FdeC, a Novel Broadly Conserved Escherichia coli Adhesin Eliciting Protection against Urinary Tract Infections

Article (PDF Available)inmBio 3(2) · February 2012with95 Reads
DOI: 10.1128/mBio.00010-12 · Source: PubMed
Abstract
Unlabelled: The increasing antibiotic resistance of pathogenic Escherichia coli species and the absence of a pan-protective vaccine pose major health concerns. We recently identified, by subtractive reverse vaccinology, nine Escherichia coli antigens that protect mice from sepsis. In this study, we characterized one of them, ECOK1_0290, named FdeC (factor adherence E. coli) for its ability to mediate E. coli adhesion to mammalian cells and extracellular matrix. This adhesive propensity was consistent with the X-ray structure of one of the FdeC domains that shows a striking structural homology to Yersinia pseudotuberculosis invasin and enteropathogenic E. coli intimin. Confocal imaging analysis revealed that expression of FdeC on the bacterial surface is triggered by interaction of E. coli with host cells. This phenotype was also observed in bladder tissue sections derived from mice infected with an extraintestinal strain. Indeed, we observed that FdeC contributes to colonization of the bladder and kidney, with the wild-type strain outcompeting the fdeC mutant in cochallenge experiments. Finally, intranasal mucosal immunization with recombinant FdeC significantly reduced kidney colonization in mice challenged transurethrally with uropathogenic E. coli, supporting a role for FdeC in urinary tract infections. Importance: Pathogenic Escherichia coli strains are involved in a diverse spectrum of diseases, including intestinal and extraintestinal infections (urinary tract infections and sepsis). The absence of a broadly protective vaccine against all these E. coli strains is a major problem for modern society due to high costs to health care systems. Here, we describe the structural and functional properties of a recently reported protective antigen, named FdeC, and elucidated its putative role during extraintestinal pathogenic E. coli infection by using both in vitro and in vivo infection models. The conservation of FdeC among strains of different E. coli pathotypes highlights its potential as a component of a broadly protective vaccine against extraintestinal and intestinal E. coli infections.

Figures

FdeC, a Novel Broadly Conserved Escherichia coli Adhesin Eliciting
Protection against Urinary Tract Infections
Barbara Nesta,
a
Glen Spraggon,
b
Christopher Alteri,
c
Danilo Gomes Moriel,
a
Roberto Rosini,
a
Daniele Veggi,
a
Sara Smith,
c
Isabella Bertoldi,
a
Ilaria Pastorello,
a
Ilaria Ferlenghi,
a
Maria Rita Fontana,
a
Gad Frankel,
d
Harry L. T. Mobley,
c
Rino Rappuoli,
a
Mariagrazia Pizza,
a
Laura Serino,
a
and Marco Soriani
a
Novartis Vaccines and Diagnostics Srl, Siena, Italy
a
; The Genomics Institute of the Novartis Research Foundation, San Diego, California, USA
b
; Department of Microbiology
and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA
c
; and Centre for Molecular Microbiology and Infection, Division of Cell and Molecular
Biology, Imperial College London, London, United Kingdom
d
ABSTRACT The increasing antibiotic resistance of pathogenic Escherichia coli species and the absence of a pan-protective vaccine
pose major health concerns. We recently identified, by subtractive reverse vaccinology, nine Escherichia coli antigens that pro-
tect mice from sepsis. In this study, we characterized one of them, ECOK1_0290, named FdeC (factor adherence E. coli) for its
ability to mediate E. coli adhesion to mammalian cells and extracellular matrix. This adhesive propensity was consistent with the
X-ray structure of one of the FdeC domains that shows a striking structural homology to Yersinia pseudotuberculosis invasin
and enteropathogenic E. coli intimin. Confocal imaging analysis revealed that expression of FdeC on the bacterial surface is trig-
gered by interaction of E. coli with host cells. This phenotype was also observed in bladder tissue sections derived from mice in-
fected with an extraintestinal strain. Indeed, we observed that FdeC contributes to colonization of the bladder and kidney, with
the wild-type strain outcompeting the fdeC mutant in cochallenge experiments. Finally, intranasal mucosal immunization with
recombinant FdeC significantly reduced kidney colonization in mice challenged transurethrally with uropathogenic E. coli, sup-
porting a role for FdeC in urinary tract infections.
IMPORTANCE Pathogenic Escherichia coli strains are involved in a diverse spectrum of diseases, including intestinal and extraint-
estinal infections (urinary tract infections and sepsis). The absence of a broadly protective vaccine against all these E. coli strains
is a major problem for modern society due to high costs to health care systems. Here, we describe the structural and functional
properties of a recently reported protective antigen, named FdeC, and elucidated its putative role during extraintestinal patho-
genic E. coli infection by using both in vitro and in vivo infection models. The conservation of FdeC among strains of different
E. coli pathotypes highlights its potential as a component of a broadly protective vaccine against extraintestinal and intestinal
E. coli infections.
Received 10 January 2012 Accepted 24 February 2012 Published 10 April 2012
Citation Nesta B, et al. 2012. FdeC, a novel broadly conserved Escherichia coli adhesin eliciting protection against urinary tract infections. mBio 3(2):e00010-12. doi:10.1128/
mBio.00010-12.
Invited Editor Soren Schubert, Max von Pettenkofer-Institut Editor Gerald Pier, Harvard Medical School
Copyright © 2012 Nesta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported
License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Address correspondence to Laura Serino, laura.serino@novartis.com.
E
scherichia coli strains are versatile microorganisms that con-
stantly acquire and lose virulence attributes, leading to the
emergence of successful new genetic combinations that can confer
an increased ability to colonize new niches and to cause a broad
spectrum of intestinal and extraintestinal diseases (1). Strains with
successful combinations of virulence factors and that cause similar
diseases have become pathotypes. Intestinal E. coli pathotypes ap-
pear to be unable to persist in the human intestine and cause
diarrheal diseases only when ingested in sufficient quantities by a
naive host. On the other hand, extraintestinal pathogenic E. coli
(ExPEC), while not inducing enteric disease, can asymptomati-
cally colonize the human intestinal tract as the predominant spe-
cies in ~20% of healthy individuals (2, 3). Extraintestinal infec-
tions resulting from these strains, however, include neonatal
meningitis, urinary tract infections (UTIs), diverse intra-
abdominal infections, pneumonia, intravascular-device infec-
tions, osteomyelitis, soft tissue infections, bacteremia, and sepsis
(4). In particular, UTIs, which can be either asymptomatic or
symptomatic, are characterized by a wide spectrum of manifesta-
tions ranging from mild dysuria to bacteremia, sepsis, or even
death (5). Uncomplicated UTI is confined to the bladder, while
severely complicated UTIs include pyelonephritis and urosepsis.
Recurrent UTIs occur as result of reinfection by bacteria from
outside the urinary tract or from persistent bacteria (6). Virulence
factors most commonly associated with uropathogenic E. coli
(UPEC) include adhesive fimbriae, iron acquisition systems, and
toxins such as hemolysin and cytotoxic necrotizing factor (7). Af-
ter bacterial attachment, UPEC may invade epithelial cells and
form small clusters of intracellular bacteria, termed intracellular
bacterial communities (IBCs) (8). Bacteria may persist in these
protective niches, creating a chronic quiescent reservoir in the
bladder.
UPEC strains contribute significantly to the burden of ExPEC-
associated diseases, being the causative agent in 70% to 95% of
RESEARCH ARTICLE
March/April 2012 Volume 3 Issue 2 e00010-12
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community-acquired UTIs and 50% of all cases of nosocomial
infections (9). Due to the emergence of an increasing number of
antibiotic-resistant strains, the development of an efficacious Ex-
PEC vaccine would have both a significant impact on public
health and great economic benefit. Recently, we determined the
genome sequence of an ExPEC K1 strain, IHE3034 (ST95), iso-
lated from a case of neonatal meningitis, and compared it to the
available genome sequences of other ExPEC strains and a few non-
pathogenic E. coli strains (10). By a subtractive reverse vaccinology
approach, nine antigens were identified and demonstrated to be
protective in a mouse model of sepsis. Their conservation in other
E. coli pathotypes indicated their potential as candidates for a uni-
versal E. coli vaccine (10). In this report, we describe the structural
and functional properties of one of these protective antigens,
ECOK1_0290, renamed FdeC (for factor adherence E. coli). In this
analysis, we elucidated its putative role during ExPEC pathogen-
esis by using both in vitro and in vivo infection models. Our find-
ings, corroborated by epidemiological data on antigen conserva-
tion in other pathotypes, strongly support the importance of FdeC
in E. coli colonization of host tissues and the relevance of the
protein as a vaccine target.
RESULTS
Genomic characterization, distribution, and conservation of
the fdeC gene. FdeC, previously annotated in the ExPEC strain
IHE3034 as bacterial immunoglobulin-like domain (group 1)
protein (ECOK1_0290) (10), is a 1,416-amino-acid (aa) protein
that has low sequence similarity with Yersinia pseudotuberculosis
invasin (11) and enteropathogenic E. coli (EPEC) intimin (12)
(conserved up to 35% over selected regions). In addition, FdeC
shares 95% identity with EaeH, a putative adhesin identified by
subtractive hybridization from the genome sequence of the en-
terotoxigenic E. coli (ETEC) strain H10407 (13). A closer exami-
nation of the region encompassing the fdeC gene showed that in
ExPEC strains (Fig. 1) and other E. coli pathotypes (see Fig. S1 in
the supplemental material) the gene resides in a locus containing
up to three putative regulatory genes and five putative reductases
and hydrolases. In the nonpathogenic K-12 strains, the fdeC gene
is disrupted and is present as a shorter pseudogene, corresponding
only to the N-terminal region of the full-length protein (Fig. 1).
To assess the distribution of the fdeC gene among different E. coli
isolates, we evaluated the prevalence of the gene in 128 sequenced
E. coli genomes available in public databases and performed PCR
amplification of the gene in a collection of 143 E. coli isolates,
including distinct pathotypes of human and animal origin. The
fdeC gene was found to be highly distributed among all strains
with an overall presence of approximately 99% in ExPEC isolates
and 92 to 100% in intestinal E. coli pathotypes (Table 1). When
present, the gene is highly conserved with 91% identity at the
amino acid sequence level among these strains.
Sequence organization and crystal structure of FdeC. Se-
quence analysis revealed that FdeC is predicted to contain a num-
ber of bacterial Ig-like domains. The N-terminal region is identi-
fied as a domain of unknown function (DUF3442) and is likely to
form a
-barrel multidomain structure. Considering this predic-
tion and the inability to purify soluble full-length recombinant
protein, FdeC was divided into three regions: region A, predicted
as the transmembrane domain, and regions B and C, predicted to
contain the extracellular Ig-like domains (Fig. 2A). While no ex-
pression was obtained for region A alone, we successfully purified
soluble recombinant polypeptides for the following fragments: AB
(FdeC
AB
), B (FdeC
B
), and C (FdeC
C
). Fragment C, however, was
unstable and rapidly degraded postpurification.
Among these fragments, we solved the crystal structure of re-
gion B (residues 597 to 1008), previously reported to be responsi-
ble for the antigenic properties of the protein (10). The FdeC
B
crystal structure was solved at 1.9-Å resolution. Electron density
could be identified only for residues 678 to 991 of the structure,
and crystal packing showed no space available to contain the first
81 residues of the construct, leading us to conclude that these
FIG 1 Comparative genome analysis of the fdeC cluster. Graphical representation of the fdeC cluster, containing the fdeC gene (red), putative transcriptional
regulators (blue), putative reductases and hydrolases (green), insertion sequences, and variable genes (gray). Regions highly conserved among all the strains are
considered core regions (black). Truncated genes are represented in light colors. Linear visualization of the fdeC cluster was generated by a noncommercial
software program for research purposes, GECO (36). NMEC, neonatal meningitis-associated E. coli.
TABLE 1 fdeC gene distribution analysis
Pathotype
a
No. positive Total no. Prevalence, %
ExPEC 116 117 99.1
ETEC 13 14 92.9
EHEC 34 38 89.5
EPEC 5 5 100.0
AIEC 3 3 100.0
EAEC 3 3 100.0
STEC 6 6 100.0
Other 53 55 96.4
Commensal/fecal 19 23 82.6
Nonpathogenic 2 7 28.6
a
The fdeC gene presence was evaluated in 128 sequenced genomes (based on 90%
identity) and in 143 clinical isolates by PCR amplification. ExPEC, avian pathogenic
E. coli; UPEC, uropathogenic E. coli; NMEC, neonatal meningitis-associated E. coli;
SEPEC, septicemic E. coli; ETEC, enterotoxigenic E. coli; EHEC, enterohemorrhagic
E. coli; EPEC, enteropathogenic E. coli; AIEC, adherent-invasive E. coli; EAEC,
enteroaggregative E. coli; STEC, Shiga toxin-producing E. coli; other, unknown
pathotypes; commensal/fecal, E. coli isolated from healthy individuals; nonpathogenic,
E. coli lab strains.
Nesta et al.
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residues were lost during crystallization via a proteolytic or deg-
radation event. The structure of residues 678 to 991 revealed an
elongated molecule of 126 Å in length and the presence of three
bacterial Ig-like domains, each of 120 amino acids in length
(Fig. 2B), similar to invasin (14). Adopting the domain nomen-
clature of Hamburger et al. (14), numbering the invasin domains
D1 to D5 from the N to the C termini, the comparison with invasin
(Protein Data Bank identification [PDB ID] 1CWV) showed that
241 out of the 319 C
atoms in the FdeC
B
fragment aligned with
invasin (PDB_ID 1CWV) with a root mean square deviation
(RMSD) of 2.67 Å. This corresponds to alignment with domains
D2 to D4 of invasin (Fig. 2C and D). Enteropathogenic E. coli
intimin (PDB_ID 1F00) aligns with the FdeC
B
structure for 139
out of the 282 C
atoms with an RMSD of 2.9 Å (Fig. 2C and D).
In the C-terminal portion of both invasin and intimin, a domain
D5, which is a C-type lectin-like moiety that is closely connected
to the D4 domain, is present. In both intimin and invasin, this
domain plays an important role in bacterial binding to their re-
spective receptors. Sequence analysis and modeling of the FdeC
structure revealed no evidence of a C-type lectin domain
(Fig. 2D).
Recombinant FdeC
AB
binds to human epithelial cells. Based
on the presence of structural features typical of bacterial surface
determinants interacting with host cells, we evaluated the in-
trinsic capacity of FdeC to bind to human bladder (UM-UC-3)
and urethral epithelial (tUEC) cells. We used an antibody-based
indirect immunofluorescence detection system supported by
fluorescence-activated cell sorting (FACS) analysis (see Materials
and Methods) to compare the adhesive properties of the two sol-
uble fragments obtained, FdeC
AB
and FdeC
B
. Recombinant Fde
-
C
AB
bound to both UM-UC-3 and tUEC cells (Fig. 3A),
reaching
a plateau at a concentration of 10
6
M. The binding constant
(K
d
) value was calculated as the FdeC
AB
concentration resulting in
saturation of 50% of the putative receptors present on the cells and
was estimated to be on the order of 10
7
M. Recombinant Fde
-
C
AB
also bound to ovary (CHO), cervix (HeLa), and kidney
(Vero) epithelial cell lines (see Fig. S2 in the supplemental mate-
rial). In contrast, FdeC
B
did not bind to human cells (data not
shown), suggesting the importance of the A region in maintaining
the molecule in a functional conformation. Epithelial cells pro-
duce a specific array of extracellular matrix (ECM) proteins that
have a fundamental role in maintaining tissue integrity. To inves-
tigate the propensity of FdeC
AB
to also interact with selected ECM
components, we performed in vitro binding assays using fibrino-
gen, fibronectin, laminin, and various types of collagen as target
molecules. Binding was detected using polyclonal anti-FdeC anti-
serum and quantified by enzyme-linked immunoabsorbent assay
(ELISA). Recombinant FdeC
AB
showed a pronounced dose-
dependent binding to collagen types I, III, V, and VI, with an
estimated K
d
on the order of 10
8
M (Fig. 3B). No binding was
observed to other ECM compounds, such as fibrinogen, fibronec-
tin, laminin, and collagen IV (data not shown).
Expression of FdeC is induced by interaction with host cells.
Since several attempts to reproduce urinary tract or intestinal con-
ditions in vitro did not result in native FdeC expression on the
bacterial surface (see the supplemental material), we hypothesized
FIG 2 FdeC regions and crystal structure. (A) Schematic of the regional organization of FdeC. (B) Ribbon diagram of FdeC
B
(amino acids 678 to 991); N and
C termini are indicated. (C) Superimposition of FdeC
B
, in blue, with intimin (PDB_ID 1F00), in purple, and invasin (PDB_ID 1CMV), in green. (D) Structural
comparison between the crystal structures of the surface-exposed domains of intimin and invasin with a model of the potential surface-exposed region of FdeC.
Modeled regions are colored in red, while the crystal structure of FdeC
B
is colored in blue.
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that expression of FdeC may be triggered upon contact with host
cells. Using confocal immunofluorescence microscopy, we exam-
ined the expression of FdeC in ExPEC strain IHE3034 (Fig. 4A)
following contact with human uroepithelial cells. We observed
that FdeC was expressed by the bacterial population that was
tightly associated with the plasma membrane of the mammalian
cell, as visualized by F-actin staining. Similar results were obtained
using the uropathogenic strain CFT073 (data not shown). No flu-
orescent signal was observed when infections were carried out
using the fdeC deletion mutant strains (Fig. 4B). These data sug-
gest that FdeC surface expression may depend on sensing or bind-
ing to host cells. To address the kinetics of FdeC expression during
interaction with host cells, we performed infection experiments
and compared the appearances of the FdeC signals on the bacterial
surface at 15, 30, 45, 60, 90, 120, 180, and 240 min. Confocal
imaging analysis of IHE3034, 536, and CFT073 strains in contact
with uroepithelial cells revealed that the switching of FdeC expres-
sion did not occur earlier than 60 min from bacterial loading and
that the proportion of bacteria expressing the protein remained
constant up to 4 h (data not shown). FdeC deletion mutant strains
were used as internal controls. Quantitative association assays
were performed using IHE3034, 536, and CFT073 strains. Infec-
tion of UM-UC-3 cells at different time points (ranging from
15 min to 4 h) revealed no significant differences in the CFU
counts between wild-type and respective isogenic fdeC mutant
strains (data not shown). These data suggest that in this in vitro
system, constitutively expressed adhesins mask the contribution
of FdeC to bacterial adhesion.
Constitutive expression of FdeC results in bacterial aggrega-
tion. To better evaluate the contribution of FdeC to adhesion, we
engineered the well-characterized, weakly adhesive E. coli K-12
strain W3110 (naturally devoid of the fdeC gene) for constitutive
FIG 3 Binding of recombinant FdeC to epithelial cells and ECM. (A) UM-UC-3 and tUEC cells were incubated for1hat4°Cwith increasing concentrations
of recombinant FdeC
AB
. Binding of FdeC
AB
to cells was assessed by flow cytometry, and data are shown as mean fluorescence intensity (MFI) values plotted in
the saturation curves. (B) Binding of increasing concentrations of FdeC
AB
to collagen I, III, V, and VI (open circles). BSA is used as a negative control (solid
circles). Binding was quantified by ELISA. Points represent the means (error bars show standard errors of the means) of measurements made in triplicate.
Nesta et al.
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surface expression of FdeC (W3110::fdeC). Localization of the
protein on the surface of the W3110::fdeC strain was confirmed by
electron microscopy analysis (Fig. 5A). Assays of the adherence of
W3110 wild-type and W3110::fdeC strains to human bladder UM-
UC-3 cells, quantified using viable CFU counts (Fig. 5B) and by
confocal microscopy (Fig. 5C), clearly showed the strong contri-
bution of FdeC in the adherence of E. coli to epithelial cells. How-
ever, gentamicin protection assays reveal a low invasion of UM-
UC-3 cells by the W3110::fdeC strain (data not shown). The use of
E. coli K-12 and its derivatives as a model to assess bacterial attach-
ment to solid surfaces has been established previously (15). By
comparing the W3110 wild-type and W3110::fdeC strains, we ob-
FIG 4 Confocal analysis of FdeC expression in contact with cultured epithelial cells. Monolayers of UM-UC-3 cells were infected with IHE3034 (A) and the
respective fdeC knockout mutant strain (B). At the end of the infection, the samples were fixed and stained for confocal immunofluorescence microscopy. FdeC
was detected using specific antibodies and visualized using a fluorescent secondary antibody (green). DAPI was used to stain the host cell nuclei and visualize
bacteria (blue). Cellular actin was stained with fluorescent phalloidin (red).
FIG 5 Constitutive surface exposure of FdeC in strain W3110::fdeC induces the formation of bacterial aggregates. (A) Immunogold transmission electron
microscopy of FdeC surface localization in W3110 wild-type (left panel) and W3110::fdeC (right panel) strains. Fixed bacteria were incubated with an anti-FdeC
serum and then with the secondary antibodies labeled with 10-nm gold particles. Bars, 200 nm. (B) Association rate of W3110 and W3110::fdeC with UM-UC-3
cells. The W3110 (white bar) and the W3110::fdeC (gray bar) strains were used to infect UM-UC-3 monolayers for3hatamultiplicity of infection of 10:1. Viable
cell-associated bacteria were quantified after cell lysis. Results are expressed as a percentage of the inoculum. The data displayed illustrate the results of three
independent experiments, including standard deviations. (C) Confocal microscopy analysis of UM-UC-3 cells infected with W3110 wild-type (left panel) and
W3110::fdeC (right panel) strains. The W3110 strain and its derivative were detected using a polyclonal rabbit anti-E. coli K-12 antibody (green fluorescence).
Actin was stained in red using phalloidin. (D) Confocal images of W3110 wild-type (left panel) and W3110::fdeC (right panel) strains adhering to glass slides
reveal the propensity of the FdeC-expressing strain to form aggregates. Bacteria were localized with DAPI (blue), and FdeC was detected using the anti-FdeC
serum and a fluorescent secondary antibody (red). (E) In vitro microcolony formation by W3110 (left) and W3110::fdeC (right) strains grown in LB medium at
RT in polystyrene tubes. The macroscopic aggregates were visualized by crystal violet staining. (F) Kinetics of microcomplex formation at up to 6 days.
Quantitative crystal violet staining was used to measure the level of the aggregation for W3110::fdeC in comparison with the W3110 wild-type strain. Data
represent means and standard deviations of three independent measurements.
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served a strong propensity of adhering bacteria to form large ag-
gregates on glass slides (Fig. 5D). ExPEC species are known to use
a number of surface-associated determinants to form in vivo mac-
rocommunities, often defined as biofilms (16). In particular, sev-
eral studies have indicated the important role of biofilms in UTIs,
notably in catheterized patients (17, 18). Using a previously estab-
lished protocol for determining the propensity of E. coli strains to
form aggregates in vitro (19), we demonstrated that, compared to
the wild-type control, the W3110::fdeC strain is able to strongly
interact with solid surfaces and produce macrocomplexes when
grown in polystyrene tubes (Fig. 5E). The capacity of the W3110::
fdeC strain to form bacterial aggregates was monitored up to
6 days and quantified by crystal violet staining (Fig. 5F).
FdeC contributes to colonization of the urinary tract. In or-
der to determine if FdeC plays a role in virulence, we performed an
independent challenge experiment in mice infected with wild-
type E. coli 536 and its isogenic fdeC mutant strain. As shown in
Fig. 6A, confocal immunofluorescence microscopy of paraffin-
embedded bladder sections revealed that FdeC is specifically ex-
pressed by wild-type bacteria closely associated with the bladder
tissue and that the number of bacteria was significantly higher
than that in tissue derived from mice inoculated with the fdeC
mutant strain (Fig. 6B). Imaging data were representative of a
qualitative imaging analysis of 10 bladder sections that collectively
revealed reduced bladder colonization by the mutant strain. To
evaluate the contribution of FdeC to bacterial fitness in vivo dur-
ing colonization of the urinary tract, female CBA/J mice were
transurethrally inoculated with a 1:1 ratio of E. coli 536 and the
fdeC, kanamycin-resistant, knockout mutant. At 48 h postinocu-
lation, bacteria from tissue homogenates were prepared in the
presence of 0.1% saponin to separate eventual aggregates. Live
bacteria were enumerated by viable counts. Bacterial fitness was
determined as competitive index (CI) for cochallenge data. We
observed that deletion of fdeC from strain 536 caused a significant
reduction in fitness during UTI in both the bladder and the kidney
(Fig. 6C). The significant difference between the mutant and wild-
type colonization levels indicated that FdeC may have an impor-
tant role in E. coli fitness during an ascending urinary tract infec-
tion.
Vaccination with recombinant FdeC confers protection
against ExPEC during experimental UTI. Since FdeC
(ECOK1_0290) was among the nine antigens shown to be protec-
tive in a mouse model of sepsis (10) and shown here to contribute
to virulence, we decided to test whether the antigen could also
induce protection in the murine model of ascending UTI. To-
wards this end, FdeC
B
was mixed with the adjuvant cholera toxin
(CT) at a ratio of 10:1 (antigen to CT) and groups of mice (n 30)
were intranasally inoculated with either the antigen-CT mixture
or CT alone. Following primary immunization (day 0) and
booster doses (days 7 and 14), animals were transurethrally chal-
lenged with the UPEC strain 536 and protection was assessed at
48 h postinfection by determining CFU in the urine, bladder, and
kidneys. FdeC
B
induced significant protection in the kidney with a
1.5-log reduction in median CFU/g (P value 0.0067) (Fig. 7A).
In addition, 17/30 vaccinated mice had undetectable levels of bac-
teria in the kidney (10
2
CFU/g tissue). We also observed a strong
trend in reduction of kidney colonization after challenge with the
UPEC strain CFT073, with a 2.5-log reduction in median CFU/g
(Fig. 7B). In this case, 50% of mice had undetectable levels of
bacteria (10
2
CFU/g tissue) in the kidney. This demonstrated
that mucosal immunization in the nares generates a protective
effect at distal sites. These data strongly support the use of FdeC
B
as a component in a vaccine able to prevent all ExPEC-associated
diseases.
DISCUSSION
The development of an efficacious, broadly cross-protective vac-
cine against most E. coli pathotypes would have significant public
health and economic benefits, considering the increasing antibi-
otic resistance among E. coli strains and the associated mortality,
morbidity, and lost productivity. We have recently compared
available genome sequences of different E. coli strains, and by us-
ing a subtractive reverse vaccinology approach, we identified a
number of antigens common to different pathotypes, capable of
inducing protection in a mouse model of sepsis, thus proposing
their use for the development of a universal E. coli vaccine (10).
Bioinformatic analysis of one of these antigen candidates, here
named FdeC, revealed an interesting structural similarity with in-
FIG 6 Role of FdeC in vivo in a model of ascending UTI. Confocal imaging analysis of FdeC expression in bladder from mice independently infected with UPEC
wild-type strain 536 (A) and the isogenic fdeC mutant strain (B). Tissue sections embedded in paraffin were stained with WGA conjugated with Alexa Fluor 647
(blue), while UPEC strain 536 was detected using a polyclonal serum raised against inactivated whole bacteria (red). FdeC was stained in green. The images are
representative of multiple observations, from at least 10 sections. (C) Cochallenge experiments using the 536 wild-type strain and the fdeC isogenic knockout
mutant. In vivo competitive indices (CI) were determined following cochallenge infections of female CBA/J mice with a 1:1 ratio of wild-type 536 and fdeC
knockout mutant strains. At 48 h postinoculation, bacteria were enumerated by plating serial dilutions from tissue homogenates. The CI from each tissue was
determined by dividing the mutant-to-wild-type ratioof CFU/g by the mutant-to-wild-type ratio of CFU from the mixed inoculum. Each dot represents bladders
and kidneys from an individual animal. Bars indicate the median CI. Significant differences in colonization (*) (P values, 0.05) were determined by the
Wilcoxon signed rank test. A CI of 1 indicates a fitness defect.
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vasin and intimin, bacterial virulence factors known to bind the
host cells. In particular, the FdeC region B (FdeC
B
) is predicted to
have the same arrangement of four bacterial immunoglobulin
folds as does the Y. pseudotuberculosis invasin structure. The X-ray
structure of FdeC
B
confirmed this structural homology, with the
truncated form of FdeC
B
(678 to 991 amino acids [aa]) sharing
both the immunoglobulin fold arrangement and the quaternary
structure of invasin (14).
Since the fdeC gene is present in different E. coli pathotypes and
has structural similarity to other pathogenic adhesion molecules,
we hypothesize that FdeC could contribute to virulence, as an
adhesin essential for infection in the host. In UPEC strains, for
instance, adhesion is vitally important to overcome host defenses
in the urinary tract and resist the flushing effect of the urine. The
role of FdeC in E. coli colonization of host tissues was supported
not only by in vivo evidence showing decreased bacterial fitness of
the fdeC mutant strain during experimental urinary tract infection
but also by the fact that the recombinant FdeC binds with strong
affinity to several epithelial cell lines and that its surface expression
on the bacteria is host cell contact dependent. In addition, its
capacity to bind different collagen types, including type V and VI
(both widely expressed in the interstitial space of kidney and blad-
der), strongly supports the hypothesis for a specific role of FdeC
during bacterial colonization.
Genome comparison of the loci containing the fdeC gene in
different E. coli strains revealed that it is present as a pseudogene in
the nonpathogenic K-12 E. coli strains, such as MG1655, W3110,
and DH10B. However, in human commensal E. coli isolates, HS
and SE11, and in many fecal strains, the full-length fdeC is present,
indicating that the protein might be functional and provide a gen-
eral advantage during host colonization. For example, E. coli HS
has been shown to stably colonize the human gastrointestinal tract
but cause no detectable disease or adverse effects (20). On the
other hand, strain SE11, a commensal E. coli isolate from the feces
of a healthy human adult, evolved to acquire and accumulate the
functions advantageous for stable colonization of intestinal cells
(21). It possesses more genes involved in adhesion than does the
K-12 strain MG1655, which is no longer able to colonize the hu-
man gut.
In general, the biogenesis of bacterial adhesive structures is a
tightly controlled process. The cost in energy and other resources
required for expression of an adhesin must be balanced with any
potential benefits that a particular adhesive organelle might pro-
vide to a bacterium. Variable expression of different adhesive or-
ganelles, such as fimbriae, may allow the bacterium to alter its
binding characteristics in response to environmental changes en-
countered within a host during the course of an infection (22). In
this context, although FdeC may share functional homology with
other adhesins localized on the E. coli surface, its host cell-
dependent expression suggests a specific role during colonization.
Nevertheless, we also observed that in vitro constitutive expression
of the protein is associated with an increased aggregative pheno-
type. We therefore suggest that the expression of FdeC at the site of
colonization may contribute to the formation of macrocolonies, a
phenotype often associated with E. coli infections. This is in agree-
ment with our observation that fdeC mutant bacteria are attenu-
ated for bladder colonization during cochallenge experiments,
confirming that FdeC is a virulence factor in mice. In fact, in vivo
competition experiments between wild-type uropathogenic E. coli
and its fdeC mutant strain revealed that the loss of FdeC caused a
significant fitness defect during extraintestinal colonization.
We recently described FdeC (ECOK1_0290) as a promising
vaccine candidate against ExPEC infections (10). In vivo protec-
tion was evaluated in mice by subcutaneous injection of the puri-
fied recombinant antigen followed by intraperitoneal challenge
with the E. coli pathogenic strain IHE3034. Using this model, we
found a good correlation between mortality and the bacterial
counts in the blood, with a significant, although rather low, pro-
tective efficacy compared to the control group. Based on the func-
tional properties of FdeC, we hypothesized that the antigen may
exert a more efficacious protective role in an experimental UTI
model. Indeed, we found that intranasal immunization with FdeC
provided considerable protection against experimental infection
with two different UPEC strains. Of interest, site-specific protec-
tion was observed, as mice immunized with the FdeC protein were
significantly protected from strain 536 colonization only in the
kidney, with a 1.5-log reduction in the median CFU/g tissue and
more than 50% of mice having undetectable levels of bacteria in
that organ. Surprisingly, despite the significant protection in the
FIG 7 FdeC
B
prevents the spread of UPEC infection into the kidney. Mice were immunized intranasally with a 10:1 ratio of FdeC
B
to CT containing 100
gof
FdeC
B
or with 10
g CT alone (mock immunized). After two boosts of 25
g antigen (10:1 ratio of antigen to CT) or CT alone given on days 7 and 14, the
individual mice were transurethrally challenged with 10
8
CFU of UPEC strain 536 (A) and CFT073 (B). After 48 h, bladder and kidneys were harvested and
homogenized. Bacteria in urine and in the tissue homogenates were enumerated by plating serial dilutions. Symbols represent CFU/g tissue or CFU/ml urine of
individual mice, and bars indicate median values. P values were determined using the nonparametric Mann-Whitney significance test.
E. coli FdeC Induces Protection against UTIs
March/April 2012 Volume 3 Issue 2 e00010-12
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mbio.asm.org 7
kidney, immunization with the FdeC protein did not reduce the
level of bacteria able to colonize the bladder.
The evidence reported in this study indicates that functional
characterization of potential vaccine candidates, and determina-
tion of their role in pathogenesis, may be pivotal to understanding
the basis for inducing protective immunity during specific stages
of disease and support the development of more efficacious vac-
cines.
MATERIALS AND METHODS
Ethics statement. All procedures were conducted according to protocol
08999 approved by the University Committee on the Care and Use of
Animals at the University of Michigan Medical School. The approved
procedures are in compliance with university guidelines, state and federal
regulations, and the standards of the Guide for the Care and Use of Labo-
ratory Animals.
Bacterial strains and culture conditions. ExPEC strain IHE3034 (se-
rotype O18: K1:H7), isolated in Finland in 1976 from a case of human
neonatal meningitis (23); RS218 (serotype O18:K1:H7); 536 (serotype
O6:K15:H31) (24); CFT073 (serotype O6:K2:H1) (25); MG1655 (K-12)
(26); and W3110 (K-12) (27) were used for comparative genome analysis.
Bacteria were cultured in Luria-Bertani (LB) liquid medium or agar plates
at 37°C. E. coli DH5
was used for cloning purposes, and E. coli
BL21(
DE3) was used for expression of His-tagged fusion proteins. The
clones carrying a specific cassette conferring antibiotic resistance were
grown in the presence of ampicillin (100
g/ml) or kanamycin (50
g/
ml).
Crystallization and data collection. Crystallization experiments were
performed in Greiner 96-well low-profile sitting-drop plates. Initial
screen conditions consisted of a sparse matrix of 480 conditions set up at
4°C and 20°C (28, 29). The reservoir volume was 50
l, and the drop
consisted of 200 nl protein with an equal volume of precipitant. Promising
protein crystals took over 4 months to grow under a condition containing
20% polyethylene glycol 6000 (PEG 6000) in 0.1 M Bicine buffer at pH 9.0.
Crystals were looped and chilled at liquid nitrogen temperature. Cryopro-
tection was achieved via the addition of 20% glycerol solution to the
mother liquor prior to harvesting the crystal. Data were collected at the
Advanced Light Source (ALS; Berkeley) on beamline 5.0.2. Data were
reduced using HKL2000 and the CCP4 suite (30). Data reduction statistics
are presented in Table 2.
Structure solution and refinement. The structure was solved by mo-
lecular replacement using the program Phaser (31) and an ensemble of
individual immunoglobulin domains from the homologues invasin and
intimin (PDB IDs 1CWV and 1F00, respectively) as probe molecules with
all data to 2.5 Å. Full-length probes of invasin and intimin failed to pro-
duce expectable solutions, in initial runs. In all the positions and orienta-
tions of three immunoglobulin domains found via this method, packing
considerations made it clear that the fourth domain, believed to be in the
construct, was missing in the crystal structure, which contained only 36%
solvent with the 3 domains fitted. Iterative rounds of building and refine-
ment with Buster (32) and Coot (33) produced a structure whose refine-
ment converged with excellent geometry (data and refinement statistics
are shown in Table 2). Superposition with structural homologues was
performed with Coot and SSM (33, 34). All crystallographic manipula-
tions other than those stated used the CCP4 package (30).
Binding assay using purified protein. Approximately 2 10
5
cells
were incubated with increasing concentrations of recombinant protein
for1hat4°C. Cells were then incubated with rabbit polyclonal anti-FdeC
serum. Subsequently, cells were incubated with a goat anti-rabbit IgG
fluorescein isothiocyanate (FITC)-conjugated antibody (Jackson Immu-
noResearch Laboratories). FACS acquisition was performed using a
fluorescence-activated cell sorter (Becton Dickinson). Data were analyzed
using FlowJo software.
Confocal microscopy analysis of E. coli strains adhering to eukary-
otic cells and tissues. Epithelial cells were grown to confluence and in-
fected with E. coli strains at a multiplicity of infection of 10:1 for different
time points. Samples were washed and fixed in 3% paraformaldehyde
(PFA) for 15 min at room temperature (RT). After 2 h of blocking in 1%
bovine serum albumin (BSA), the rabbit anti-FdeC serum was used to
detect the protein, using a donkey anti-rabbit IgG Rhodamine Red
X-conjugated antibody as secondary antibody. Alexa Fluor dye-labeled
phalloidins (Invitrogen) were used to stain the actin cytoskeleton accord-
ing to the manufacturer’s instructions. Bacterial and cellular DNA were
stained with 4=,6-diamidino-2-phenylindole (DAPI). The ProLong Gold
reagent (Invitrogen) was used as a liquid mountant. The W3110 strain was
detected using the polyclonal rabbit anti-E. coli K-12 (Dako) and the
Alexa-Fluor 488 goat anti-rabbit IgG secondary antibody. The analysis
was done with a Zeiss LSM 710 laser scanning microscope. Confocal im-
aging on tissues was performed on bladder sections derived from ExPEC-
infected animals. In detail, the bladder was cut transversely, preserved in
10% formalin (pH 7.2), and embedded in paraffin. Tissue sections were
cut and mounted onto slides. Samples were dewaxed and subjected to
antigen retrieval before the immunofluorescence labeling. Staining of the
sections was done as described for in vitro staining, except for tissues that
were labeled with wheat germ agglutinin (WGA) (Alexa Fluor 647 conju-
gated; Invitrogen).
Vaccination in a murine model of ascending UTI. Female CBA/J
mice, 6 to 8 weeks old, were transurethrally inoculated as previously de-
scribed (35). Purified antigen was mixed with cholera toxin (CT) (Sigma)
at a ratio of 10:1. The vaccine was administered intranasally in a total
volume of 20
l/animal (10
l/nare). Animals received a primary dose on
day 0 of 100
g antigen (containing 10
g CT) or 10
g CT alone. Two
boosts of 25
g antigen (mixed with 2.5
g CT) or 2.5
g CT alone were
given on days 7 and 14, and mice were challenged on day 21. E. coli
CFT073 and 536 suspensions in phosphate-buffered saline (PBS) (50
l/
mouse) were delivered transurethrally using a sterile 0.28-mm-inner-
diameter polyethylene catheter connected to an infusion pump (Harvard
Apparatus), with a total inoculum of 10
8
CFU/mouse. For determination
of CFU, organs were aseptically removed from euthanized animals at 48 h
postinoculation and homogenized in PBS with a GLH homogenizer
(Omni International). Bacteria in tissue homogenates were enumerated
TABLE 2 Crystallographic and refinement statistics
Parameter FdeC
Protein
Space group P2
1
2
1
2
No. of molecules in ASU
d
1
Unit cell (Å) 44.6, 150.93, 47.40
Wavelength (Å) 0.9202
Resolution (Å) 50.0-1.9
Total no. of unique reflections 24,734
Completeness, % (highest shell) 95.6 (67.0)
R
merge
, % (highest shell)
0.084 (0.634)
Highest-resolution shell, Å 1.97–1.90
Mean I/
(I) (highest shell) 23.9 (1.7)
Refinement
No. of references, working set 23,413
No. of references, test set 1,265
R
cryst
(R
free
a
)
b
(%)
17.1 (20.5)
RMSD bonds (Å) 0.017
RMSD angles (°) 1.44
Avg B (Å)
2
26.27
ESU
e
based on R
free
(Å)
c
0.135
a
R
free
as for R
cryst
, but for 5.0% of the total reflections chosen at random and
omitted from refinement.
b
R factor ⫽⌺|I
i
⫺⬍I
i
||/|I
i
|, where I
i
is the scaled intensity of the ith measurement
and I
i
is the mean intensity for that reflection.
c
Estimated overall coordinate error.
d
ASU, asymmetric unit.
e
ESU, estimated standard uncertainty.
Nesta et al.
8
®
mbio.asm.org March/April 2012 Volume 3 Issue 2 e00010-12
by being plated on LB agar containing 0.5 g/liter NaCl using an Autoplate
4000 spiral plater (Spiral Biotech), and CFU were determined using a
QCount automated plate counter (Spiral Biotech). Blood was collected as
necessary from anesthetized mice by an infraorbital bleed using 1.1- to
1.2-mm Micro-Hematocrit capillary tubes (Fisher), and serum was sepa-
rated using Microtainer serum separator tubes (Becton Dickinson). The
animals were 15 weeks old at the conclusion of all experiments.
Protein structure accession number. The structure has been depos-
ited in the structure database of the Genomics Institute of the Novartis
Research Foundation (San Diego, CA), and entry code in the Protein Data
Bank (PDB) is 4E9L.
ACKNOWLEDGMENTS
This work was supported by internal funding from Novartis Vaccines and
Diagnostics.
We are grateful to Marina Cerquetti (Istituto Superiore di Sanita
`
,
Rome) for kindly providing ExPEC clinical isolates and to Lothar H.
Wieler (Freie Universität, Berlin), Ulrich Vogel (Universität Würzburg),
and Karin Schnetz (Universität Köln) for providing other E. coli isolates.
We thank Angela Spagnuolo for technical assistance in the septic mouse
model, Anna Rita Taddei for contributing to the electron microscopy
studies, and Paolo Ruggiero and Laura Pancotto for assistance in prepa-
ration of sections from tissue samples. We are indebted with gratitude to
Kate Seib for critical reading of the manuscript.
The crystallization experiments were conducted at beamline 5.0.2 of
the Advanced Light Source (ALS). The ALS is supported by the Director,
Office of Science, Office of Basic Energy Sciences, Material Sciences Divi-
sion of the U.S. Department of Energy, under contract no. DE-AC03-
76SF00098, at Lawrence Berkeley National Laboratory. We thank all of the
staff of these beamlines for their continued support.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://mbio.asm.org
/lookup/suppl/doi:10.1128/mBio.00010-12/-/DCSupplemental.
Text S1, DOCX file, 0.1 MB.
Text S2, DOCX file, 0.1 MB.
Figure S1, TIF file, 5.5 MB.
Figure S2, DOCX file, 1.4 MB.
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E. coli FdeC Induces Protection against UTIs
March/April 2012 Volume 3 Issue 2 e00010-12
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mbio.asm.org 9
    • "Although our recent sequencing of APEC indicates that E. coli evolve to cause avian disease from varied lineages via acquisition of distinct sets of virulence genes, we also revealed a core genome encoding of over 3000 conserved factors [4] . Mining of the core genome of human extraintestinal pathogenic E. coli strains has yielded a subset of factors that show promise as crossprotective vaccines for control of sepsis and urinary tract infections [53,54] , and a similar approach for avian colibacillosis has merit. The models and data presented here will help to inform and evaluate improved vaccines in target hosts. "
    [Show abstract] [Hide abstract] ABSTRACT: Avian pathogenic Escherichia coli (APEC) infections are a serious impediment to sustainable poultry production worldwide. Licensed vaccines are available, but the immunological basis of protection is ill-defined and a need exists to extend cross-serotype efficacy. Here, we analysed innate and adaptive responses induced by commercial vaccines in turkeys. Both a live-attenuated APEC O78 ΔaroA vaccine (Poulvac® E. coli) and a formalin-inactivated APEC O78 bacterin conferred significant protection against homologous intra-airsac challenge in a model of acute colibacillosis. Analysis of expression levels of signature cytokine mRNAs indicated that both vaccines induced a predominantly Th2 response in the spleen. Both vaccines resulted in increased levels of serum O78-specific IgY detected by ELISA and significant splenocyte recall responses to soluble APEC antigens at post-vaccination and post-challenge periods. Supplementing a non-adjuvanted inactivated vaccine with Th2-biasing (Titermax® Gold or aluminium hydroxide) or Th1-biasing (CASAC or CpG motifs) adjuvants, suggested that Th2-biasing adjuvants may give more protection. However, all adjuvants tested augmented humoral responses and protection relative to controls. Our data highlight the importance of both cell-mediated and antibody responses in APEC vaccine-mediated protection toward the control of a key avian endemic disease.
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    • "Although, bladder is the common site of UTI in about 95% of cases [7], the infection can cause pyelonephritis , bacteremia, sepsis and sometimes death [3] [7] [8]. Uropathogenic Escherichia coli (UPEC) are isolated from 50% to 90% of all reported cases [9] [10] [11]. Among different virulence factors of UPEC [3] [7] [12], Type 1 pili which has the adhesin FimH is one of the most important UPEC virulence factors [13] [14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Urinary tract infection (UTI) caused by Uropathogenic Escherichia coli (UPEC) is one of the most common infections in the world. Despite extensive efforts, a vaccine that confers protection against UTIs in human is currently lacking. In this study, the ability of flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist of UPEC strain, and the conventional adjuvant Montanide ISA 206 to enhance the protective immune responses of FimH against urinary tract infection have been compared. Mice immunized with the fused FimH.FliC protein induced significantly higher humoral (IgG1 and IgG2a) and cellular (IFN-γ and IL-4) immune responses than with FimH alone or FimH admixed with FliC. The immune responses of Montanide formulations were comparable to that of the fusion protein and were significantly higher than that of FimH alone. Our results showed that based on the IgG1/IgG2a ratios, FliC directed the anti-FimH responses preferentially toward Th2 and Montanide toward Th1. The FimH.FliC fusion and FimH admixed with FliC and Montanide formulations gave the best results in protection of bladder colonization, compared to the control mice. The results propose new promising vaccine formulation based on the adjuvant properties of FliC and Montanide against UTI caused by UPEC strains.
    Full-text · Article · Jan 2013
  • [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Escherichia coli outbreak in Germany, which resulted in more than 4,000 cases, including 908 cases of hemolytic-uremic syndrome (HUS) and at least 50 deaths, highlighted the genome plasticity of E. coli and the potential for new virulent strains to emerge. The analysis of 170 E. coli genome sequences for the presence of nine previously identified protective extraintestinal pathogenic E. coli antigens suggested the feasibility of a combination vaccine as a universal intervention against all pathogenic E. coli strains. Importance: This article reports on the feasibility of a combination vaccine as a universal intervention against all pathogenic Escherichia coli strains.
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