INFECTION AND IMMUNITY, May 1988, p. 1023-1029
Copyright © 1988, American Society for Microbiology
Hyperadhesive Mutant of Type 1-Fimbriated Escherichia coli
Associated with Formation of FimH Organelles (Fimbriosomes)
SOMAN N. ABRAHAM,l2* JON D. GOGUEN,3 AND EDWIN H. BEACHEY"2'4
Departments of Medicine1 and Microbiology and Immunology,2 University of Tennessee, and Veterans Administration
Medical Center,4 Memphis, Tennessee 38163, and Department ofMolecular Genetics and Microbiology, University of
Massachusetts, Worcester, Massachusetts 016053
Received 5 November 1987/Accepted 19 January 1988
The relationships of the genes and gene-products mediating D-mannose-specific attachment of type 1 fimbriae
of Escherichia coli to eucaryotic cells were investigated by deletion mutation analysis of recombinant plasmid
pSH2, which carries the genetic information for the synthesis and expression of functional type 1 fimbriae.
Mutant pUT2004 was derived by a deletion remote from the structural gene encoding the 17-kilodalton (kDa)
subunit protein of type 1 fimbriae. Phenotypically, the mutant demonstrated an eightfold-higher mannose-
specific hemagglutination titer than the parent strain. On electron microscopy, the mutant strain expressed the
same number of fimbriae as the parent strain. However, numerous 10-nm-diameter rounded structures
(fimbriosomes) were observed both closely associated with fimbriae and in the culture medium. Fimbriosomes
isolated from the medium agglutinated guinea pig erythrocytes in a mannose-sensitive manner. Dissociation of
the fimbriosomes yielded a single 29-kDa protein, as demonstrated by sodium dodecyl sulfate gel electropho-
resis. Antibodies raised against fimbriosomes reacted with a 29-kDa protein on immunoelectroblots of
dissociated type 1 fimbriae and also blocked the adherence of other strains of type
eucaryotic cells. These findings suggest that the enhanced adhesive properties of the mutant pUT2004 strain are
associated with overproduction of the 29-kDa FimH in the form of fimbriosomes which contain the determinant
of the D-mannose-sensitive adhesion of type 1 fimbriae.
1 fimbriated E. coli to
Type 1 fimbriae are hairlike appendages that are produced
by Escherichia coli and by other members of the family
Enterobacteriaceae. These fimbriae are characterized by
their ability to bind to D-mannose-containing receptors on
eucaryotic cells (7, 21). The role of type 1 fimbriae in
mediating adherence of bacteria to mucosal surfaces of
animals has led to the conclusion that these adhesins are
important determinants of bacterial virulence (1, 8). The
biosynthesis, assembly, and expression of type 1 fimbriae
are determined by a cluster of genes which map at 98 min on
the E. coli chromosome. At least eight genes have been
identified in the fim cluster, and the functions of some of
them are now becoming clear. The gene fimA encodes a
17-kilodalton (kDa) subunit which makes up most of the
fimbrial structure (9). fimC and fimD are thought to be
involved in fimbrial assembly, because mutants lacking these
genes fail to produce fimbriae even though they synthesize
and process the fimA gene product (12, 20). fimB and fimE
determine fimbrial phase variation; products of these genes
control the orientation of a 300-base-pair invertable phase
switch that contains the promoter for fimA (5, 10). Recent
studies have demonstrated that the intact type 1 fimbria is
composed of at least two additional but minor 14-kDa
(FimG) and 29-kDa (FimH) subunits (3). These proteins
are encoded by genes which also map in the fim cluster
Recently we undertook a study to identify the genetic
responsible for mediating the
binding properties of type 1 fimbriae ofE. coli. These studies
involved deletion mutation analysis of a recombinant plas-
mid, pSH2, which carries the genes
expression offunctional fimbriae, followed by immunochem-
necessary for the
ical analysis of the resulting mutants (18). We found that a
mutant of pSH2, pUT2002, containing a deletion remote
from the fimA gene failed to mediate D-mannose-specific
binding even though fimbriae morphologically identical to
those of the wild type were expressed (18). Immunochemical
analysis of the nonadhesive mutant fimbriae revealed no
change in the immunological and chemical properties of
FimA, but the two ancillary FimG and FimH proteins were
found to be missing (3, 18). This finding indicated that the
D-mannose-binding property of type
depend on FimA, the major subunit, but on one or both
ancillary subunits. Similar conclusions were reached inde-
pendently by Maurer and Orndorff (16, 17), who recently
provided additional evidence, from analysis of nonhemag-
glutinating mutants, that the fimH gene product (PilE) is
responsible for mannose binding (16, 17).
In this communication, we describe the physical, biologi-
cal, and immunological characteristics of a hyperadhesive
mutant of pSH2 and report evidence that its hyperadhesive
properties are due to overproduction of the ancillary fimbrial
1 fimbriae did not
MATERIALS AND METHODS
Bacterial strains and culture conditions. E. coli ORN103
[thr-J leu-6 thiA(argF-lac)U169 xyl-7 ara-13 mtl-2 gal-6 rpsL
tonA2fhuA2 minA minB recA13A(fimABCDEFGH)], which
is deleted for all of thefim genes, was used as the host strain
for plasmid pSH2 and its derivative pUT2004. This strain
was the kind gift of Paul Orndorff. For best expression of
fimbriae, ORN103(pSH2) and ORN103(pUT2004) were cul-
tured in Luria broth under static conditions for 48 h. To
maintain the plasmid in the host, chloramphenicol (30 ,ug/ml)
was used in the culture medium. Other E. coli strains
expressing type 1 fimbriae used in this study were CSH50
Vol. 56, No. 5
ABRAHAM ET AL.
[araA&(lac-pro) rps L thil, a well-characterized K-12 strain
(4-6), and C15, isolated from a patient with a urinary tract
Recombinant DNA techniques. Plasmid purification, re-
striction endonuclease digestion, mapping, isolation of re-
striction fragments, ligation, transformation, and agarose gel
electrophoresis of plasmid DNA were performed by stan-
dard methods (15).
Isolation and purification of type 1 fimbriae. Type 1 fim-
briae were isolated from all strains of E. coli by the method
of Dodd and Eisenstein (6). The purity of the fimbrial
preparations was verified by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) and by electron
Immunization of rabbits. New Zealand White rabbits were
injected subcutaneously with 100 ,ug of the fimbriosomes
(see Results) which had been emulsified in complete Freund
adjuvant. Blood was obtained before the initial injection and
at 2-week intervals thereafter. At 4 and 10 weeks the animals
were given booster injections of 100 jigof the purified
protein in phosphate-buffered saline.
Assays of antibody reactivity. Detection and titration of
antibodies were performed with an enzyme-linked immuno-
sorbent assay (ELISA) of the antiserum. The assay was
performed as described previously (2) except that purified
fimbriosomes (see Results) were absorbed to ELISA trays as
the solid-phase antigen. Because these antibodies demon-
strated weak cross-reactivity with the structural subunit
FimA, the antiserum was preabsorbed with FimA as de-
scribed previously (3).
SDS-PAGE. SDS-PAGE was performed in a 1.5-mm thick
15% acrylamide slab gel by the system of Laemmli (13).
Samples of fimbriae were dissociated before electrophoresis
by heating in acid (6).
Immunoelectroblots of fimbriae. After SDS-PAGE, the
bands of fimbrial protein were electrophoretically trans-
ferred onto nitrocellulose paper strips as previously de-
scribed (4). The nitrocellulose strips were then incubated
with a 1:200 dilution of antiserum, followed by peroxidase-
labeled goat anti-rabbit immunoglobulin G antibody and
finally by the substrate (2).
Electron microscopy. Samples ofpurified type 1 fimbriae or
type 1 fimbriated bacteria were applied to Formvar-coated
copper grids and placed in a drop of phosphotungstic acid.
The samples were viewed in a Zeiss electron microscope. To
examine the binding of antibodies to the fimbrial structure,
grids coated with type 1 fimbriae were incubated in a drop of
0.5% bovine serum albumin for 15 min and then incubated in
a drop of antiserum, diluted 1:40, for 20 min at ambient
temperature. The grids were then washed several times in
deionized water and incubated for another 20 min at ambient
temperature in a drop of gold-conjugated protein A, diluted
1:30. The grids were then washed several times and finally
stained with phosphotungstic acid and viewed under the
Adherence inhibition assays. The ability of antibodies
evoked against the fimbriosomes to block attachment of type
1 fimbriated E. coli to human buccal epithelial cells was
examined with a previously described ELISA procedure
Hemagglutination assays. Twofold serial dilutions of a
bacterial suspension were mixed with an equal volume of2%
guinea pig erythrocytes. The hemagglutination titer was
determined after 2 h of incubation at 4°C.
FIG. 1. Restriction map of thefim locus in E. coli. Thefim genes
are indicated as heavily stippled rectangles, and their location is
based on the DNA sequence determined by Klemm and Christian-
sen (11). Arrowheads P, C, and B indicate the locations of recogni-
tion sites for the restriction endonucleases PvuII, ClaI, and BamHI,
respectively. The heavy line below the bar indicates the region
deleted in pUT2004. kb, Kilobase. A through F, Products of the
genes indicated by the heavily shaded areas.
Origin of pUT2004. pUT2004 (18) was constructed by
subjecting pSH2 to partial digestion with PvuII, followed by
religation and transformation. One of the resulting trans-
formants was found to carry a plasmid with a restriction map
identical to that of pSH2 except for the loss of a single PvuII
fragment (Fig. 1). This plasmid was designated pUT2004 and
used in the following studies.
Hyperadhesive properties of E. coli ORN103(pUT2004). In
static culture, ORN103(pUT2004) grew in large aggregates
and formed a massive pellicle on the surface of the culture
medium. Although formation of a pellicle on the surface of a
liquid culture is characteristic of all type 1-fimbriated bacte-
ria, the size of the pellicle formed by the mutant was much
greater than that produced by the parent strain, ORN103
(pSH2). The formation of both bacterial clumps and surface
pellicle in culture by the mutant could be neutralized by the
incorporation of 1% ot-methyl-D-mannoside but not by a-
methyl-D-glucoside, indicating that these traits were mani-
festations of the D-mannose-binding moiety of type 1 fim-
briae. Consistent with this observation, the mutant had an
eightfold-higher hemagglutination titer than its parent.
When plasmid pUT2004 was introduced into other E. coli
K-12 strains, such as LE392 and HB101, bacterial clumping
and massive pellicle formation were again observed, indicat-
ing that the hyperadhesive property was determined by the
plasmid and not by factors associated with the bacterial host.
It would appear that the mutant ORN103(pUT2004) Was
exhibiting the same D-mannose-binding property as type
1-fimbriated ORN103(pSH2) E. coli but at a much higher
level than the parent strain.
Electron microscopy of ORN103(pUT2004). Electron mi-
croscopic examination of ORN103(pUT2004) revealed a
normal number, length, and diameter of type 1 fimbriae,
although many of them were slightly kinked or broken (Fig.
2A and B) compared with the wild-type pSH2-encoded
fimbriae (Fig. 2C). However, numerous spherical structures
of various diameters appeared closely associated with the
fimbriae and in the surrounding medium (Fig. 2). For con-
venience, we designated these spherical structures fimbrio-
somes. These observations suggested that the hyperadhesive
phenotype of the mutant was related to the presence of
Isolation and purification offimbriosomes. After removal of
the bacteria by sedimentation, the culture supernatant was
collected and the free fimbriosomes were sedimented by
centrifugation at 110,000 x g for 20 h. We found that the
fimbriosomes could be further purified by absorption to
D-MANNOSE-BINDING MOIETY OF TYPE 1 FIMBRIAE
FIG. 2. (A and B) Electron micrographs of negatively stained hyperadhesive mutant ORN103(pUT2004) expressing rounded fimbrio-
somes. On higher magnification (B), note the close association of fimbriosomes to the kinked and broken fimbrial filaments. (C) Electron
micrograph of ORN103 containing the parent plasmid pSH2, showing normal fimbria and absence of fimbriosomes. Magnifications: A,
x30,000; B and C, x100,000.
VOL. 56i 1988
ABRAHAM ET AL.
FIG. 3. Electron micrograph of purified fimbriosomes. Magnifi-
yeast cells that contained surface mannan rich in D-man-
nose. The crude suspension of fimbriosomes was absorbed
with three batches of yeast cells, followed by elution of the
mannoside. After each elution step and before absorption
with the next batch of yeast cells, the preparation of fimbrio-
somes was dialyzed against phosphate-buffered saline to
remove all traces of a-methyl-D-mannoside. The material
obtained after three cycles of absorption consisted of a
homogeneous preparation of fimbriosomes, as determined
by electron microscopy (Fig. 3) and by SDS-PAGE. No
residual fimbriae were detected in the preparation.
Like type 1 fimbriae, fimbriosomes did not easily pene-
trate SDS gels and had to be boiled at pH 1.0 for 10 min to
dissociate into subunits. SDS-PAGE of the fimbriosome
preparation revealed a single 29-kDa band (Fig. 4), suggest-
ing that the fimbriosomes were composed entirely of FimH.
Immunological cross-reactivity between fimbriosomes and
the FimH subunit of type 1 fimbriae. To see whether the
29-kDa fimbriosome protein was similar to FimH, we re-
acted antibodies raised against fimbriosomes with dissoci-
ated fimbriae from E. coli ORN103(pSH2). We found that
the antibody reacted with a doublet band of protein in the
region of29 kDa in a Western blot (immunoblot) (Fig. 5, lane
A); whether the lower-molecular-weight band represents a
truncated form of the 29-kDa protein was not determined.
Similar reactions were seen when type 1 fimbrial prepara-
tions from a K-12 (CSH50) and a clinical strain (CI5) of E.
coli were exposed to antibodies against fimbriosomes (Fig. 5,
lanes B and C). Moreover, antibodies raised against syn-
thetic peptides corresponding to the amino-terminal region
FIG. 4. SDS-PAGE of dissociated fimbriosomes, showing a sin-
gle Coomassie blue-staining band at 29 kDa.
of FimH (3) reacted with a 29-kDa protein band of dissoci-
ated fimbriosomes on a Western blot (data not shown).
These findings suggest that these fimbriosomes have molec-
ular masses and antigenic epitopes similar to those of the
FimH protein from different type 1-fimbriated strains of E.
Antiadhesive properties of antibodies evoked against fim-
briosomes. We had previously demonstrated that antibodies
directed at a synthetic peptide of the FimH protein inhibited
binding of type 1-fimbriated E. coli to human epithelial cells
(3). Antibodies raised against the fimbriosomes were simi-
larly antiadhesive (Table 1). The antifimbriosome antiserum
inhibited E. coli ORN103(pSH2) binding by 87%. Further-
more, the antibodies also inhibited the binding of two
additional strains, CSH50 and CI5, of type 1-fimbriated E.
coli by 89 and 85%, respectively. The antiserum had no
FIG. 5. Westemn blots of dissociated type 1 fimbriae. Lane A, E.
coli ORN103(pSH2); lane B, CSH50; lane C, C15. Each lane was
probed with antiserum raised against fimbriosomes. The antiserum
reacted strongly with a 29-kDa and weakly with a 28-kDa band in
each of the fimbrial preparations.
D-MANNOSE-BINDING MOIETY OF TYPE 1 FIMBRIAE
TABLE 1. Inhibition by antibodies to fimbriosomes' of adhesion
of type 1-fimbriated E. coli to human buccal epithelial cells
E. coli strain
C16 (Pap-fimbriated control)..................................
aSubagglutinating concentrations of antibodies were added to the test E.
coli strains (all were type 1 fimbriated except the control strain) and incubated
in the microtiter well coated with human buccal epithelial cells. Adherence
inhibition was measured as described in Materials and Methods.
effect on the binding of a Pap-fimbriated strain of E. coli C16
(Table 1). Thus, antibodies to fimbriosomes were potent
inhibitors of the adherence of type 1-fimbriated E. coli, and
our findings are consistent with the notion that fimbriosomes
are composed, at least in part, of FimH and that FimH
mediates the D-mannose-binding property of type 1 fimbriae
Determination ofthe stochiometric ratio ofFimH to FimA in
fimbriae. Fimbriae from the mutant
ORN103(pUT2004) appeared to be crooked and often bro-
ken, indicating an abnormality in the structure of these
fimbriae. To determine whether this abnormality was caused
by a difference in the relative abundance of fimbrial proteins
and in particular by an increase in the amount of FimH, we
estimated the relative amounts of fimbrial proteins in SDS
FIG. 6. Densitometric
ORN103(pUT2004) (A) and ORN103(pSH2) (B) after SDS-PAGE.
*. ;l r .
s!,oAt. ;';.w :s:e
*. dF .,
FIG. 7. Immunoelectron micrographs of fimbrial filaments of
ORN103(pUT2004) (A) and ORN103(pSH2) (B) reacted with anti-
bodies directed against fimbriosomes, followed by gold-conjugated
protein A. Notice the crooked ORN103(pUT2004) fimbria and the
large number of gold particles associated with it. Magnification,
gels by densitometry of approximately similar amounts of
dissociated fimbriae. The ratio of FimH subunits to FimA
subunits in the wild-type pSH2 fimbriae was 1:60 (Fig. 6). In
marked contrast, the ratio was as high as 1:15 in the
pUT2004 mutant fimbriae (Fig. 6). Thus, the mutant fimbriae
contained at least a fourfold increase in FimH.
Localization of FimH on E. coli ORN103(pUT2004) fim-
briae. The binding of antibodies evoked by fimbriosomes to
ORN103(pUT2004) and ORN103(pSH2) fimbriae was exam-
ined by immunoelectron microscopy. We found that a higher
number of antibody-binding sites were associated with
pUT2004-encoded fimbriae, asjudged by the relative amount
of protein A-conjugated gold particles that were associated
with each fimbrial filament (Fig.
appeared to bind along the fimbrial filament at intervals,
suggesting that FimH is interspersed periodic
17-kDa major subunits. Interestingly, the rati., between the
average interval of the mutant pUT2004 fimbriae and that of
the wild-type pSH2 fimbriae was approximately 6:1, in
approximate agreement with the ratio found by gel scan
analysis (see above).
7). The gold particles
lly among the
VOL. 56, 1988
ABRAHAM ET AL.
Recent studies have provided evidence that the fimbrial
appendages ofE. coli possess minor components within their
quaternary structures that mediate adhesion to sugar-con-
taining receptors on eucaryotic cells. Whereas evidence las
been presented that these minor proteins reside at the tips of
Pap fimbriae (14), we have recently shown that the minor
protein FimH resides not only at the tips but also at long
intervals along the length of type 1 fimbriae (3). Antibodies
against both these minor proteins have proved to be antiad-
hesive (3, 14).
Although considerable knowledge has been gained on the
regulation of the major fimbrial subunits, the regulation of
the minor fimbrial proteins remains unclear. Our data sug-
gest that a negative regulatory element resides beyond the
known fimbrial gene cluster within
fragment adjacent to FimH; Deletion of this fragment mark-
edly increased the production of FimH out of proportion to
the production of FimA, the major structural subunit of type
1 fimbriae. However, we cannot discount the possibility
that, in addition to the deletion, pUT2004 has a mutation at
another site, for example, in the ribosomal binding site for
fimH, which could account for this phenomenon.
The overproduction of FimH is associated with the pro-
duction of peculiar structures that we have named fimbrio-
somes because of their association with fimbriae and their
rounded shape. From our data thus far, fimbriosomes appear
to be composed entirely of the 29-kDa FimH protein. The
structures appear to be associated rather loosely with the
fimbriae because of the ease by which they can be separated
from the fimbriae. According to Klemm and Christensen,
fimH appears to require either of two ancillary genes, fimF
or fimG, to mediate adhesion (11). Although we did not
detect thefimF or fimG gene products in our fimbriosome
preparation, it is possible that they were also secreted in the
culture medium by the mutant but failed to adsorb to yeast
cells in the isolation process. Interestingly, no increase in
either of these gene products was detected in the compara-
tive gel scans of dissociated mutant and wild-type fimbriae.
evoked anti-FimH antibodies in high titer. It should be noted
that the immune sera also contained antibodies that reacted
with FimA, but such cross-reactions can be explained on
the basis that FimA and FimH contain certain sequence
homologies (11). Cross-absorption of the antifimbriosome
antibodies with mutant nonadhesive fimbriae lacking FimH
rendered the immune sera FimH specific. That the fimbri-
osomes were c!ntaminated with minute amounts of FimA or
other fimbrial components such as FimG cannot be com-
pletely excluded. However, only the 29-kDa FimH protein
could be detected in dissociated fimbriosomes separated on
SDS gels and stained with silver nitrate or in Western blots
reacted with polyclonal antifimbriosome antibodies.
rhe finding that antibodies raised against the fimbriosomes
were antiadhesive for other type 1-fimbriated strains of E.
coli is in agreement with our previous findings with anti-
FimH antibodies (3) and has important implications for the
development of antiadhesive vaccines. In contrast to FimA,
FimH appears to be antigenically conserved, and therefore
vaccines composed of FimH may be broadly protective
against the colonization of mucosal surfaces by a variety of
type 1-fimbriated strains of E. coli. Fimbriosomes composed
entirely of FimH may prove useful in the formulation of such
a 1.5-kilobase PvuII
We thank Loretta Hatmaker for excellent electron microscopy
and Pearly Howard for excellent secretarial assistance.
The study was supported by research funds from the U.S.
Veterans Administration and Public Health Service grants AI-23821,
AI-13550, and AI-10085 from the National Institutes of Health.
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