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Infect Immun, June 1998, p. 2887-2894, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of the Type 3 Fimbrial Adhesins of
Klebsiella Strains
Tricia A. Schurtz
Sebghati,1
Timo K.
Korhonen,2
Douglas B.
Hornick,3 and
Steven
Clegg1,*
Departments of
Microbiology1 and
Internal
Medicine,3 University of Iowa College of
Medicine, Iowa City, Iowa 52242, and
Department of General
Microbiology, University of Helsinki, Helsinki,
Finland2
Received 5 December 1997/Returned for modification 10 February
1998/Accepted 27 March 1998
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ABSTRACT |
The Klebsiella pneumoniae fimbrial adhesin, MrkD,
mediates adherence to the basolateral surfaces of renal and pulmonary
epithelia and to the basement membranes of tissues. Although all
isolates possessing the MrkD adhesin mediate the agglutination, in
vitro, of erythrocytes treated with tannic acid, the mrkD
gene is not conserved within species. The ability of a plasmid-borne
mrkD gene product to mediate binding to type V collagen is
associated frequently with strains of K. oxytoca and rarely
with strains of K. pneumoniae. In K. pneumoniae, the MrkD adhesin is located within a chromosomally
borne gene cluster and mediates binding to collagen types IV and V. The
plasmid-borne determinant, mrkD1P, and the
chromosomally borne gene, mrkD1C, are not
genetically related. Some strains of enterobacteria possess a
mrkD1C allele that is associated with
hemagglutinating activity but does not bind to either type IV or type V
collagen.
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INTRODUCTION |
Type 3 fimbriae are produced by many
members of the Enterobacteriaceae, including
Klebsiella, Enterobacter, Proteus,
Providencia, and Serratia species (5, 7, 10,
19, 22, 23, 25). This fimbrial type is detected by agglutination,
in vitro, of erythrocytes treated with tannic acid, and
hemagglutination can occur in the presence or absence of
D-mannose (7, 21). This characteristic was
originally demonstrated in Klebsiella, and the associated
adherence phenotype is often referred to as the mannose-resistant
Klebsiella-like hemagglutination (MR/KHA) reaction (7,
24, 26). MR/KHA activity is mediated by the MrkD adhesin polypeptide of the type 3 fimbrial gene cluster (1, 5, 16), and the adhesin facilitates binding to the basement membranes of human
tissues (14, 33).
The expression of type 3 fimbriae in Klebsiella requires the
presence of at least six mrk genes which have been cloned
and sequenced (1, 5). The gene (mrkD) encoding
the fimbrial adhesin is distinct from that which encodes the major
fimbrial subunit (mrkA) (1, 5). In one strain of
Klebsiella pneumoniae, the MrkD adhesin has been shown to
mediate adherence to human basement membrane and basolateral surfaces
of renal and pulmonary epithelia (14, 16). Specifically,
this adhesin has been shown to bind to type V collagen and is an
extracellular matrix binding protein (32). However, we have
previously demonstrated that the mrkD gene is not conserved
among all fimbriate and hemagglutinating strains of
Klebsiella (28). Southern hybridization analysis indicated that in one isolate of K. pneumoniae, the
mrkD gene is present on a large native plasmid
(16). It has not been determined whether most isolates of
K. pneumoniae possess a plasmid-borne copy of the
mrk gene cluster. Also, since all mrkD genes are
not identical, the ability of different mrkD gene products
to bind to type V collagen has not been investigated. In the studies
described below, the MrkD-mediated receptor-binding specificity of type 3 fimbria-associated adhesins encoded by mrkD alleles is
reported. Differences in receptor-binding specificities can be
associated with variability in the amino acid sequences of the MrkD
adhesin.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
The sources of the
clinical and environmental isolates of Klebsiella strains
used in this study have previously been described (14, 15,
28). Table 1 lists the recombinant
plasmids used, and these plasmids were maintained in Escherichia
coli HB101 (4), JM109 (36), or DH12S
(20). Transformations were performed by electroporation with
an ECM600 pulse generator (BTX Inc., San Diego, Calif.), and
transformants were selected following growth in L-broth supplemented
with the appropriate antibiotics.
Optimal expression of type 3 fimbriae in
Klebsiella strains
was achieved by cultivation on glycerol-Casamino Acids agar as
previously described (
15,
16). All strains were incubated
at
37°C for 18 to 24 h. Large, native plasmids from
Klebsiella isolates were purified by a commercially
available technique (Bigger
Prep; 5 prime-3 prime, Inc., Boulder,
Colo.). Plasmids were restricted
with endonucleases from commercial
sources according to the manufacturers'
instructions. Restriction
fragments were analyzed by agarose gel
electrophoresis as described
elsewhere (
27).
Southern hybridization analysis.
Total genomic DNA was
prepared by standard procedures (3), and Southern
hybridization analysis with both total genomic DNA and plasmid DNA
preparations was performed as previously described (28). DNA
probes were constructed by PCR amplification with relevant plasmids as
the template (Table 1) and primers derived from sequences within
appropriate mrk genes. The construction and use of the
mrkA gene probe and one of the mrkD probes have been described elsewhere (15, 28). All DNA probes were
nonradioactively labeled according to standard procedures (Genius
System; Boehringer Mannheim, Indianapolis, Ind.), and hybridization was
carried out under high-stringency conditions as previously described
(28).
Isolation of mrkD1C genes.
The
mrkD genes from K. pneumoniae UIR079 and 43816 were isolated with a gene probe derived from K. pneumoniae
IApc35 (Table 1) (16). The probe was prepared with the
primers 5'-TTCTGCACAGCGGTCCC-3' and
5'-GATACCCGGCGTTTTCGTTAC-3' and comprises 581 bp within the region flanked by mrkC and mrkF on the chromosome
of K. pneumoniae IApc35 (Fig.
1, probe B). Following isolation of
genomic DNA from the two K. pneumoniae strains
(3), the DNA was partially digested with EcoRI,
and DNA fragments of approximately 5 to 7 kb in size were isolated
(27). Subsequently, these fragments were ligated into the
EcoRI site of pBluescript KS (Stratagene, La Jolla, Calif.), and transformants in E. coli JM109 or DH12S were isolated by
conventional techniques (27). Recombinants possessing
Klebsiella-derived DNA were identified with the
mrkD gene probe and by colony hybridization techniques
described in detail elsewhere (3, 28).
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FIG. 1.
Genetic organization of the mrk gene
cluster. The Klebsiella-derived DNA fragments of recombinant
plasmids are shown by the solid lines. The regions and sources of the
mrkD1P-specific gene probe (A) and the
mrkD1C-specific gene probe (B) are as indicated.
The function of the mrk gene products has previously been
reviewed (5).
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The nucleotide sequences of the
mrkD genes from
K. pneumoniae UIR079 and 43816 were determined with the fmol
sequencing reagents
(Promega Corporation, Madison, Wis.) according to
the manufacturer's
instructions. The plasmids pTS83 and pTS84 (Table
1
and Fig.
1) were used as templates for these reactions, and the DNA
sequences
of both strands were determined. The predicted amino acid
sequences
of the
mrkD determinants were derived, and all
amino acid sequence
comparisons were performed with the basic local
alignment search
tool (BLAST) program (
2) or with Eugene
software from UNIX
systems (Molecular Biology Information Resource,
Baylor College
of Medicine, Houston, Tex.).
Purification of type 3 fimbriae.
Fimbriae were purified from
Klebsiella isolates as previously reported (11).
Briefly, bacteria were grown under conditions to optimize fimbrial
production, harvested, resuspended in 5 mM Tris-HCl (pH 7.5), and
homogenized in a Waring blender at 4°C. After homogenization, the
bacteria were removed by centrifugation, and following a second
homogenization and centrifugation step, ammonium sulfate (10%
[wt/vol]) was added to the supernatant. After 30 min at ambient
temperature, the precipitate was collected by centrifugation and
discarded. Ammonium sulfate (30% [wt/vol]) was added to the
supernatant, and the suspension was allowed to stand at 4°C for
18 h. The precipitate was subsequently collected and dissolved in
distilled water. Cesium chloride was added (42% [wt/vol]), and the
protein solution was centrifuged in a vertical angle rotor for 6 h
at 55,000 × g. The fimbriae were collected, concentrated, and resuspended in sterile distilled water.
Detection of type 3 fimbriae and fimbria-associated
proteins.
MR/KHA activity was determined as previously described
with tanned erythrocytes (26). The presence of type 3 fimbriae on the surface of bacteria was detected with fimbria-specific
antiserum as described elsewhere (22, 26). Transmission
electron microscopy was used to confirm phenotypic expression of type 3 fimbriae by bacteria (24, 26).
The purity and size of fimbrial polypeptides were determined by sodium
dodecyl sulfate-polyacrylamide electrophoresis. Western
blotting was
performed by standard procedures with either anti-type
3 fimbrial serum
or serum raised against a synthetic oligopeptide
representing the first
10 amino acids of the mature MrkD adhesin
(
12,
28).
Binding to ECMPs.
An enzyme-linked immunosorbent assay was
developed to demonstrate specific binding mediated by type 3 fimbriae.
The wells of flat-bottom microtiter plates were coated following
incubation overnight at 4°C with optimal concentrations of
extracellular matrix proteins (ECMPs) diluted in carbonate-bicarbonate
buffer, pH 9.6 (34). Stock solutions of commercially
available purified types I, IV, V, and X collagens, fibronectin, and
laminin were prepared as recommended by the manufacturer, and the
optimal coating concentrations were determined as described elsewhere
(32). Prior to incubation with bacteria or purified
fimbriae, nonspecific binding sites were blocked by incubation for 2 h
at 22°C with a 1% (wt/vol) solution of bovine serum albumin.
Subsequently, 100 µl of serial twofold dilutions of either bacterial
suspensions (1010 bacteria/ml) or purified type 3 fimbriae
(50 µg), prepared in phosphate-buffered saline-Tween 20 (PBS-T) (pH
7.4; 0.5 ml Tween 20 in 1 liter of PBS), was added to the wells.
Following incubation for 2 h at 22°C with gentle shaking,
unattached bacteria were removed by washing three times in PBS-T. For
each well, the adherence assay was developed with a rabbit monospecific
antifimbrial serum (100 µl) diluted in PBS-T. Then, after being
washed in PBS-T, 100 µl of goat anti-rabbit immunoglobulin G
conjugated to alkaline phosphatase was added to the wells and allowed
to incubate for 1 h at 37°C. Finally, the plates were washed
thoroughly, p-nitrophenyl phosphate (5 mg/ml in
diethanolamine buffer) (Sigma Chemical Co., St. Louis, Mo.) was added,
and the reaction was allowed to proceed for 40 min at 37°C. All tests
were performed in triplicate, and color development was determined with
an enzyme-linked immunosorbent assay plate reader set to an optical
density of 405 nm.
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RESULTS |
Plasmid-borne and chromosomally borne mrkD genes.
Of 44 strains of Klebsiella examined, all 10 isolates of
K. oxytoca and 6 of 34 K. pneumoniae strains
possessed a gene related to the mrkD determinant carried on
pFK10 (Fig. 1 and Table 2). All of these
strains express type 3 fimbriae and demonstrated the characteristic
MR/KHA activity. Seven of the K. oxytoca strains were shown
to possess a plasmid-borne mrkD gene, designated
mrkD1P, since plasmid preparations from these
strains possessed sequences homologous to the gene probe (Fig. 1, probe
A). Three K. pneumoniae strains also possessed an
mrkD1P determinant on a plasmid isolated from
these strains. The remaining 28 isolates of K. pneumoniae were MR/KHA positive and produce type 3 fimbriae but do not possess sequences related to the mrkD gene carried on pFK10.
K. pneumoniae IApc35 is a derivative of
K. pneumoniae IA565 that has lost its plasmid-borne copy of the
mrk gene cluster but
retains a copy on the chromosome
(
16). Strain IApc35 was used
to prepare an
mrkD
gene probe comprising sequences from within
the chromosomally borne
mrkD (Fig.
1, probe B). Plasmid pTS
T3
carries the
mrkD gene, designated
mrkD1C, cloned
from
K. pneumoniae IApc35 (Table
1 and Fig.
1). A 581-bp DNA
fragment consisting
solely of
mrkD-derived nucleotides was
used as a gene probe to
detect homologous sequences among the
Klebsiella strains. With
this gene probe, 31 of 34
K. pneumoniae isolates could be shown
to carry homologous sequences,
whereas none of the type 3 fimbria-producing
strains of
K. oxytoca possess related nucleotide sequences (Table
2). Three of
the 34 isolates of
K. pneumoniae did not possess
nucleotides
related to the probe derived from pTS
T3, although
these three
strains do retain an
mrkD1P gene homologous to
that
carried on pFK10 (Table
2). Also, in all three of these strains,
the
mrkD1P gene was detected in plasmid DNA
preparations. Three
strains of
K. pneumoniae, including
strain IA565, from which the
mrk gene cluster was originally
cloned (
9,
12), possess sequences
related to both
mrkD gene probes.
A total of eight fimbriate isolates of
Enterobacter cloacae
were also examined for the presence of
mrkD genes because
this
genus expresses type 3 fimbriae related to those of
Klebsiella (
22). Seven isolates possess a gene
comprised of sequences related
to those derived from
mrkD1C, whereas one strain carried a gene
similar to the
mrkD1P DNA probe. In this latter
strain, the
mrkD gene was carried on DNA isolated either as
total genomic DNA or
from plasmid preparations.
Characterization of mrkD sequences and their gene
products.
K. pneumoniae IApc35 is a nonhemagglutinating
derivative of IA565 (16). Therefore, this strain and
K. pneumoniae UIR079, an MR/KHA-positive isolate, were
used as a source of DNA to determine the nucleotide sequences of
their mrkD1C genes. The mrkD gene of
K. pneumoniae 43816 was also isolated, since this gene is
closely related to that of strain IApc35 as determined by Southern
hybridization, and the bacteria are hemagglutinating but do not bind to
collagen (see below).
The plasmid pTS83 (Table
1 and Fig.
1) contains a 6.2-kb DNA fragment
derived from
K. pneumoniae UIR079 and possesses an
mrkD determinant that is 978 bp in length. The predicted
size
of the MrkD polypeptide is 34.9 kDa, which is slightly larger
than
the 33.8-kDa gene product encoded by the
mrkD1P
gene carried
on pFK10 (Fig.
1) (
5). The
mrkD gene
of
K. pneumoniae 43816
is carried on a 6.0-kb DNA fragment
of plasmid pTS84 and comprises
an open reading frame of 987 bp. The
predicted size of the MrkD
polypeptide encoded by this region is 35.4 kDa, and a comparison
of the MrkD proteins of
K. pneumoniae
UIR079 and 43816 with that
of the previously described plasmid-borne
MrkD adhesin (
5,
12) is shown in Fig.
2. At the amino acid level,
MrkD
1C of
K. pneumoniae UIR079 is 55% identical
to the MrkD
1P polypeptide,
and the two genes encoding these
molecules possess 62.5% identity
at the nucleotide level. A comparison
of the amino acid and nucleotide
sequences of the MrkD
1C
polypeptides and their genes from
K. pneumoniae UIR079 and
43816 indicates 74 and 86% identity, respectively.
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FIG. 2.
Comparison of the deduced amino acid sequences of the
MrkD polypeptides. MrkD1P is the gene product encoded by a
plasmid of K. pneumoniae IA565; MrkD1C1 is
encoded by a chromosomally borne gene of K. pneumoniae
UIR079; MrkD1C2 is the gene product of the mrkD
allele located on the chromosome of K. pneumoniae 43816. Identical amino acids are indicated by solid lines, and isofunctional
amino acids are represented by dots.
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Bacterial binding to ECMPs.
The results of the ECMP-binding
assays for strains possessing the mrkD genes are summarized
in Table 3 and Fig.
3. None of the Klebsiella
isolates adhere, in vitro, to type I or type X collagen, fibronectin,
laminin, or bovine serum albumin. All nine Klebsiella
strains tested that possess the mrkD1P gene
related to that carried on pFK10 bound only to type V collagen (Table 3). Nineteen of 28 K. pneumoniae strains that are type 3 fimbriate and exhibit MR/KHA but do not possess the
mrkD1P gene bind to type IV and type V collagen.
All of these strains possess mrkD1C sequences
related to those isolated from K. pneumoniae IApc35 and
UIR079. E. coli transformants possessing pTS83 also adhere to type IV and type V collagens, whereas transformants lacking the
mrk gene cluster do not. Nine hemagglutinating and fimbriate isolates of K. pneumoniae, including strain 43816, possess
nucleotide sequences homologous to those of strain IApc35 but did not
bind to any of the ECMPs used in the assays. Six of the 10 K. oxytoca strains that were found to possess sequences homologous to
the pFK10-derived mrkD gene probe were also examined for
their ability to bind collagens. All six exhibited binding only to type
V collagen.
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FIG. 3.
Binding of Klebsiella strains to collagen.
Binding to type V collagen is indicated by the solid bars, and type IV
collagen binding is represented by the striped bars. All bacterial
suspensions were normalized to an optical density at 600 nm
(OD600) of 0.3 corresponding to approximately 6 × 108 bacteria/ml. (A) K. pneumoniae IA565 and its
derivative, IApc35, lacking mrkD1P. (B)
Representative strains possessing mrkD1C without
mrkD1P. (C) Klebsiella isolates
possessing mrkD1P but no
mrkD1C. (D) Strains of Klebsiella
with mrkD1C and no mrkD1P
but not binding to collagen. For each column, the value is expressed as
the mean ± standard error of the mean; all tests were performed
at least three times.
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As shown in Table
3, six of the seven
Enterobacter strains
examined possess sequences homologous to IApc35-derived
mrkD1C and bound to type IV and type V collagen.
One
E. cloacae isolate
demonstrated binding only to type V
collagen, and DNA from this
strain hybridized to the pFK10-derived
mrkD1P gene probe.
Binding of purified fimbriae to ECMPs.
Cell-free fimbriae from
four representative isolates of Klebsiella were prepared.
Fimbriae from K. pneumoniae IA565 and its nonhemagglutinating derivative, IApc35, were used in the binding assay.
Also, purified type 3 fimbriae from K. pneumoniae UIR079 and
43816 were prepared. The fimbriae of K. pneumoniae UIR079 binds to type IV and type V collagen, whereas fimbriae from 43816 do
not bind to either collagen type, and both strains are strongly hemagglutinating. Also, both strains possess
mrkD1C sequences related to those carried on the
chromosome of K. pneumoniae IApc35. The pattern of collagen
binding by the purified fimbriae was identical to that found for whole
bacteria (Fig. 4). Fimbriae from strain IA565 bound only to type V collagen, whereas those isolated from K. pneumoniae IApc35 did not adhere to either collagen type.
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FIG. 4.
Binding of purified type 3 fimbriae to collagen. Binding
of type V and type IV collagen is shown by the striped and solid bars,
respectively. All assays were performed with 12.5 µg of fimbrial
protein. Standard errors of the means were calculated after each assay
was performed at least three times.
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The presence of a 34-kDa polypeptide in the fimbrial preparations,
encoded by the
mrkD adhesin carried on pFK10, was determined
with a mrkD-specific antiserum (
28). Western blot analyses
indicated
the presence of this polypeptide in fimbrial preparations
from
K. pneumoniae IA565 but not in those from other strains
(Fig.
5). All fimbrial preparations were
recognized by serum raised
against the MrkA major fimbrial subunit
protein of strain IA565.
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FIG. 5.
Immunoblots of purified fimbriae with anti-MrkD (A) and
anti-MrkA (B) sera. Fimbriae were purified from K. pneumoniae IA565 (lane 1), K. pneumoniae IApc35 (lane
2), and K. pneumoniae UIR079 (lane 3). The anti-MrkD serum
was raised against a synthetic polypeptide representing the first 10 amino acids of MrkD1P, and the anti-MrkA serum was raised
against fimbriae purified from E. coli (pFK10).
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DISCUSSION |
We have previously demonstrated that K. pneumoniae
IA565 possesses two mrk gene clusters that are located on a
plasmid and the chromosome (16). Also, K. pneumoniae IApc35, a derivative of strain IA565, has lost the
plasmid-borne mrk sequences but retained the chromosomal
copy of the mrk gene cluster (16). Using an
mrkD gene probe derived from the plasmid-borne gene cluster, we demonstrated that this determinant was not highly conserved among
isolates of K. pneumoniae, even though most of these strains express type 3 fimbriae. However, this plasmid-borne mrkD
gene was found with a high frequency among fimbriate strains of
K. oxytoca and more rarely among K. pneumoniae
isolates (28). Therefore, even though most strains of
K. pneumoniae express the type 3 fimbrial MR/KHA, they must
possess an MrkD adhesin that is not closely related to that encoded by
the plasmid of K. pneumoniae IA565. In order to determine
whether strains of K. pneumoniae possess a highly conserved
mrkD gene, this determinant was isolated and characterized.
Using an mrkD gene probe derived from K. pneumoniae IApc35, a strain lacking the plasmid-borne
mrk gene cluster (16), we demonstrated that most
isolates of K. pneumoniae possess sequences related to those
of the gene probe and that these sequences were not associated with
plasmids. The results of Southern hybridization analysis indicate that
fimbriate K. pneumoniae isolates do retain a highly
conserved mrkD determinant that is different from that associated with the plasmid-borne adhesin gene. The plasmid-borne adhesin gene is carried more frequently by strains of K. oxytoca, often on large native plasmids. Similarly, in the small
number of K. pneumoniae isolates that do possess an
mrkD gene related to that carried on the recombinant plasmid
pFK10, the gene is commonly carried on a plasmid. Therefore, the
plasmid-borne gene found in most isolates of K. oxytoca has
been designated mrkD1P, and that associated with
the chromosome of K. pneumoniae is termed mrkD1C. The presence of the
mrkD1P gene in a small number of K. pneumoniae strains may be due to horizontal transfer from K. oxytoca. We have previously demonstrated that the remaining
mrk genes of the fimbrial gene cluster are highly conserved
regardless of whether they are present on the chromosome or a plasmid
in Klebsiella (15, 16). Therefore, the two genes
encoding the MR/KHA of the type 3 fimbriae have undergone evolutionary
divergence. Similarly, in fimbriate isolates of E. cloacae,
the two distinct mrkD genes can be found, and in the strain
possessing mrkD1p, this determinant is carried
on a plasmid.
Although it could be demonstrated by PCR analysis that K. pneumoniae IApc35 retains a region of DNA between its
chromosomally borne mrkC and mrkF genes that is
approximately 1,200 nucleotides in length, this strain expresses
nonadhesive fimbriae (16). If, in fact, the mrkD
adhesin gene is present in this location, K. pneumoniae
IApc35 should be hemagglutinating. However, nucleotide sequencing
demonstrated that a translation termination codon was present in the
middle of this region of DNA, and therefore this strain will synthesize
a truncated MrkD polypeptide. Consequently, a hemagglutinating isolate
of K. pneumoniae, strain UIR079, that possessed DNA
sequences related to that of the mrkD1C gene
probe was used to clone and subsequently to determine the nucleotide sequence of a functional MrkD1C adhesin. The
mrkD1C and mrkD1P genes
are not closely related, and this lack of similarity explains the
inability of each gene probe to detect sequences from the heterologous
gene. A comparison of the MrkD1C and MrkD1P
polypeptides indicates significant differences in the amino acid
sequences of these two molecules (Fig. 2), particularly in the
N-terminal regions. A greater degree of amino acid sequence
conservation is found within the C termini of the two polypeptides. The
C-terminal domain of various fimbrial adhesins has been reported to
possess four conserved sequence motifs that are thought to be necessary for folding and assembly (13). The MrkD adhesins share these motifs in hydrophobic clusters, which display a typical configuration strongly associated with amphipathic 
-strands. As discussed below, K. pneumoniae 43816 demonstrates a distinct binding
specificity associated with its type 3 fimbriae, and therefore the
mrkD determinant from this isolate was also cloned and
characterized. This specific gene is closely related to that of
K. pneumoniae UIR079 and IApc35, and a comparison of the
amino sequences of the gene products indicates a close similarity (Fig.
2). In fact, the mrkD1C genes are allelic variants, and their gene products differ at only two small regions within the N terminus of the polypeptides. This close relatedness explains why the K. pneumoniae 43816 mrkD gene is
recognized by the gene probe derived from strain IApc35. The lack of
reactivity of the K. pneumoniae UIR079 and 43816 MrkD
proteins with serum raised against the N terminus of MrkD from strain
IA565 is consistent with the observed differences in the amino acid
sequences at this region. Serum raised against the major fimbrial
subunit, MrkA, reacts with the MrkA polypeptides of all fimbriate
strains examined (Fig. 5), confirming the serologic relatedness of the
type 3 fimbriae reported by Old and Adegbola (22).
We have previously demonstrated that type V collagen-binding
specificity is a function of the MrkD1P polypeptide and
that all strains of Klebsiella possessing this adhesin
exhibit identical binding properties (16, 32). We have now
demonstrated that the possession of the MrkD1C
fimbria-associated protein is frequently correlated with the ability of
bacteria to bind to both type IV and type V collagen. Both fimbriate
bacteria and cell-free, purified fimbriae exhibited identical binding
specificities with those appendages possessing the MrkD1c
polypeptide adhering to collagen type IV and type V. Interestingly, the
fimbriae isolated from K. pneumoniae 43816 could not be
shown to bind to any of the extracellular matrix proteins used in our
assays. However, the bacteria and purified fimbriae are strongly
hemagglutinating, suggesting that these appendages do function in vitro
as adhesins. In fact, using the mrkD1P gene, we
have previously shown that the MrkD polypeptide is responsible for
MR/KHA activity and binding to type V collagen (12).
Therefore, hemagglutination by K. pneumoniae 43816 is most
likely a function of MrkD1C. Since the only major
differences in the MrkD1C polypeptides of K. pneumoniae UIR079 and 43816 are found at two sites within the N
termini of these molecules, it is possible that the observed
receptor-binding specificity is a function of these regions. Currently,
we are investigating this hypothesis, using site-specific mutagenesis
of the two mrkD genes.
A small number of the Klebsiella isolates examined in this
study, as exemplified by K. pneumoniae 43816, did not
mediate binding to any of the target proteins used in our assays. It is
not possible to conclude that these strains represent one group
exhibiting an identical binding pattern. However, all of these strains
clearly possess an mrkD gene that is closely related to
mrkD1C, since these strains hybridize to this
gene probe. Differences in binding specificity have been associated
with allelic variation in the adhesin gene of the type 1 fimbrial gene
cluster of E. coli (29-31). Also, the PapG
adhesin of P fimbriae demonstrates variability in binding activity
dependent upon the papG gene present in the gene cluster
(17, 18). The mrkD1C genes of the
type 3 fimbrial gene cluster are also comprised of at least two allelic
variants. However, the plasmid-borne mrkD1p gene
does not demonstrate a relatedness to the mrkD1C
alleles, and the nucleotide sequences of these two genes have diverged
such that the gene probes do not cross-hybridize.
In summary, we have demonstrated that most strains of K. pneumoniae produce type 3 fimbriae possessing an MrkD polypeptide different from that associated with the plasmid-encoded,
type-V-collagen-binding molecule (16). This latter MrkD
adhesin is found primarily in fimbriate strains of K. oxytoca and less frequently among K. pneumoniae isolates. Most strains of K. pneumoniae produce an MrkD
molecule that is not encoded by a plasmid. In these strains, fimbriae
carrying one type of MrkD adhesin can be shown to mediate binding to
type IV and type V collagen. However, in some Klebsiella
strains, another MrkD variant present in fimbriae exhibits the
characteristic MR/KHA activity but cannot be shown to bind in vitro to
a range of ECMPs. Investigations into the molecular biology of these
naturally occurring MrkD polypeptides could provide information on the
nature of the receptors recognized by type 3 fimbriae. Since type 3 fimbriate enterobacteria are frequent opportunistic pathogens of
immunocompromised individuals (6, 8, 15, 35), the role of
MrkD molecules in binding to damaged epithelial surfaces is currently
being investigated.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, 3403 Bowen Science Building, University of Iowa College of Medicine, Iowa City, IA 52242-1109. Phone: (319) 335-7778. Fax:
(319) 335-9006. E-mail: steven-clegg{at}uiowa.edu.
Editor: R. N. Moore
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Infect Immun, June 1998, p. 2887-2894, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
