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Infect Immun, June 1998, p. 2803-2808, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Multiple Fimbrial Adhesins Are Required for Full
Virulence of Salmonella typhimurium in Mice
Adrianus W. M.
van der
Velden,1
Andreas J.
Bäumler,1,2
Renée M.
Tsolis,1,3 and
Fred
Heffron1,*
Department of Molecular Microbiology and
Immunology, Oregon Health Sciences University, Portland, Oregon
97201,1
Department of Medical
Microbiology and Immunology, Texas A&M University Health Science
Center, College Station, Texas 77843-1114,2
and
Department of Veterinary Pathobiology, Texas A&M
University, College Station, Texas 77843-44673
Received 1 December 1997/Returned for modification 14 January
1998/Accepted 31 March 1998
 |
ABSTRACT |
Adhesion is an important initial step during bacterial colonization
of the intestinal mucosa. However, mutations in the Salmonella typhimurium fimbrial operons lpf, pef, or
fim only moderately alter mouse virulence. The respective
adhesins may thus play only a minor role during infection or S. typhimurium may encode alternative virulence factors
that can functionally compensate for their loss. To address this
question, we constructed mutations in all four known fimbrial
operons of S. typhimurium: fim,
lpf, pef, and agf. A mutation in
the agfB gene resulted in a threefold increase in the oral
50% lethal dose (LD50) of S. typhimurium for
mice. In contrast, an S. typhimurium strain carrying
mutations in all four fimbrial operons (quadruple mutant) had a 26-fold
increased oral LD50. The quadruple mutant, but not the
agfB mutant, was recovered in reduced numbers from
murine fecal pellets, suggesting that a reduced ability to colonize the
intestinal lumen contributed to its attenuation. These data are
evidence for a synergistic action of fimbrial operons during
colonization of the mouse intestine and the development of murine
typhoid fever.
 |
INTRODUCTION |
Salmonella enterica
serotype Typhimurium (S. typhimurium) causes murine typhoid
fever. This systemic infection is initiated by colonization and
penetration of the intestinal mucosa, which is commonly
accepted as a necessary first step in the establishment of infection.
Indeed, recent evidence suggests that fimbrial adhesins of
S. typhimurium play a role during bacterial attachment
to and invasion of the intestinal mucosa in vitro and in vivo (3, 5, 6, 16). For instance, attachment mediated by fimbrial adhesins
appears to be important for invasion of cultured epithelial cell lines
in vitro (4, 10, 11). In addition, a mutation in
pefC, encoding the putative outer membrane usher of
plasmid-encoded (PE) fimbriae, reduces the ability of S. typhimurium to attach to the murine villous small intestine
(3). Furthermore, insertional inactivation of
lpfC, encoding the putative outer membrane usher of long
polar (LP) fimbriae, impairs colonization of murine Peyer's patches by
S. typhimurium (5, 6). However, since
mutations in fimbrial biosynthesis genes cause only a subtle decrease
(3, 5) or even a slight increase (16) in mouse
virulence, it is not evident from these data that adhesion
mediated by fimbriae is essential during the development of murine
typhoid.
Since blockage of individual adhesins does not strongly reduce mouse
virulence of S. typhimurium, it has been speculated
that attachment is not essential during murine typhoid (14).
However, more recent evidence suggests an alternative interpretation of these data, namely that S. typhimurium encodes
alternate pathways for intestinal penetration (6, 16). The
presence of additional entry mechanisms may mask the effect of
mutations in individual virulence genes of a single pathway. For
example, a synergy of virulence factors involved in penetrating the
intestinal mucosa is suggested by the fact that an S. typhimurium lpfC invA double mutant has a 150-fold increased oral
50% lethal dose (LD50). In contrast, isogenic strains
carrying a single insertion in either lpfC or
invA are only 5- or 15-fold attenuated in mouse virulence, respectively (6). In addition, a similar synergistic effect has been observed for motility and type I fimbriation. Loss of motility
has no effect on mouse virulence, and deletion of the fim
operon, encoding type I fimbriae, results in a modest decrease in
LD50. However, an S. typhimurium mutant
that is both nonmotile and lacks type I fimbriae is 150-fold attenuated
(16).
The presence of at least four distinct fimbrial operons in
S. typhimurium, fim (8),
lpf (2), pef (12), and
agf (9), raises the possibility that
S. typhimurium compensates for a functional defect of
any individual fimbrial adhesin by producing alternate attachment
elements. Redundancy in virulence determinants involved in intestinal
colonization may explain why mutations that affect the expression of
only one fimbrial structure have little to no effect on the ability of
S. typhimurium to cause a lethal systemic infection in
mice. Thus, a simultaneous loss of several fimbrial adhesins would be
expected to reduce S. typhimurium virulence to a
greater degree than mutations in individual fimbrial operons. To
investigate whether inactivation of the genes essential to assembling
distinct fimbrial adhesins has a synergistic effect on the ability of
S. typhimurium to cause murine typhoid, we determined the virulence properties of strains carrying mutations in one or more
fimbrial operons.
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MATERIALS AND METHODS |
Bacterial strains, bacteriophages, and recombinant DNA
techniques.
Bacteria were grown overnight in Luria-Bertani broth
at 37°C. Antibiotics, when required, were incorporated into the
medium at the following concentrations: naladixic acid, 50 mg/liter; kanamycin, 60 mg/liter; chloramphenicol, 30 mg/liter; and
carbenicillin, 100 mg/liter. Analytical-grade chemicals were purchased
from Sigma (St. Louis, Mo.) or Boehringer Mannheim (Indianapolis,
Ind.). AJB3 is a fully mouse virulent naladixic acid-resistant
derivative of S. typhimurium SR-11 (3).
SR-11 derivatives carrying a
pefC::Tetr allele (AJB9) or a deletion
of the fim operon (AJB4) have been described previously
(3, 4). Bacteriophage KB1int or
P22HTint was used to transduce a
pefC::Tetr or
lpfC::Kanr mutation from S. typhimurium AJB7 (3) or AJB1 (5),
respectively, into the desired SR-11 background. Recombinant DNA
techniques and Southern hybridizations were performed by using standard
protocols (1).
A 927-bp fragment internal to agfB was amplified from
4252 (wild-type SR-11 [16]) with primers
5'-CTGACAGATGTTGCACTGCTGTG-3' and
5'-TTCGCCCGATTATTTCCTCC-3'. This PCR product was cloned into the EcoRV site of pBluescript SK to yield plasmid pAV326.
The agfB allele was inactivated upon insertion of a
chloramphenicol resistance gene (a 1.2-kb SmaI fragment from
pCMXX [6]) into a unique NruI site
(nucleotide 466). This plasmid was digested with SacI and
KpnI, and a 2.2-kb fragment was cloned into suicide vector
pGP704 (19). The resulting plasmid (pAV328) was transformed into Escherichia coli S17
pir (15)
and conjugated into S. typhimurium AJB3 (wild type) and
AJB12 (
fim lpfC pefC). A double cross-over was obtained
by homologous recombination. Chloramphenicol-resistant, carbenicillin-sensitive (loss of vector pGP704) exconjugants were screened for and named AWM394 (agfB) and AWM401
(
fim lpfC pefC agfB).
DNA probes specific for
fim,
lpf,
pef,
and
agf were used as probes for Southern hybridization. In
brief, a
SphI fragment of
pISF101 (
7) and a
SacI-
KpnI fragment of pMS1054 (
2)
served
as probes to detect
fim- and
lpf-specific
loci, respectively.
A 520-bp fragment internal to
pefA was
amplified by PCR with primers
5'-GGGAATTCTTGCTTCCATTATTGCACTGGG-3'
and 5'-TCTGTCGACGGGGGATTATTTGTAAGCCACT-3'
and cloned
into the
EcoRV site of pBluescript (
21) to give
rise
to plasmid pAV323. The
EcoRI- and
ClaI-restricted insert of pAV323
was labeled and used as a
pef-specific probe. A
SacI-
KpnI
fragment
of pAV326 was used to generate an
agf-specific
probe. Restriction
enzyme-digested chromosomal DNA was separated on an
agarose gel
and transferred onto a positively charged membrane
(Boehringer
Mannheim). The predicted sizes of hybridizing fragments
were as
follows. A
fim-specific probe detected a 13.7-kb
fragment in
SphI-restricted
chromosomal DNA of
fim+ strains (AJB3, AJB5, AJB9, AJB11, AWM394,
and AWM400) and 10.5-
and 3.1-kb fragments in
SphI-restricted chromosomal DNA of
fim mutants
(AJB4, AJB6, AJB12, and AWM401). An
lpf-specific probe
detected a 3.7-kb fragment in
PstI-restricted chromosomal
DNA
of
lpfC+ strains (AJB3, AJB4, AJB9, and
AWM394) and 2.8- and 1.7-kb fragments
in
PstI-restricted
chromosomal DNA of
lpfC mutants (AJB5, AJB6,
AJB11, AJB12,
AWM400, and AWM401). A
pef-specific probe detected
a 3.6-kb
fragment in
EcoRI- and
HindIII-restricted
chromosomal
DNA of
pefC+ strains (AJB3, AJB4,
AJB5, AJB6, and AWM394) and a 2.8-kb fragment
in
EcoRI- and
HindIII-restricted chromosomal DNA of
pefC
mutants
(AJB9, AJB11, AJB12, AWM400, and AWM401). An
agf-specific probe
detected a 1.8-kb fragment in
EcoRI- and
SalI-restricted chromosomal
DNA of
agfB+ strains (AJB3, AJB4, AJB5, AJB6, AJB9,
AJB11, and AJB12) and
a 3.0-kb fragment in
EcoRI- and
SalI-restricted chromosomal DNA
of
agfB mutants
(AWM394, AWM400, and AWM401). Detection was performed
by using the
Renaissance random primer fluorescein dUTP labeling
and detection
system from DuPont NEN (Boston, Mass.).
Mouse experiments.
Six- to eight-week-old female BALB/c mice
(Jackson Laboratories, Bar Harbor, Maine) were used throughout this
study. To determine the (two-step) LD50, a series of
10-fold dilutions of overnight cultures in a 0.2-ml volume were
injected intragastrically into groups of four mice. The
LD50s were calculated 28 days postinfection by the method
of Reed and Muench (20). For course of infection studies,
approximately 108 bacteria were administered to groups of
four mice by intragastric injection. Five days postinfection, the
animals were sacrificed, after which internal organs (Peyer's patches,
villous intestinal tissues, mesenteric lymph nodes, spleens, and
livers) were collected and homogenized in 5 ml of phosphate-buffered
saline (PBS) by using a stomacher (Tekmar, Cincinnati, Ohio). To test
the ability of a particular strain to colonize the intestinal lumen,
fecal pellets were collected at days 1, 3, and 5 postinfection and
homogenized in 5 ml of PBS. A 10-fold dilution series was plated on
Luria-Bertani agar plates containing the appropriate antibiotics to
determine the number of CFU. Results are reported in CFU per organ or
per gram of feces (single strain infections) or as percentages of the
total number of bacteria recovered (mixed infections). A paired t test was used to calculate statistical differences between
arithmetic means.
Electron microscopy.
Bacterial strains were grown as 3-ml
static broth cultures to promote expression of fimbrial
structures. Subsequently, 15 µl of bacterial suspension was
pipetted onto a Formvar-coated grid (Ted Pella Inc., Redding,
Calif.). Bacteria were allowed to adhere for 2 min and then
were fixed for 1 min with 1.5% glutaraldehyde in sodium cacodylate
buffer (100 mM, pH 7.4). The grids were rinsed twice with water
and negatively stained with 0.75% (wt/vol) uranyl acetate (pH 6.4) for
1 min. The grids were drained and subjected to microscopic studies.
 |
RESULTS |
Construction of S. typhimurium fimbrial
mutants.
Mutations in three fimbrial operons, fim,
lpf, and pef, have been reported previously
(3, 5, 16) and were used to construct a set of isogenic
S. typhimurium mutants that carried deletions of and/or
insertions in essential fimbrial biosynthesis genes (Table
1). The
lpfC::Kanr allele of S. typhimurium ATCC 14028 derivative AJB1 (5) was transduced into SR-11 derivatives AJB3 (wild type) and AJB4
(
fim) (3), yielding strains AJB5
(lpfC) and AJB6 (
fim lpfC), respectively. The
pefC::Tetr allele of strain AJB7
(3) was transduced into AJB3 and AJB6 to give rise to
strains AJB9 (pefC) and AJB12 (
fim lpfC pefC), respectively (Table 1). The lpfC::Kanr
allele of AJB1 was then transduced into AJB9 to give rise to AJB11
(lpfC pefC) (Table 1). All mutants were confirmed by
Southern blot analysis with the appropriate DNA probes (Fig.
1).

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FIG. 1.
Southern blot analyses of chromosomal DNA digested with
SphI using a fim-specific probe (A), of
chromosomal DNA digested with PstI using an
lpf-specific probe (B), of chromosomal DNA digested with
EcoRI and HindIII using a
pef-specific probe (C), and of chromosomal DNA digested with
EcoRI and SalI using an agf-specific
probe (D). For further details, see Materials and Methods. Molecular
sizes in kilobases (kb) are shown at right.
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Thin aggregative fimbriae, which are encoded by the
S. typhimurium agf operon, are assembled by an export machinery that
is
distinct from the chaperone- and usher-dependent transport systems
of type 1 fimbriae, PE fimbriae, or LP fimbriae. Curli, encoded
by the
csg operon in
E. coli, is the prototypic member
of this
novel pilus assembly class. Recent evidence by Hammar et al.
suggests
that CsgB, a membrane-associated nucleator protein, is
required
for the assembly of curli fimbriae on the bacterial cell
surface
(
13). It was therefore decided to inactivate
agfB, the
csgB homolog in
S. typhimurium (
9). An
agfB allele (carried on
plasmid
pAV328) was inactivated by insertion of a 1.2-kb
chloramphenicol
resistance cassette and introduced into strains AJB3
(wild type),
AJB11 (
lpC pefC), and AJB12 (
fim lpfC
pefC). Double cross-over
events were obtained by homologous
recombination, and the resulting
strains were designated AWM394
(
agfB), AWM400 (
lpfC pefC agfB),
and AWM401
(
fim lpfC pefC agfB), respectively (Table
1). All
three
mutants were confirmed by Southern blot analysis with an
agfB-specific DNA probe (Fig.
1).
Synergistic effect of mutations in fimbrial operons on
mouse virulence.
LD50 studies were conducted to
investigate the effect of mutations in fimbrial operons on
mouse virulence (Table 2)
(20). Strains carrying mutations in a single fimbrial
operon were either more virulent (AJB4,
fim) or
less than fivefold attenuated (AJB5, lpfC; AJB9,
pefC; and AWM394, agfB) in comparison with the
wild type (AJB3). Strain AJB12 (
fim lpfC pefC) also
exhibited slightly increased virulence, suggesting that the
phenotype of a fim deletion mutant is dominant over the
attenuating effect of mutations in lpf and pef.
Interestingly, AWM400 (lpfC pefC agfB) is more strongly attenuated (>29-fold) than AWM401 (
fim lpfC pefC agfB)
(see below and Table 2). We and others have observed that all
fim mutants tested had a slight increase in virulence
compared to that of the wild type (16) (Table 2 and
our unpublished results). These data suggest a dominant phenotype for
mutant fim alleles.
AWM401 (
fim lpfC pefC agfB), a quadruple fimbrial mutant,
was more strongly attenuated (26-fold) than AJB12 (
fim lpfC
pefC)
or any of the strains carrying a single fimbrial mutation.
This
result suggested an additive attenuating effect of these mutations
on the ability of
S. typhimurium to cause murine
typhoid (Table
2). Furthermore, the increased virulence of strain AJB12
(
fim lpfC pefC) compared to that of AWM401 (
fim
lpfC pefC agfB) supports
the idea that the insertional
inactivation of
agfB is one of the
mutations responsible for
the strong attenuation of the quadruple
mutant.
The quadruple mutant has a reduced ability to colonize liver,
spleen, and intestine.
To investigate at which step during the
infection process AWM401 (
fim lpfC pefC agfB) is
impaired, course of infection studies were conducted. Since our mouse
virulence data (Table 2) suggested that a mutation in agfB
in combination with a mutation in at least one other fimbrial
operon is responsible for the attenuation of AWM401
(
fim lpfC pefC agfB), strain AWM394 (agfB) was
included in these studies. Groups of four mice were orally infected
with 108 CFU, and bacteria were recovered from Peyer's
patches, mesenteric lymph nodes, and spleens on days 3 (data not shown)
and 5 postinfection (Fig. 2A). In
addition, bacteria were recovered from the feces on days 1, 3, and 5 postinfection to monitor intestinal colonization (Fig. 2B). Compared to
the wild type (AJB3), reduced numbers of both AWM401 (
fim lpfC
pefC agfB) and AWM394 (agfB) were recovered from
internal organs and fecal pellets. However, these differences proved
not to be statistically significant (P > 0.05). As
bacterial numbers recovered from individual animals may vary greatly
during infection, small differences between wild type and mutant may go
undetected. In order to control for the variability between experimental animals, mixed infections with AJB3 (wild type), AWM394
(agfB), and AWM401 (
fim lpfC pefC agfB) were
performed, permitting a direct comparison between wild type and
mutants. A group of four mice was orally infected with 108
CFU of a mixture containing approximately equal amounts of AJB3 (wild
type), AWM394 (agfB), and AWM401 (
fim lpfC pefC
agfB). On day 5 postinfection, CFU in internal organs (Peyer's
patches, villous intestinal tissues, mesenteric lymphs, spleens, and
livers) were determined. In addition, bacteria were recovered from the feces up to 5 days postinfection to monitor intestinal colonization (Fig. 3). Both AWM394 (agfB)
and AWM401 (
fim lpfC pefC agfB) were able to compete with
the wild type (AJB3) for colonization of Peyer's patches and villous
intestinal tissues in the terminal ileum. Interestingly, increased
numbers of both the quadruple mutant (AWM401) and the agfB
mutant (AWM394) were recovered from the mesenteric lymph nodes compared
to that of the wild type (AJB3). These differences were not
statistically significant (P > 0.05). However,
both AWM394 (agfB) and AWM401 (
fim lpfC pefC
agfB) were outcompeted by the wild type (AJB3) for colonization of
the liver and spleen (P < 0.05 and P < 0.01, respectively). In addition, AWM401 (
fim lpfC pefC
agfB) failed to compete with the wild type for colonization of the
intestine, as suggested by recovery of significantly reduced numbers of
AWM401 from fecal pellets (P < 0.05). These results
provide evidence that fimbrial adhesins act synergistically
during colonization of the mouse intestinal tract.

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FIG. 2.
Bacterial recovery from internal organs 5 days
postinfection (p.i.) (A) and feces 1, 3, and 5 days postinfection (B)
reported in CFU per organ (three Peyer's patches in the terminal
ileum, close to the cecum, were collected and pooled for each mouse) or
CFU per gram of feces. Three groups of four mice each were orally
infected with 108 CFU of AJB3 (wild type [wt]), AWM394
(agfB), or AWM401 ( fim lpfC pefC agfB). Data
are arithmetic means. Error bars indicate standard deviations.
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FIG. 3.
Bacterial recovery from internal organs and feces 5 days
postinfection (p.i.) reported as percentages of the total number of
bacteria recovered. Three groups of four mice each were orally infected
with a 1:1:1 mixture of three strains, AJB3 (wild type [wt]), AWM394
(agfB), and AWM401 ( fim lpfC pefC agfB),
respectively, for a total of 108 CFU per mouse. Three
Peyer's patches in the terminal ileum, close to the cecum, were
collected and pooled for each mouse. Data are arithmetic means. Error
bars indicate standard deviations. *, P < 0.05 (paired t test); **, P < 0.01 (paired
t test).
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Identification of new fimbrial structures.
Although we have
demonstrated that fimbrial adhesins of S. typhimurium
play an important role during infection (Table 2), AWM401 (
fim
lpfC pefC agfB) was still able to cause a lethal systemic illness
in mice when administered at higher doses. These data suggest that
AWM401 (
fim lpfC pefC agfB) may express yet other factors
for intestinal attachment. To investigate this possibility, we examined
strain AWM401 (
fim lpfC pefC agfB) by electron microscopy for the presence of fimbriae. Interestingly, this mutant (AWM401) expressed thus far uncharacterized fimbrial structures (Fig.
4A), which could easily be distinguished
from flagellar filaments required for cell motility (Fig. 4). Flagellar
filaments varied in length from 5 to 10 µm, with a diameter of
approximately 20 nm (18). Fimbriae could be distinguished
from flagella by means of morphology and diameter (typically between 2 and 8 nm [17]). These data provide direct evidence for
the expression of at least one yet uncharacterized fimbrial structure
in S. typhimurium which may contribute to the
redundancy of virulence factors involved in colonization of the
intestinal mucosa.

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FIG. 4.
Electron micrographs of AWM401, which harbors mutations
in the fim, lpf, pef, and
agf fimbrial operons. This quadruple mutant
expresses a thus far uncharacterized fimbrial structure (A, arrows)
that can be distinguished from flagellar filaments (A and B,
arrowheads). Magnification, ×35,000 (A) and ×8,000 (B).
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|
 |
DISCUSSION |
Our results demonstrate that despite the moderate effect on mouse
virulence of individual mutations in fimbrial operons, the simultaneous inactivation of genes involved in the biosynthesis of four
distinct fimbrial adhesins markedly attenuates S. typhimurium. To our knowledge, this is the first study to
provide direct evidence for a synergistic effect of fimbrial
adhesins during infection. Previous studies have shown that
inactivation of biosynthetic genes for type 1 fimbriae, LP fimbriae, or
PE fimbriae attenuate S. typhimurium mouse virulence
only fivefold or less (3, 5, 16). Here, we report that a
mutation in a fourth S. typhimurium fimbrial
operon, agf, resulted in a threefold reduction in
mouse virulence. A recent study indicated that thin aggregative
fimbriae mediate adhesion to murine small intestinal epithelial cells
(23). We have observed that strains carrying the
agfB mutation have an altered colony morphology (data not
shown). A pleiotropic effect for agf mutants regarding
colony morphology has also been reported by others (23).
However, our virulence data strongly suggests that this pleiotropic
effect does not reduce the ability to cause murine typhoid (Table 2 and
Fig. 2, AWM394 [agfB]). Furthermore, from these data it is
evident that inactivation of individual adhesins does not strongly
reduce the ability of S. typhimurium to cause a lethal
systemic infection in mice. However, strain AWM401, in which all four
known fimbrial operons are inactivated, was 26-fold attenuated
when orally administered to mice. These results are consistent with the
idea that mutations in individual S. typhimurium
fimbrial operons have only moderate effects on mouse virulence
because the lack of a single attachment factor can be compensated for
by the presence of other adhesins.
Because a strain carrying mutations in fim,
lpf, and pef (AJB12) was not attenuated,
insertional inactivation of agfB must be partly
responsible for the strong attenuation of AWM401 (
fim lpfC
pefC agfB). Neither the agfB mutant (AWM394) nor
the quadruple mutant (AWM401) were able to compete with the wild type
(AJB3) for colonization of the liver and spleen (P < 0.05 and P < 0.01, respectively). However, during
mixed infection experiments, only AWM401 (
fim lpfC pefC
agfB) was recovered in reduced numbers from fecal pellets
(P < 0.05), suggesting that the decreased
virulence of AWM401, compared to that of AWM394
(agfB), is caused by a defect in intestinal colonization.
From these results, we conclude that the absence of at least two
fimbrial structures may significantly decrease adherence to
murine intestinal tissue and further reduce mouse virulence.
Additional studies are needed to identify which combination of
mutations in fimbrial operons reduces virulence.
The ability of AWM401 (
fim lpfC pefC agfB) to cause a
lethal systemic infection in mice upon intragastric injection of large inocula suggested that a quadruple mutant might express additional means of adhesion and colonization. Electron microscopic studies demonstrated that, in addition to flagellar filaments, AWM401 (
fim lpfC pefC agfB) expresses at least one additional,
yet uncharacterized, fimbrial structure. Thus, this fimbrial structure,
and possibly others, may be the adhesive organelle(s) that allows
residual colonization of the mouse intestine by S. typhimurium in the absence of type 1, LP, PE, and thin aggregative
fimbriae.
 |
ACKNOWLEDGMENTS |
We are indebted to Roy Curtiss III for providing strain
4252
and Steven Clegg for providing plasmid pISF101. We acknowledge Paula
Stenberg and Robert J. Kayton for assistance with the electron microscopy studies and thank Peter J. Valentine for many helpful discussions.
This work was supported by Public Health Service grant AI22933 to F.H.
from the National Institutes of Health. A.W.M.v.d.V. is a recipient of
a Tarter Trust Fellowship.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology, Oregon Health Sciences
University, 3181 SW Sam Jackson Park Rd., L220, Portland, OR 97201. Phone: (503) 494-6841. Fax: (503) 494-6862. E-mail:
heffronf{at}ohsu.edu.
Editor: P. E. Orndorff
 |
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