enhanced lethality of E. coli remains ill defined. It is now clear, however, that fatty acid acylation of the lysine residues of Hly, via expression
of the hlyC locus, is absolutely critical to
facilitating both its toxicity and its rendering of IL-1 secretion. It
is perplexing that despite the correlation between Hly expression
and IL-1 secretion, the prevention of IL-1 and/or TNF signaling does
not protect animals from acute hemolytic E. coli infection
as it has in other models of nonhemolytic E. coli infection
(1) or Listeria infection (46).
Necropsy studies of animals infected with WAF270 have not been helpful
in identifying a cause of death, demonstrating only a mild-to-moderate
hepatic infiltration of neutrophils (unpublished results).
We have learned that animals infected with WAF270 and a sterile stool
adjuvant actually recruit significantly fewer leukocytes to the
peritoneum during WAF270 infection than during WAF108 infection. A much
greater percentage of the leukocytes that are recovered from
WAF270-infected animals are nonviable compared to those isolated from
WAF108-infected animals (unpublished data). Thus, the mechanism by
which Hly enhances the lethality of E. coli may be
explained, in part, by its effects on neutrophil recruitment,
degranulation, and cell viability. In vitro studies corroborate the
hypothesis that Hly has profound effects on neutrophils (e.g.,
increased granule formation [3], increased
neutrophilic chemiluminescence [16], increased release
of toxic free radicals [4], and diminished bactericidal activity of neutrophils [50]), although a
relationship between Hly's effect on neutrophils and
Hly-induced mortality remains to be determined. Finally, we have
demonstrated that pretreatment with killed bacteria well before the
generation of an anamnestic response (as early as 2 days before live
bacterial challenge) completely protects animals from an
otherwise-lethal WAF270 challenge, again suggesting that
upregulating nonspecific immune responses (e.g., neutrophilic
recruitment and function) may protect against this rapidly fatal
bacterial infection (25). Collectively, these studies
provoke the suspicion that nonspecific immune mechanisms like
granulocyte recruitment and effector functions may be particularly relevant during hemolytic E. coli infections, and they
support the notion that the deleterious effects of hemolysin and
hemolytic bacteria may be preventable or suppressible in clinical
settings.
T. G. Gleason was supported by Public Health Service,
National Research Service Award 1F32AI09482-01A1.
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Abstract
Introduction
