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* Unité de Pathogénie Microbienne Moléculaire and Institut National de la Santé et de la Recherche Médicale, Unité 389, and
Unité de Recherche et d’Expertise Histotechnologie et Pathologie, Institut Pasteur, Paris, France; and
Department of Clinical Veterinary Medicine, Center for Veterinary Science, University of Cambridge, Cambridge, United Kingdom;
Karolinska Institute, Clinical Research Center, Huddinge University Hospital, Stockholm, Sweden; and
¶ Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by
human monocytes and to cause rupture and inflammatory destruction of
the epithelial barrier in the rabbit ligated intestinal loop model of
shigellosis, indicating that lipid A plays a significant role in
aggravating inflammation that eventually destroys the intestinal
barrier. In addition, neutralization of TNF- during invasion by the
wild-type strain strongly impaired its ability to cause rupture and
inflammatory destruction of the epithelial lining, thus indicating that
TNF- is a major effector of epithelial destruction by
Shigella. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current physiopathological scheme of shigellosis proposes that
luminal bacteria initially cross the epithelial barrier, predominantly
via M cells of the follicle-associated epithelium, and quickly invade
resident macrophages that undergo apoptosis due to IpaB-mediated
activation of caspase-1 (6, 7, 8). This leads to the release
of IL-1
, which, in conjunction with a dramatic drop in IL-1R
antagonist, is likely to play a major role at the early stage of
Shigella pathogenesis by causing inflammation-mediated
disruption of epithelial permeability that facilitates extension of
bacterial translocation (9, 10, 11). Bacteria that have
reached subepithelial tissues and survived macrophage killing may
efficiently invade epithelial cells basolaterally and proceed to
cell-to-cell spread. LPS delivered by Shigella in the
cytoplasmic compartment of infected epithelial cells activates the
translocation of NF-
B (12) via a Nod1-dependent pathway
(13) and causes these cells to produce proinflammatory
cytokines and chemokines such as IL-8 (12, 14, 15). This
places epithelial cells at the front line (16) in the
elicitation of the inflammatory cascade that leads to destruction of
the epithelial barrier. IL-8 plays a major role by inducing massive
influx of polymorphonuclear leukocytes
(PMN)3 that
participate in mucosal destruction (17).
As the signals leading to acute intestinal inflammation in shigellosis
are being deciphered, it appears that the actual impact of the
bacterial endotoxin (i.e., the lipid A moiety of LPS) has remained
largely unexplored. It may participate in triggering or enhancing
inflammation upon interaction with Toll-like receptor 4 expressed,
along with CD14, by monocyte-macrophages (18) and upon
invasion of epithelial cells through Nod1 (19). In an
attempt to define this role, we previously conducted experimental
Shigella infection in ligated intestinal loops of rabbits
injected with a neutralizing anti-CD14 mAb (20).
Compared with animals receiving a control Ab, rabbits in which CD14 was
neutralized showed a dramatic increase in the quantity of bacteria
invading the intestinal mucosa and more severe tissue destruction.
Considering that these paradoxical results did not shed light on the
actual role of lipid A in the development of intestinal inflammation,
we decided to take a more straightforward approach, relying on the
recent possibility to genetically attenuate the endotoxin activity of
lipid A by constructing mutants in the genes responsible for the last
two steps of its biosynthesis (21, 22), during which the
12-carbon fatty acid laurate and the 14-carbon fatty acid myristate are
acyl-oxyacyl-linked to two of the four 3-OH-myristic acids available on
keto-doxy-octonate 2 lipid A (Fig. 1
). The htrB gene encodes the
transferase that catalyzes the acyl-oxyacyl linkage of laurate to the
3' hydroxy-myristate that is itself linked to the 2' position of the
glucosamine. Inactivation of this gene results in a conditionally
lethal phenotype (21). The msbB gene encodes
the transferase that catalyzes the acyl-oxyacyl linkage of myristate on
the hydroxy-myristate that is itself linked to the 3' position of the
glucosamine disaccharide. The msbB mutations are not lethal
(23). The acyl-oxyacyl-linked secondary myristate chain is
essential for full host recognition of lipid A of a living
microorganism (22), because an Escherichia coli
msbB mutant shows dramatic decrease in proinflammatory potential
as monitored in vitro by induction of selectin expression by
endothelial cells and production of TNF- by monocytes.
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in the course of an experimental
infection by the wild-type strain led to similar protection against
rupture of the epithelial barrier. In consequence, the degree of lipid
A endotoxicity plays a major role in the rupture of epithelial
continuity and mucosal tissue destruction in experimental shigellosis,
and TNF- appears to be a major effector. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S. flexneri strains used in this study derived from
the wild-type strain M90T (serotype 5a), including the
streptomycin-resistant strain M90T-Sm and the virulence plasmid-cured
strain BS176 (3, 24). E. coli strains
were derivatives of E. coli K-12: DH5 (25)
was used for plasmid constructions, DH5 
pir, SM10 pir
(26), and 2155.88-(pir RP4 DAP) were used to
construct derivatives of the suicide vector pLAC2 (24) and
to transfer these plasmids to S. flexneri, and CC118pir
and S17-1(pNJ5000) were used to construct derivatives of the suicide
vector pCVD442 (27) and to transfer these plasmids to
S. flexneri. Bacteria were grown in Luria-Bertani medium or
tryptic soy broth. Antibiotics were used at the following
concentrations: ampicillin, 100 µg/ml; kanamycin (Km), 40 µg/ml;
streptomycin, 100 µg/ml; and chloramphenicol, 30 µg/ml. When
indicated, the growth medium was supplemented with Congo red (0.01%)
and diaminopimelic acid (0.3 mM).
Inactivation of the chromosomal msbB1 gene
We used PCR to amplify a DNA fragment corresponding to the 5'
portion and the upstream region of the msbB1 gene, the
aphA gene from pUC4K (28), and the 3' portion
and downstream region of the msbB1 gene. PCR fragments were
digested by appropriate restriction enzymes, the sites for which had
been incorporated within the PCR primers, mixed, and ligated together
with SphI- and XbaI-digested pUC19 DNA (Fig. 2). In the pUC19
msbB1::Km recombinant plasmid thus constructed,
the 3.4-kb SphI-XbaI insert corresponded to the
aphA gene flanked by 1) the 5' region of msbB1,
from nucleotide -859 (coordinates from the ATG of msbB1) to
nucleotide +145, and 2) the 3' region of msbB1, from
nucleotide +866 to nucleotide +2016 (coordinates from the ATG of
msbB1). This insert was then cloned between the
SphI and XbaI sites of the suicide vector pCVD442
(27) to construct pCVD442 msbB1::Km.
The E. coli CC118pir derivative harboring pCVD442
msbB1::Km was then mated with E. coli
S17-1 (pNJ5000), which was used to mobilize the suicide plasmid into
S. flexneri M90T. Transconjugants were selected by plating
onto plates that contained sucrose (10%) and Km. The allelic exchange
between the mutated msbB1 gene carried by the suicide
plasmid and the wild-type msbB1 gene carried by the
chromosome was confirmed by PCR using appropriate primers. The
msbB1 mutant thus constructed was designated
SFmsbB1::Km. It appeared that strain
SFmsbB1::Km had lost the capacities to bind Congo
red and invade HeLa cells, probably as a consequence of rearrangements
within the virulence plasmid. Therefore, the
msbB1::Km mutation was then transduced from strain
SFmsbB1::Km to the wild-type strain M90T to
construct the strain SC574 (msbB1). Similarly, the
msbB1::Km mutation was transduced to the virulence
plasmid-cured strain BS176 to construct strain SC575
(VP-msbB1). Both recombinant strains were then tested for
the presence of the mutated msbB1 gene using PCR. In these
msbB1 mutants, the msbB1 gene was interrupted
after codon 48 (CGT).
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A 0.46-kb fragment extending from nucleotide +146 to nucleotide
+606 of the msbB2 gene was amplified by PCR from the
virulence plasmid pWR100 and cloned between the BamHI and
SphI sites of the vector pK19 (29) to construct
the plasmid pHS50. The insert carried by pHS50 was then cloned between
the SphI and XbaI sites of the suicide vector
plasmid pLAC2 (24) to construct the plasmid pHS53. In this
later plasmid, the msbB2 internal fragment was cloned
upstream from and in the same orientation as the promoterless
lacZ gene carried by the vector. Plasmid pHS53 was then
introduced by transformation into E. coli strains Sm10pir
or 2155.88-(pirRP4), and transformants were used to transfer the
plasmid into S. flexneri strains M90T-Sm (wild-type) and
SC574 (msbB1), respectively, by conjugation. Transconjugants
were selected on plates containing ampicillin and either streptomycin
or Km. The structure of the derivatives of pWR100 carrying the
integrated pHS53 plasmid was confirmed by PCR, and the resulting
strains were designated SC576 (msbB2) and SC577
(msbB1 msbB2). In the msbB2 mutants,
the msbB2 gene was interrupted after codon 202 (GGG). To
complement the msbB1 msbB2 mutant, we constructed
a derivative of the vector pSU2718 carrying a complete msbB2
gene under the control of a lac promoter. Briefly, a DNA
fragment encompassing the region from nucleotide -37 to nucleotide
+950 of msbB2 was amplified and cloned between the
BamHI and HindIII sites of pSU2718 to construct
the plasmid pHS54. The sequence of the insert cloned into pHS54 was
verified by sequencing, and the plasmid was introduced by
transformation into the strain SC577 to construct strain SC578
(msbB1 msbB2 pHS54/MsbB2+).
Analysis of lipid A by MS
Preparation of LPS. The strains were grown in submerged culture to late exponential phase in 22 L of Luria broth containing 1% glucose, using a 30-L fermenter (Belach, Stockholm, Sweden) under constant aeration at 37°C (pH 7). Depending on the antibiotic cassette used to select the mutations, either Km (40 µg/ml) or ampicillin (100 µg/ml) was added to the medium. Strain M90T was grown in batch culture of 3 L. A preculture in the same medium was used to inoculate the fermenter. The medium was inoculated with a loop directly from a Congo red-positive clone on Congo red-agar plates. All cultures were checked for purity at the end of the growth cycle. Bacteria were then killed with 1% (mass/volume) formaldehyde. After incubation overnight at 4°C, cells were separated from the medium by continuous-flow centrifugation using a CEPA model LE centrifuge (C. Padberg, Centrifugenbau, Lahr, Germany) at a cylinder speed of 35,000 rpm and a flow of 25 L/h. The culture of M90T strain was centrifuged at 8,000 x g for 20 min at 4°C and finally resuspended in distilled water. LPS was extracted by the hot phenol/water method (30). The aqueous phase was dialyzed for 3–5 days against tap water, then overnight against distilled water, concentrated against diminished pressure, and lyophilized. Contaminating nucleic acids were removed by ultracentrifugation (100,000 x g for 4 h at 4°C). The nucleic acid content was determined spectrophotometrically as described (31), and the protein content was estimated with BSA as a standard (32). The presence of remaining nucleic acids and proteins was found to be <5 and <0.5%, respectively.
Preparation and purification of lipid A. S. flexneri LPS was subjected to mild acid hydrolysis using aqueous 1% acetic acid at 100°C for 2 h. Lipid A was isolated using centrifugation and pellets were washed twice with water with intermediate centrifugations. The washed lipid A was further purified by partition using chloroform/methanol/water (2/1/1) for 10 min. Following centrifugation, the lower chloroform phase was evaporated to dryness using a stream of nitrogen.
Mass spectrometry.
Electrospray ionization (ESI) mass spectrometry (MS) in negative mode
was performed using an LCQ ion trap mass spectrometer (Thermo Finnigan,
San Jose, CA). Lipid A was dissolved in 0.5 ml chloroform/methanol
(1/1) to a concentration of 
0.2–0.4 mg/ml. This dilution of lipid A
was introduced into the mass spectrometer at a flow of 5 µl/ml.
Nitrogen was used as sheath gas, the needle voltage was set to -4 kV,
and the temperature of the heated capillary was set at 185°C.
Matrix-assisted laser desorption ionization (MALDI)-MS in the negative
mode was performed using a Lasermat 2000 time-of-flight mass
spectrometer (Thermo Finnigan). For MALDI-MS, lipid A was dissolved in
chloroform at a concentration of 50–500 µg/ml and a saturated
solution of norharmane (Sigma-Aldrich, St. Louis, MO) in chloroform was
used as matrix. An equal volume of sample and matrix was mixed and 1
µl was loaded onto the sample plate. The plate was then dried at room
temperature and introduced into the mass spectrometer. Spectra are the
average from 10 pulses from a nitrogen laser (337 nm), and the
extraction potential used was 20 kV.
Virulence assays
Virulence of the various strains was evaluated by testing their ability to invade HeLa cells, to form plaques on confluent Caco2 cells (33, 34), and to induce keratoconjunctivitis in Guinea pigs (35).
In addition, a total of 16 New Zealand White rabbits weighing 2.5–3 kg (Charles River Breeding Laboratories, Wilmington, MA) were used for experimental infections. From each of these animals, nine intestinal ligated loops, each of 5 cm, were prepared, as previously described (36, 37). Within each loop, 109 bacteria were injected in 0.5 ml of isotonic saline. Following 8 h of infection, animals were sacrificed.
In the eight rabbits that were used to test the msbB mutants
in comparison to the wild-type M90T strain and the noninvasive BS176
strain, the exudates contained within the loops were aspirated,
measured, and frozen at -80°C before assay for TNF- activity. The
loops were dissected, longitudinally opened, and fixed in 4% buffered
formalin or zinc sulfate buffer before histopathological analysis.
According to this protocol, each strain was tested an average of 12
times (i.e., 9 loops x 8 rabbits/6 different strains).
Among the eight rabbits that were used to evaluate the effect of
TNF- neutralization, 30 min before the laparotomy was started four
animals were injected i.v. with a murine IgG1 mAb (23H1) that
neutralizes rabbit TNF- activity at a concentration of 5 mg/kg. When
injected at this concentration in rabbits, 23H1 completely neutralizes
TNF- cytotoxic activity appearing in serum 1–2 h following
injection of 10 mg of LPS from Salmonella enterica Minnesota
Re595 (J. Mathison and R. J. Ulevitch, unpublished data). The four
control rabbits received 5 mg/kg i.v. of a nonrelated, anti-yeast
glutathione-reductase, murine monoclonal IgG1 (Oriental Yeast, Azuzawa,
Japan). Following sacrifice of the animals after an 8-h infection,
collection and measurement of intestinal fluid and of tissue samples
were conducted as above. According to this protocol, the wild-type
strain M90T was tested an average of 25 times in each of the two
situations (i.e., TNF- neutralization and control), because the
noninvasive strain BS176 was introduced in at least two loops per
rabbit as a negative control.
Histopathological analysis of tissue samples
Intestinal biopsies fixed in 4% buffered formalin were
dehydrated, embedded in paraffin, and sectioned in 5-µm slices. As
previously described (20), sections were used for H&E
staining or for LPS immunostaining. Bacterial LPS was labeled by using
a primary murine mAb anti-S. flexneri 5a LPS (6 µg/ml;
A. Phalipon, Institut Pasteur, Paris, France). Intestinal biopsies
fixed in zinc sulfate buffer at 4°C for 5 days were embedded in
low-temperature paraffin (melting point, 37°C) from which 4-µm
sections were prepared and labeled with primary murine mAb
anti-rabbit CD14 clone 1116, 16 (8 µg/ml) (38).
Slides were washed three times in PBS, then the secondary biotinylated
goat-anti mouse Ig (AO433; DAKO, Glostrup, Denmark) diluted 1/400 was
added for 1 h. Slides were washed in PBS and incubated in the
presence of streptavidin-HRP conjugate (DAKO) for 45 min. For
anti-CD14 immunochemistry, a pyramide signal amplification
step was added (NEN Life Sciences, Boston, MA). The color reaction was
developed by addition of
H2O2 and
aminoethylcarbazole (Sigma-Aldrich). Sections were counterstained with
hematoxylin.
TNF- production by stimulated human adherent monocytes
Human PBMCs were isolated by density gradient centrifugation
with low-endotoxin Ficoll-Hypaque (Amersham Pharmacia Biotech, Orsay,
France). The mononuclear cells were resuspended at 2 x
106 cells/ml in 0.5% FCS-RPMI 1640 medium and 10
ml of the cell suspension were added to petri dishes and left to adhere
for 1 h at 37°C. The nonadherent cells were washed out with RPMI
1640, and 10 ml of 5% FCS-RPMI medium were added to the adherent
cells. These cells were then incubated for 18 h at 37°C.
Adherent monocytes were scraped using EDTA (10 mM), centrifuged,
and resuspended in RPMI. After counting, cells were diluted to 5
x 105/ml, and 1-ml aliquots of this suspension
were added to each well of 24-well tissue culture plates coated with
100 µl of human normal serum type AB (Sigma-Aldrich). Cells were
allowed to adhere for 2 h at 37°C. Bacteria were grown in
tryptic soy broth to stationary phase and adjusted to
108 CFU/ml in 2% human normal serum-RPMI medium,
and dilutions of the bacterial suspensions were used to infect adherent
monocytes. After 4 h of infection at 37°C, the culture media
were harvested and centrifuged, and the supernatants were assayed for
the presence of TNF- using a specific ELISA (Amersham Pharmacia
Biotech) (22).
TNF- bioassay
Supernatants recovered from centrifuged intestinal exudates were
filtered and assayed for bioactivity using the TNF--sensitive murine
fibrosarcoma cell line WEHI 164, clone 13, as previously described
(39, 40). The cytotoxic activity of the tested
supernatants could be inhibited by coincubation with a polyclonal Ab
directed against rabbit TNF- (38800; BD PharMingen, Le Pont de
Claix, France).
Statistical analyses
The nonparametric Mann-Whitney test was used for two-way comparison and determination of the statistical significance of the differences in absolute values. Values of p < 0.05 were considered statistically significant and are marked on figures as * or **.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of the lipid A modifications on the
intensity of the inflammatory response induced by S.
flexneri upon infection of an epithelium, we decided to use lipid
A mutants in which the hydroxy-myristate linked to the 3' position of
the glucosamine disaccharide would not be modified by acyl-oxyacyl
linkage of a myristate molecule. Analysis of the complete sequence of
the virulence plasmid pWR100 had revealed the presence of an open
reading frame, the product of which exhibited 69 and 30% sequence
identity with the MsbB and HtrB proteins of E. coli,
respectively (4). This suggested that the virulence
plasmid encodes a protein endowed with an MsbB-like activity. This
plasmidic gene was designated msbB2 and the chromosomal gene
was designated msbB1. To analyze the respective
contributions of each of these two genes in the modification of lipid
A, we constructed strains in which either msbB1 or
msbB2, or both genes, had been inactivated and used the
following strains: M90T, the wild-type strain; BS176, a derivative of
M90T that had been cured of pWR100 and therefore lacked the
msbB2 gene; SC575, a derivative of BS176 in which the
msbB1 chromosomal gene was inactivated; SC574 and SC576, two
derivatives of M90T in which either the chromosomal msbB1 or
the plasmidic msbB2 genes, respectively, were inactivated;
SC577, a derivative of M90T in which both msbB1 and
msbB2 were inactivated; and SC578, a derivative of SC577
carrying a plasmid expressing a wild-type copy of msbB2 from
a constitutive promoter. The lipid A obtained by mild acidic hydrolysis
of the LPS prepared from the different strains was analyzed by ESI-MS
and MALDI-MS. The wild-type strain M90T had a dominant (M-H)- ion at
m/z 1716 that corresponds to a monophosphorylated lipid A containing
six acyl groups linked to the disaccharide. The structure of this ion
was assumed to contain hydroxymyristic acid at C2', C3', C2, and C3,
which was further esterified with myristic acid at C3' and lauric acid
at C2' (Fig. 1
), as previously proposed (41). In contrast
to that of the wild-type strain, the lipid A of strain SC575
(msbB1 pWR100-) showed a dominant (M-H)- ion at m/z 1506
and a smaller ion at m/z 1279. The ion at m/z 1506 corresponds to a
lipid A containing five acyl groups, with hydroxymyristic acids at C2',
C3', C2, and C3, where one hydroxymyristic acid is further esterified
with lauric acid. The ion at m/z 1279 corresponds to a
lipid A containing four acyl groups. As indicated in Table I, strains SC574 (msbB1),
SC576 (msbB2), and BS176 (pWR100-) had a dominant (M-H)-
ion at m/z 1716 and ions with lower relative intensity at m/z 1506 and
1279, whereas SC577 (msbB1 msbB2) had a dominant
(M-H)- ion at m/z 1506. The absence of
N-acetyl-glucosamine
(GlcN)2(HPO3)(14:O(3-OH))2(14:O(3-O(14:O)))(14:O(3-O(12:O))) in
the lipid A of SC575 (msbB1 pWR100-) reflected the lack of
acyl-oxy-acylation by myristic acid in C3' of lipid A. This indicated
that there was a single copy of the msbB gene on the
chromosome of S. flexneri. The relative proportions of
(GlcN)2(HPO3)(14:O(3-OH))2(14:O(3-O(14:O)))(14:O(3-O(12:O))) among the
different fatty-acyl groups produced by M90T (wild-type) and BS176
(pWR100-) were 93 and 63%, respectively, which indicated that
saturation of the lipid A was almost complete in the presence of the
virulence plasmid and only partial in its absence. This suggested that
the msbB2 gene carried by the virulence plasmid was
expressed and encoded a protein endowed with acyl-oxy-acyl-transferase
activity. Indeed, the lipid A of the msbB1 derivative of
M90T (SC574) still contained 59% of
(GlcN)2(HPO3)(14:O(3-OH))2(14:O(3-O(14:O)))(14:O(3-O(12:O))), a product
that was longer present in the lipid A produced by SC577
(msbB1 msbB2). Furthermore, SC576
(msbB2) exhibited a significant decrease in the percentage
of (GlcN)2(HPO3)(14:O(3-OH))2(14:O(3-O(14:O)))(14:O(3-O(12:O))) as
compared with the wild-type strain. The presence of the ion (M-H)- at
m/z 1716 in the lipid A extracted from strains SC574
(msbB1), SC576 (msbB2), and BS176 (pWR100-) and
its absence in the lipid A extracted from strains SC577
(msbB1 msbB2) and SC575 (msbB1
pWR100-) indicated that both MsbB1 and MsbB2 are endowed with some
activity carrying out acylation by (14:O(3-OH))3(14:O(3-O(12:O))) in
position 3'. In conclusion, the lipid A of SC577 (msbB1 and
msbB2) and SC575 (msbB1 pWR100-) showed no
acyl-oxy-acylation of the myristate in 3', whereas that of SC574
(msbB1), SC576 (msbB2), and BS176 (pWR100-) had
partial acyl-oxy-acylation and that of the wild-type strain M90T was
fully modified.
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Effect of msbB mutations on S. flexneri interaction with epithelial cells and monocytes
To investigate the effect of the lipid A modification on the
efficiency of entry of S. flexneri, the various mutants were
tested in the plaque assay on Caco-2 cells (34).
Derivatives of M90T in which either msbB1 or
msbB2, or both genes, were inactivated induced the formation
of the same number of plaques as compared with the wild-type strain,
which indicated that the mutants had no defect in their ability to
enter into epithelial cells. Likewise, the size of the plaques produced
by the mutants was similar to the size of those produced by the
wild-type strain, indicating that the mutants were not impaired for
cell-to-cell spread (data not shown). The production of TNF- by
human monocytes from peripheral blood was used as a read-out for
expression of endotoxicity. Monocytes were incubated in the presence of
bacteria at various multiplicities of infection (MOI) for 4 h, and
the amount of TNF- released in the medium was measured by ELISA as a
read-out for stimulation. The dose-response curves obtained with the
invasive strains M90T (wild-type) and SC577 (msbB1
msbB2) and the noninvasive strains BS176 (pWR100-) and
SC575 (msbB1 pWR100-) are shown in Fig. 3. At MOI 2 (106
CFU), all the strains induced the release of similar amounts of TNF-
by the infected monocytes; however, at lower MOI, differences appeared
between the strains that produced a modified lipid A, such as M90T
(wild-type) and BS176 (pWR100-), and those that produced unsaturated
lipid A, such as SC575 (msbB1 pWR100-) and SC577
(msbB1 msb2), and reached statistical
significances for MOI 0.02 (104 CFU) and 0.002
(103 CFU). This indicated that differences in
modification of the lipid A rather than in the invasive abilities of
the mutants were responsible for the decreased stimulation. For each
strain, the stimulating dose 50 (SD50), i.e., the
number of bacteria that was required to cause 50% of the maximal
release of TNF-, was calculated (see insert in Fig. 3). The various
strains could thus be grouped into three classes: 1) M90T (wild type),
BS176 (pWR100-), and SC578 (msb1 msbB2 pMsbB2), with
SD50 values of 4–7 x
103; 2) SC574 (msbB1) and SC576
(msbB2), with SD50 values of
20–30 x 103; and 3) SC575
(msbB1 pWR100-) and SC577 (msbB1
msbB2), with SD50 values of 80 x
103.
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Before proceeding to rabbit infections, msbB mutants were tested in the classical keratoconjunctivitis assay in Guinea pigs (Sereny test, Ref. 35). Although detailed quantification of inflammation is difficult in this test, compared with the wild-type strain M90T, which caused severe keratoconjunctivitis in 48 h, the msbB mutants, particularly SC577 (msbB1 msbB2), caused delayed (i.e., 72–96 h) and significantly weaker tissue reaction. This indicated that endotoxin affects the degree of inflammation in this test. However, compared with the negative reaction observed with the noninvasive strain BS176, it was clear that these mutants were still able to cause significant damage to the invaded conjunctival and corneal epithelium, thus the need for further analysis in the rabbit ligated intestinal loop model of experimental shigellosis.
In this assay, severity of tissue inflammation can be evaluated by
recording qualitative and quantitative alterations of the intestinal
mucosa. Infections of ligated loops by each of the strains described
above were conducted for 8 h. The amount of fluid exudate in
infected loops, shown as volume/length (V/L) and the concentrations of
TNF- in these samples were recorded. Histopathological alterations
were quantified and recorded as the average ratio between the length
and the width of the villi that measures villus atrophy on
hematoxylin-eosin-safranin (HES)-stained tissue sections
(9) and the average number of abscesses disrupting the
epithelial lining on tissue sections stained with an anti-LPS serum
(9). These data are shown in Fig. 4. The wild-type strain M90T caused
maximum fluid accumulation (0.59 ± 0.2 ml/cm), whereas the
noninvasive strain BS176 barely caused fluid production (0.05 ±
0.01 ml/cm), and the amount of fluid elicited by SC577 (msbB1
msbB2) was one-third of that elicited by M90T (both values were
significantly different from M90T), while the mutants SC574
(msbB1) and SC576 (msbB2) and the complemented
strain SC578 (msbB1 msbB2 pMsbB2) elicited intermediate
values. The acute villus atrophy caused by M90T (wild type) was
reflected by a length-width (L:W) ratio of 1.7, in contrast to
the absence of atrophy caused by BS176 (pWR100-), which was reflected
by a L:W ratio of 8. SC577 (msbB1 msbB2) appeared strongly
attenuated, as reflected by a L:W value of 5.8, and the degrees of
atrophy caused by SC574 (msbB1), SC576 (msbB2),
and SC578 (msbB1 msbB2 pMsbB2) were intermediate between
those caused by M90T and SC577 (msbB1 msbB2). Compared with
M90T, L:W ratios reached significant difference for BS176, SC576,
SC577, and SC578. Similarly, the density of foci of epithelial rupture
by abscesses was highest (80%) following infection by M90T, whereas no
significant lesions were observed with BS176. SC577 (msbB1
msbB2) showed low density of lesions, and the single mutants, as
well as SC578 (msbB1 msbB2 pMsbB2), again caused
intermediate values. All values appeared significantly different from
M90T, with SC577 itself being significantly lower than single mutants.
Qualitatively, as detailed below, lesions caused by SC577 (msbB1
msbB2), SC574 (msbB1), and SC576 (msbB2)
corresponded essentially to limited areas of rupture of the epithelial
lining and not to extensively destructive mucosal abscesses, as
observed with M90T. Finally, M90T caused the highest concentration of
TNF- in the collected fluid samples and SC577 caused the lowest
concentration (one-third of M90T, sole statistically significant
difference), while the single mutants and SC578 caused intermediate
concentrations.
|
. In the characteristic lesions observed
after infection by the wild-type strain M90T (Fig. 5A),
the villi were shortened and enlarged in cases with tissue
necrosis, leaving crypts as the only identifiable epithelial structure.
Often, the lamina propria appeared hypercellular and hemorrhagic. In
addition, submucosal tissues were strongly edematous, with a large area
infiltrated by inflammatory cells between the residual muscularis
mucosae and the muscular layer. The most salient feature, in all
circumstances, was the rupture of the epithelial lining. In contrast,
intestinal villi of loops infected with the noninvasive strain BS176
appeared long and narrow, with no evidence of rupture of the epithelial
lining, no significant infiltrate of the lamina propria, and no
submucosal edema (Fig. 5B). The msbB1 and
msbB2 mutants caused an intermediate phenotype with regard
to tissue alterations. In intestinal loops infected by the
msbB1 mutant (SC574, Fig. 5C) or the
msbB2 mutant (SC576, Fig. 5D), the villi appeared
shortened and enlarged with the lamina propria swollen by a significant
inflammatory infiltrate. The epithelial lining was rarely disrupted;
however, it was very often indented, leading to multiple zones of
constriction of the villi. Variable submucosal edema was observed. The
msbB1 msbB2 mutant (SC577, Fig. 5E)
showed a dramatic decrease in the pattern of pathology. Villi remained
in general of a length and a width very similar to those observed
following infection with the noninvasive strain BS176. A limited edema
was observed with few cells infiltrating the lamina propria and, most
importantly, no rupture of the epithelial lining was ever observed.
Conversely, the complemented strain SC578 recovered its capacity to
cause major alteration in the villi characterized by areas of rupture
of the epithelial lining (Fig. 5F), although these lesions
were rather scarce.
|
, A and
B). In contrast, as shown in Fig. 6, G and
H, infection by the noninvasive strain BS176 did not elicit
detectable recruitment of CD14-positive cells. Minor staining was
associated with vessel walls in the lamina propria, but the intact
epithelial lining was devoid of any inflammatory infiltrate.
Interestingly, as shown in Fig. 6, C and D, in
intestinal tissues infected by SC577 (msbB1
msbB2), CD14-positive cells were recruited in the lamina
propria and immediate subepithelial areas of infected villi. However,
two salient features characterized the lesions: 1) the almost total
lack of recruited PMNs and 2) the lack of epithelial rupture. This
attenuated phenotype was not observed with strain SC578
(msbB1 msbB2 pMsbB2) and, although the epithelial
and mucosal destruction did not reach the level observed with M90T, the
two characteristic features were reacquired (Fig. 6, E and
F): 1) epithelial rupture and, in some villi, large areas of
epithelial necrosis, and 2) presence of PMNs in the inflammatory
infiltrate (arrowheads) associated with epithelial rupture or
destruction.
|
, A and B, a
massive amount of bacteria and bacterial LPS was associated with the
lamina propria and the epithelium, particularly in areas of
rupture/destruction of the epithelial lining following infection with
M90T. Under similar conditions, BS176 showed no invasion at all (data
not shown). In intestinal loops infected either by SC574
(msbB1) (Fig. 7, C and D) or by SC576
(msbB2) (Fig. 7, E and F), an
interesting pattern of staining was observed that matched the previous
observations and provided better characterization of the impact of
lipid A toxicity on the development of the infectious process. Single
msbB1 or msbB2 mutants caused less tissue damage,
as reported above, but the numbers of invasive bacteria appeared
similar, compared with the wild-type strain M90T. Bacteria were seen in
the lamina propria but also in large amounts inside epithelial cells.
Indentation of the epithelium that was reported above (i.e., HES
staining) corresponded to the development of infectious foci. This
indicates that infected tissues, both the epithelial layer and the
lamina propria, are more tolerant to the presence of invasive
microorganisms, thus limiting the rupture/destruction of the intestinal
barrier. Moreover, following invasion by SC577 (msbB1
msbB2), an even more striking pattern appeared (Fig. 7, G and H) in which bacteria were observed both in
the lamina propria and in epithelial cells, in the absence of
significant lesion, indicating dramatic tolerance of the infected
intestinal barrier to invasive pathogens. As shown in Fig. 7, I and J, this phenotype of tolerance was reversed
when the trans-complemented strain SC578 (msbB1
msbB2 pMsbB2) was used. In conclusion, histopathological analysis
of infected rabbit intestinal tissues indicates that genetic
attenuation of lipid A endotoxicity causes dramatic attenuation of
pathogenicity that is particularly characterized by the lack of
rupture/destruction of the epithelial lining.
|
as a major effector of endotoxin-mediated rupture of the
epithelial barrier
To evaluate the importance of TNF- in the rupture and
destruction of the epithelial barrier, we compared the parameters of
fluid production (V/L), acute villi atrophy (L/W), average number of
areas of epithelial rupture/destruction per villus unit, and
histopathological lesions in control rabbits infected for 8 h with
M90T and rabbits also infected with M90T, in which TNF- was
neutralized. Fig. 8 shows data from four
control rabbits and an equivalent number of rabbits in which TNF-
was neutralized. In both groups, BS176 induced neither fluid in the
loops nor significant acute villi atrophy or epithelial alteration.
Control animals infected with M90T showed V/L values ranging between
1.14 ± 0.14 and 0.38 ± 0.09 ml/cm, L/W values ranging
between 1 and 2.3, and the average number of epithelial
rupture/destruction per individual villus ranging between 40 and 100%.
In contrast, animals in which TNF- was neutralized showed dramatic
attenuation of the symptoms of mucosal injury with V/L values ranging
between 0.058 ± 0.04 and 0.29 ± 0.13, L/W values ranging
between 6.5 and 7.2, and the average number of epithelial
rupture/destruction per individual villus ranging between 15 and
3%.
|
,
intestinal lesions were limited compared with control conditions.
Interestingly, the pattern of lesions observed was very similar to that
observed following infection with strain SC577. HES staining confirmed
that, in the absence of TNF- neutralization, villi were shortened
and enlarged, with massive cellular infiltrate of the lamina propria
and many areas of epithelial rupture or destruction (Fig. 9, A and B,
arrowheads). In addition, the submucosal tissue appeared
massively edematous with numerous mono- and polymorphonuclear cells
adhering to the vessel wall on their way to diapedesis (Fig. 9A, arrow). In contrast, following infection with M90T in
animals treated with the anti-TNF--neutralizing mAb, villi
showed the typical appearance observed with msbB mutants,
i.e., the epithelial lining was rarely disrupted, although often
indented, leading to multiple constrictions of the villi (Fig. 9, C and D, arrowheads). The lamina propria in villi
was consistently edematous and variable submucosal edema was
observed.
|
and with msbB
mutants. As seen in Fig. 10, A and B, bacterial invasion was associated with
rupture/destruction of the epithelial lining (arrowheads), whereas in
animals in which TNF- has been neutralized continuity of the
epithelial lining was preserved and, again, both the lamina propria and
infected epithelial cells appear to tolerate the bacteria without
eliciting destructive inflammation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
upon
caspase-1 activation (11, 48) and early drop in production
of IL-1R antagonist (10). Endotoxin may also signal
through pathways independent of CD14, particularly once the increased,
unchecked mass of shigellae has reached the cytoplasmic compartment of
epithelial cells (12, 13). This result emphasized the
complexity of the signaling cascades that support the innate response
and was a strong incentive to directly alter the endotoxin level of the
lipid A. This has been made possible following the identification of
the genes and enzymes that carry out lipid A synthesis (21, 22).
However, despite several contributions comparing the effect of purified
LPS or synthetic lipid A preparations of variable endotoxin level in in
vitro assays or in various animal models of septic shock or organ
damage (49, 50, 51), still few studies have reported the
effect of genetically attenuating lipid A endotoxin activity on the
pathogenicity of the mutant bacteria in animal models. A mutation in
the waaM (htrB) gene of Salmonella
typhimurium severely altered the capacity to cause systemic
dissemination and to colonize organs in mice. However, the conditional,
temperature-dependent lethality of this mutation may have accounted for
impairment (52). More recently, following discovery of the
msbB gene function (23), a S. typhimurium
waaN (msbB) mutant administered systemically to mice
showed higher LD50 and reached higher numbers in
organs, confirming that death in the murine typhoid fever model depends
greatly on the endotoxin activity of lipid A (53).
TNF-, IL-1, and inducible NO synthase are likely mediators of
lipid A-dependent lethality. Similarly, a msbB mutant of
E. coli was tested systemically in mice. In an E.
coli K-12 background the attenuating effect of the mutation was
limited. When transduced into a wild-type, encapsulated isolate, the
msbB mutation considerably increased the
LD50, showing that the msbB gene
product is an important virulence factor. However, it should be
emphasized that the msbB mutation also resulted in
filamentation at 37°C and reduction in expression of the capsule,
indicating that this mutation has a pleiotropic effect that may
contribute to virulence attenuation (54). In contrast,
another msbB mutant of E. coli showed no
alteration in membrane permeability barrier (55).
Similarly, none of our Shigella msbB mutants expressed
significant defect in growth or division and no filamentous phenotype
was observed. Furthermore, the mutation did not affect their pathogenic
behavior in in vitro assay systems, unlike mutations in the LPS core
and O side chains, which severely affect actin-dependent intracellular
motility and cell-to-cell spread (56). In vitro, LPS from
an E. coli htrB msbB mutant induces a high level
of macrophage-inflammatory protein-1, without induction of
TNF- and IL-1 (57), indicating that, in addition to
attenuating the endotoxicity, these attenuated LPS may also modulate
the innate response.
Only one study has evaluated the impact of a msbB mutation on the capacity of S. typhimurium to cause enteritis in the rabbit ligated ileal loop model (58). This study showed that introduction of the msbB mutation had no significant effect on the mutant’s capacity to cause enteritis because neither the amount of fluid secretion nor the severity of villus alterations was affected, compared with the wild-type control. Similarly, a ssrA mutation (i.e., SPI2 mutation) did not significantly affect the severity of enteritis. In contrast, an invA mutation (i.e., SPI1 mutation) caused a dramatic decrease in the severity of enteritis. This indicated that, in Salmonella, SPI1, but not SPI2, is essential to cause enteritis, and that there is a very little role for the lipid A. These results are inconsistent with the results observed with Shigella, because we have shown a clear attenuating effect of the msbB mutation on the major parameters of enteritis: fluid production, acute villus atrophy, and inflammatory rupture/destruction of the epithelium. This difference may reflect the different kinetics of infection used in the respective studies, because Salmonella infection was conducted for 18 h, whereas Shigella infection was conducted for only 8 h. It is likely that an 18-h infection course introduces a time effect that minimizes the effect of mutations that only partially affect enteritis.
Alternatively, the discrepancy may point to intrinsic differences
between Shigella and Salmonella. In the course of
Salmonella infection, flagella that can be recognized
through Toll-like receptor 5, thereby activating NF-B
(59), may be a front-line effector inducing enteritis,
whereas in the course of infection by Shigella which do not
produce flagella, lipid A may be a front line effector (13, 60). However, other options may be envisioned: 1) the recent
demonstration that Nod1 in epithelial cells (13, 16)
recognizes intracellular LPS suggests that, because it remains inside a
vacuolar compartment (unlike Shigella that quickly escape
into the cytoplasm) (34), Salmonella may
present LPS more slowly to Nod; and 2) macrophage apoptosis is also a
proinflammatory event caused by Shigella infection
(11) following the activation of caspase-1 that leads to
the release of mature IL-1. Pretreatment of macrophage with LPS
before challenge with invasive Shigella considerably
increases the release of mature IL-1 (48).
Shigella possesses two msbB genes,
msbB1 located on the chromosome and msbB2 located
on the 214-kb virulence plasmid. This is also observed in
enterohemorrhagic E. coli (EHEC), where a second
msbB gene is present on the virulence plasmid of serotype
O-157 (61, 62). No function has been attributed so far to
this extra copy of the msbB gene, which is not present on
the enteropathogenic E. coli virulence plasmid
(63). EHEC are extracellular microorganisms that express
their pathogenic potential by binding intimately to the apical brush
border of intestinal epithelial cells and secreting Shiga-like toxins
(64). Therefore, the presence of an additional
msbB gene is not related to an intracellular mode of life.
In contrast, both Shigella and EHEC, unlike enteropathogenic
E. coli, express their pathogenic potential in the colon and
cause infection at very low inoculum. Future research will tell whether
this particular ecosystem represents a selective pressure for
maintenance of an extra copy of this gene and whether its regulation
responds to particular environmental conditions similarly to
Salmonella, in which msbB expression is regulated
by phoP/Q (65). The tolerance of
important amounts of invasive shigellae harboring the two
msbB1 and msbB2 mutations (SC577) without major
inflammatory rupture/destruction of the epithelial lining suggested a
significant role for lipid A endotoxicity in the disease process and
raised the question of which factor produced in the presence of
wild-type Shigella may account primarily for intestinal
destruction. There is growing evidence in murine models
(66) as well as in new therapeutic approaches of
inflammatory bowel diseases such as Crohn’s disease, using receptor or
neutralizing mAb therapy (67), that TNF- plays a
central role in causing the destructive inflammatory lesions. In
addition, high concentrations of TNF- have been found in the stools
of children with Shigella infection (68).
Therefore, we studied whether inhibition of TNF-, in the course of
experimental Shigella infection in the rabbit, could control
the lesions. We observed that neutralization led to a decrease in the
lesions that mimicked the situation observed when infection was
conducted with the msbB1 msbB2 mutant of S.
flexneri, essentially the loss of epithelial rupture/destruction,
despite the presence of numerous foci of infection, thus indicating
that TNF- is a major mediator of lipid A-induced epithelial rupture
in the course of experimental shigellosis.
These observations have several implications. Anti-TNF therapy may be considered an option to control the severe forms of acute bacterial colitis such as shigellosis. This is a theoretical concept, as shigellosis occurs essentially in the poorest areas of the world where costly immuno-interventions are not available. In contrast, we are currently introducing msbB mutations in live attenuated vaccine candidates against Shigella, as phase I trials have shown 9% reactivity in naive western volunteers, essentially short-term fever and intestinal discomfort, that are likely to reflect residual proinflammatory capacity of these strains (69). The phenotype of virulence attenuation that has been observed in this study following introduction of mutations in the two msbB genes may respond to the need of a less reactogenic oral vaccine.
|
Acknowledgments |
|---|
bioactivity, Josette Arondel for expert technical
help, Michel Huerre for permanent interest in this work, and Colette
Jacquemin for editing this manuscript. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Philippe J. Sansonetti, Unité de Pathogénie Microbienne Moléculaire and Institut National de la Santé et de la Recherche Médicale, Unité 389, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France. E-mail address: psanson{at}pasteur.fr
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization; Km, kanamycin; MS, mass spectrometry; SD50, stimulating dose 50; MOI, multiplicity of infection; GlcN, N-acetyl-glucosamine; EHEC, enterohemorrhagic Escherichia coli; HES, hematoxylin-eosin-safranin; L:W, length-width.
Received for publication December 18, 2001. Accepted for publication March 15, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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