Cellular Microbiology (2007) 9(9), 2230–2241
doi:10.1111/j.1462-5822.2007.00952.x
First published online 8 May 2007
Development of intracellular bacterial communities of
uropathogenic Escherichia coli depends on type 1 pili
Kelly J. Wright,1 Patrick C. Seed2 and
Scott J. Hultgren1*
1
Department of Molecular Microbiology, Box 8230,
Washington University School of Medicine, 660 S.
Euclid Avenue, St. Louis, MO 63110, USA.
2
Departments of Pediatrics, Molecular Genetics and
Microbiology, Box 103100, Duke University Medical
School, Durham, NC 27710, USA.
Summary
Uropathogenic Escherichia coli, the predominant
causative agent of urinary tract infections, use type 1
pili to bind and invade bladder epithelial cells. Upon
entry, the bacteria rapidly replicate and enter a
complex developmental pathway ultimately forming
intracellular bacterial communities (IBCs), a niche
with biofilm-like properties protected from innate
defences and antibiotics. Paradoxically, bacteria
within IBCs produce type 1 pili, an organelle thought
only to be an extracellular colonization factor. Thus,
we investigated the function of type 1 pili in IBC
development. The cystitis isolate, UTI89, was genetically manipulated for conditional fim expression
under control of the tet promoter. In this strain, UTI89tetR/Ptet fim, piliation is constitutively inhibited by the
tetracycline repressor, TetR. Repression is relieved
by anhydrotetracycline (AHT) treatment. UTI89-tetR/
Ptet fim and the isogenic control strain, UTI89-tetR,
grown in the presence of AHT, colonized the bladder
and invaded the superficial umbrella cells at similar
levels at early times in a murine model of infection.
However, after invasion UTI89-tetR/Ptet fim became
non-piliated and was unable to form typical IBCs comprised of tightly packed, coccoid-shaped bacteria in
contrast to the control strain, UTI89-tetR. Thus, this
work changes the extracellular colonization functional paradigm of pili by demonstrating their intracellular role in biofilm formation.
Received 2 October, 2006; revised 10 March, 2007; accepted 12
March, 2007. *For correspondence. E-mail hultgren@borcim.wustl.
edu; Tel. (+1) 314 362 6772; Fax (+1) 314 362 1998.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Introduction
Uropathogenic Escherichia coli (UPEC) are the predominant cause of urinary tract infections (UTI), one of the
most common bacterial infections today (Foxman, 2002;
Ronald, 2002). UPEC harbour an arsenal of virulence
determinants to overcome innate host defences including
the essential cystitis determinant, type 1 pili (Hultgren
et al., 1985; Langermann et al., 1997; Bahrani-Mougeot
et al., 2002; Hung et al., 2002; Snyder et al., 2004; 2006).
Type 1 pili are adhesive surface fibres which mediate
intimate contact between UPEC and host urothelium via
FimH, the type 1 pilus tip adhesin (Abraham et al., 1988).
FimH binds to mannosylated uroplakin plaques lining the
bladder lumen leading to invasion of the underlying superficial umbrella cells (Wu et al., 1996; Mulvey et al., 1998;
Martinez et al., 2000; Min et al., 2002). Once within the
intracellular milieu, UPEC rapidly replicate and undergo a
complex developmental pathway leading to the formation
of intracellular bacterial communities (IBCs), morphologically distinct structures with biofilm-like properties that
ultimately protect UPEC from host surveillance (Mulvey
et al., 1998; Anderson et al., 2003).
Recently, major events of the IBC pathway were visualized by high resolution, time lapse video microscopy in
a murine model of infection (Justice et al., 2004). Initially,
bacteria invade into superficial umbrella cells and replicate into loosely organized, rod-shaped bacteria. IBCs
mature into an organized mass of compact, dense,
coccoid bacteria. These mature IBCs are transient in
nature in that unknown signals trigger their dispersal
within hours after they form (Justice et al., 2004). During
the dispersal stage, the bacteria become highly filamentous and flux from the IBC periphery and out of the superficial umbrella cell. This occurs in order to disseminate
UPEC throughout the bladder to initiate subsequent
rounds of IBC formation in naïve superficial umbrella
cells, thus perpetuating acute cystitis. Cycles of IBC formation continue at progressively slower rates eventually
ceasing and forming a quiescent intracellular reservoir
(QIR) comprised of Lamp1-positive, membrane enclosed
rosettes of intracellular UPEC undetectable by the host
immune system (Mulvey et al., 2001; Mysorekar and Hultgren, 2006). It has been shown that QIRs dispersed
throughout the bladder epithelium can be activated by
stimulating superficial cell exfoliation which in turn
Type 1 pili-mediated IBC development during acute cystitis 2231
Table 1. Bacterial strains and plasmids.
Strains or plasmids
Strain name
MG1655
MG1655 DfimH
UTI89
UTI89/pVC
UTI89 Dfim
UTI89 Dfim/pfim
UTI89 DfimH
UTI89 DfimH/pfimH
UTI89-tetR
UTI89-tetR/Ptet fim
DH5aPRO
Plasmid
pVC
pfim
pTRYC
pfimH
pKD13
Relevant genotype (phenotype)
Resistance
K-12 Escherichia coli
MG1655 DfimH
Cystitis Escherichia coli isolate
UTI89/pCR®-Blunt II-TOPO®
UTI89 DfimA (fim null strain/Phase OFF by
PCR analysis (Schwan et al., 1992))
UTI89 Dfim/pCR®-Blunt II-TOPO®-fimBEAICDFGH
UTI89 DfimH
UTI89 DfimH/pTRYC-fimH
UTI89 chromosomal::PtetR -TetR
UTI89 DPfim::PLtetO-1/fimAICDFGH, chromosomal::PtetR /TetR
deoR, endA1, gyrA96, hsdR17(rk–mk+), recA1, relA1,
supE44, thi-1, D(lacZYA-argF) U169, f80 dL
acZDM15, F–, l–, PN25/tetR, Placi q/laci, Spr; Source of PN25/tetR
Features
pCR®-Blunt II-TOPO®; Control vector
pCR®-Blunt II-TOPO® with fimBEAICDFGH native expression
Hybrid pTRC99a-pACYC IPTG-inducible expression vector
pTRYC with IPTG-inducible fimH expression
Kanamycin cassette template for red-recombinase-mediated knockout
activates differentiation and proliferation cascades. These
events activate the QIR so that bacterial replication
begins again initiating new rounds of IBC formation and
inflammation (Mysorekar and Hultgren, 2006).
Recent reports demonstrated the presence of type 1 pili
within IBCs (Anderson et al., 2003) which is suggestive of
a yet to be demonstrated structural role downstream of
binding and invasion of urothelium. Furthermore, UTI89
deficient in the cis–trans prolyl isomerase, SurA (UTI89
surA::kan), exhibited extracellular and intracellular colonization defects concomitant with reduced type 1 piliation
(Justice et al., 2006). Finally, type 1 pili are likely necessary
for subsequent rounds of IBC formation because FimHmediated binding and invasion is a prerequisite for IBC
pathway initiation. We therefore investigated whether type
1 pili play an intracellular function critical in IBC formation
and maturation. We directly investigated the role of type 1
pili in IBC formation using fim and conditional fim mutant
strains in a well characterized murine model of UTI (Mulvey
et al., 1998). Our experiments demonstrated that type 1 pili
were necessary for intracellular aggregation into an IBC
and that the inability of UPEC to express type 1 pili, postinvasion, dramatically attenuated virulence.
Results
fim mutants are deficient in extracellular and intracellular
bladder colonization
Type 1 pili confer upon UPEC the ability to bind and
invade bladder epithelial cells (Mulvey et al., 1998; Martinez et al., 2000; Anderson et al., 2003; Justice et al.,
2004) and are considered to be an essential virulence
KanR
KanR
CmR
CmR
SpecR
SpecR
KanR
CmR
CmR
KanR
Reference
Metcalf et al. (1990)
This work.
Mulvey et al. (2001)
This work
This work
This work
This work
This work
This work
This work
Clontech Laboratories
Invitrogen Life Technologies
This work
This work
This work
Datsenko and Wanner (2000)
determinant of cystitis-causing UPEC (Hultgren et al.,
1985; Langermann et al., 1997; Bahrani-Mougeot et al.,
2002; Hung et al., 2002; Snyder et al., 2004; 2006).
UTI89, a prototypic cystitis strain (Mulvey et al., 2001),
produces type 1 pili (Justice et al., 2006) and was used in
these studies. UTI89 colonizes the luminal surface of the
bladder and invades superficial umbrella cells where they
go on to develop into IBCs (Anderson et al., 2003; Justice
et al., 2004; 2006). Type 1 pili are known to mediate
colonization of the bladder and invasion into bladder epithelial cells, however, an intracellular role for pili, including
a role in the IBC pathway, has never been investigated.
UTI89 contains 10 chaperone-usher gene clusters which
encode for known and putative adhesive fimbriae. Thus,
fim mutants were characterized in vitro and in vivo to
invade and/or be phagocytosed by superficial umbrella
cells of the bladder epithelium to ensure loss of invasion
and hence bladder colonization in a genetic milieu of other
potential adhesive systems yet to be characterized in this
or other uropathogenic strains. fim- (lacks the entire type
1 pilus operon) and fimH-negative (lacks only the fimH
adhesin gene) UTI89 mutant strains (Table 1), were deficient in guinea pig erythrocyte agglutination and binding
to 5637 cultured bladder epithelial cells (Martinez et al.,
2000; Mulvey et al., 2001) relative to the isogenic wt
UTI89 control strain (data not shown). In vivo, both UTI89
Dfim (Fig. 1A) and UTI89 DfimH (Fig. 1B) were unable to
colonize (data not shown) and invade bladder epithelium
by 6 h post infection relative to UTI89 (Fig. 1; *P = 0.0079;
Mann–Whitney U-test). These colonization and invasion
defects could be complemented by supplying the entire
fim operon (pfim) or fimH (pfimH) in trans to UTI89 Dfim
and UTI89 DfimH, respectively.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2230–2241
2232 K. J. Wright, P. C. Seed and S. J. Hultgren
not shown) (Schwan et al., 1992). Thus, UTI89 DfimA in
effect is a fim null strain because the entire operon is
locked in a phase OFF condition and this construct is
referred to as UTI89 Dfim.
Conditional fim strain characterization
Fig. 1. fim mutants are deficient in intracellular bladder colonization
in an ex vivo gentamicin protection assay. C3H/HeN female mice
were transurethrallly inoculated with (A) UTI89/pVC, UTI89
Dfim/pVC, UTI89 Dfim/pfim, or (B) UTI89, UTI89 DfimH and UTI89
DfimH/pfimH. At 6 h post infection, bladders were aseptically
harvested and processed as described. Intracellular/invaded
bacteria were enumerated by serial dilution and plating. Horizontal
bars indicate the geometric mean titre. Recovery of intracellular
UTI89 Dfim/pVC and UTI89 DfimH bacteria was significantly lower
than for UTI89 or the complemented UTI89 strains (*P = 0.0079;
#P < 0.05 Mann–Whitney U-test) indicating a failure to invade and
establish cystitis in absence of functional type 1 pili.
In this study, we used UTI89 DfimA as our model mutant
strain, as it was completely deficient in producing fim
subunits despite that we only targeted fimA for partial or
complete deletion. The fim promoter is part of an invertible
element that switches from phase ON to phase OFF via
the action of various recombinases (Gally et al., 1996;
Wolf and Arkin, 2002). We discovered that fimA deletions
resulted in a phase locked OFF phenotype by polymerase
chain reaction (PCR) analysis of the fim promoter (data
UTI89 Dfim and UTI89 DfimH were unable to colonize the
bladder (data not shown). Accordingly, these strains were
also defective in invasion into bladder epithelial cells
(Fig. 1) therefore preventing us from studying intracellular
contributions of type 1 pili. Thus, it was necessary to
engineer a strain that would transiently express type 1 pili,
to allow attachment and invasion, but which would subsequently suppress the fim operon once in an intracellular
niche in order to investigate whether type 1 pili function
intracellularly in IBC maturation. To do this we engineered
a conditional fim strain, UTI89-tetR/Ptet fim (Table 1). The
native, chromosomal fim promoter was exchanged with
the tetracycline-inducible promoter, PLtetO-1 (Lutz and
Bujard, 1997), by homologous recombination in order to
achieve exogenous control of fim expression (Fig. 2A).
The gene encoding the tetracycline repressor, TetR,
was constitutively expressed from the chromosome in
single-copy from its native promoter such that fim transcription occurred only with relief of TetR repression by
anhydrotetracycline (AHT) treatment. As evidenced by
FimH immunoblot of whole cell lysates (Fig. 2B), guinea
pig erythrocyte agglutination (Table 2), and electron
microscopy (Fig. 2D), type 1 piliation of UTI89-tetR/Ptet fim
occurred only in the presence of an optimized dose of
AHT. The wt isogenic control strain, UTI89-tetR, produced
type 1 pili regardless of AHT treatment when grown statically at 37°C (Fig. 2B and C, Table 2). Upon removal of
AHT from culture media, UTI89-tetR/Ptet fim type 1 piliation diminished to undetectable levels by 6 h as demonstrated by haemagglutination (Table 2). Of note, with or
without AHT treatment, UTI89-tetR and UTI89-tetR/Ptet fim
did not differ significantly in growth rate in vitro (data not
shown). These in vitro data validated our experimental
design aiming to inoculate type 1 piliated bacteria into the
bladder to allow colonization and invasion followed by
repression of piliation once the bacteria established an
intracellular niche.
UTI89-tetR/Ptet fim is deficient in establishing
murine cystitis
Prior to addressing an intracellular role for type 1 pili,
UTI89-tetR and UTI89-tetR/Ptet fim were evaluated for
their ability to invade superficial umbrella cells in transurethrally infected C3H/HeN mice using an ex vivo gentamicin protection assay that assesses levels of extracellular
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2230–2241
Type 1 pili-mediated IBC development during acute cystitis 2233
Fig. 2. Conditional fim strain construction and
characterization.
A. Schematic diagram of the isogenic control
strain, UTI89-tetR and the conditional fim
strain, UTI89-tetR/Ptet fim.
B. FimCH immunoblot analysis. Lane 1,
UTI89-tetR, without AHT; lane 2,
UTI89-tetR/Ptet fim, without AHT; lane 3,
UTI89-tetR with AHT; lane 4, UTI89-tetR/Ptet
fim with AHT.
C and D. Representative images of negatively
stained electron micrographs of UTI89-tetR
(C) and UTI89-tetR/Ptet fim (D) grown in the
presence of AHT. Scale bar is equal to
200 nm.
Table 2. Guinea pig erythrocyte haemagglutination titres.
HA titres
Strain
AHT
No mannose
+2% Mannose
UTI89-tetR
UTI89-tetR/Ptet fim
UTI89-tetR
UTI89-tetR/Ptet fim
UTI89-tetR/Ptet fim
–
–
+
+
+→ –a
256–512
16b
256–512
128–512
0–2d
0–4b
16–32b
0b
16–32c
0–2d
a. AHT-induced cultures were subcultured into fresh media without
AHT, grown to late log phase (~3 h), cultured statically for 3 h, and
assayed for haemagglutination.
b. Significantly different from UTI89-tetR, No Mannose (P < 0.001,
Student’s t-test).
c. Significantly different from UTI89-tetR/Ptet fim, No Mannose
(P < 0.001, Student’s t-test).
d. Significantly different from AHT-induced, type 1 pili-positive,
UTI89-tetR/Ptet fim (P < 0.001, Student’s t-test).
and intracellular, or gentamicin-protected bacteria
(Justice et al., 2006). UTI89-tetR/Ptet fim was fully piliated
upon inoculation (Table 2), and consequently, this strain
colonized (Fig. 3; P = 1.000; Mann–Whitney U-test) and
invaded (Fig. 3; P = 0.2222; Mann–Whitney U-test) the
bladder tissue to equivalent levels as UTI89-tetR at 1 h
post-infection. Thus, UTI89-tetR/Ptet fim was able to carry
out early colonization and invasion events when the bacteria were grown in the presence of the inducing agent,
AHT. Later time points were next evaluated in order to
determine whether the lack of type 1 pili was detrimental
to persistent cystitis. UTI89-tetR colonization levels
ranged from 105 to 106 over a 2 week period of time,
whereas UTI89-tetR/Ptet fim was significantly lower at all
time points examined (Fig. 4; *P < 0.0001, Mann–Whitney
U-test). The inability of UTI89-tetR/Ptet fim to persist and
establish wt infection levels was presumably attributed to
the loss of type 1 piliation following invasion since
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2230–2241
2234 K. J. Wright, P. C. Seed and S. J. Hultgren
Fig. 3. In vivo invasion of murine bladder
epithelium by UTI89-tetR/Ptet fim relative to
UTI89-tetR in an ex vivo gentamicin
protection assay. C3H/HeN female mice were
transurethrallly inoculated with UTI89-tetR or
UTI89-tetR/Ptet fim, the bladders aseptically
harvested at 1 h post-infection, and processed
as described. Luminal (Luminal) and
intracellular/invaded bacteria (Intracellular)
were enumerated by serial dilution and
plating. Horizontal bars indicate the geometric
mean titre. Similar intracellular numbers of
UTI89-tetR/Ptet fim bacteria were present
relative to UTI89-tetR indicating that the
conditional fim strain is not deficient in
invasion.
exogenous AHT was absent. Loss of type 1 piliation
resulted from simultaneous repression of transcription at
the tetracycline promoter and dilution of pili upon each cell
division event during intracellular growth (Table 2).
Wt IBCs stain robustly for type 1 pili
To determine whether type 1 pili were produced by UTI89tetR within an IBC, immunohistochemical analysis of
UTI89- and UTI89-tetR-infected bladders was performed
as described in the Experimental procedures. Using antiFimH antisera, both wt UTI89 and UTI89-tetR were shown
to produce type 1 pili (green) within IBCs located within
uroplakin-positive (red) superficial facet cells (Fig. 5)
which is consistent with previous results (Anderson et al.,
2003). Uroplakin staining demonstrated that the IBCs
were correctly localized within superficial umbrella cells.
UTI89-tetR/Ptet fim is deficient in forming IBCs
Uropathogenic E. coli use the IBC developmental
pathway as a defense mechanism against innate host
defenses to establish and cause disease within the
murine urinary tract (Mulvey et al., 1998; Anderson et al.,
2003; Justice et al., 2004). The effect of the inability of
UTI89-tetR/Ptet fim to produce type 1 pili intracellularly, on
IBC formation and maturation, was determined by two
independent methods at 6 h post-infection. IBC number
was initially assessed by LacZ staining (Justice et al.,
2006), a high throughput but low resolution colorimetric
technique which stains IBCs purple for quantification by
light microscopy. IBC number was subsequently confirmed by high resolution laser scanning confocal microscopy of bladders stained with the membrane permeable
nucleic acid dye, TO-PRO®-3 iodide as previously
described (Wright et al., 2005). Consistent with previous
LacZ staining studies (Justice et al., 2006), UTI89-tetRinfected bladders yielded a range of 11–93 IBCs per
bladder (n = 3 mice; mean = 51; median = 50; data not
shown). Infection with UTI89-tetR/Ptet fim produced
diffuse, poorly staining collections of bacteria in all bladders examined (n = 5 mice; data not shown) that were not
scored as IBCs because an IBC is defined as a dense,
compact collection of intracellular bacteria with biofilm-like
properties (Anderson et al., 2003; Justice et al., 2004).
Confocal microscopy examination of UTI89-tetR/Ptet fimFig. 4. The conditional fim strain,
UTI89-tetR/Ptet fim, is deficient in establishing
murine cystitis. C3H/HeN female mice were
transurethrallly inoculated with UTI89-tetR or
UTI89-tetR/Ptet fim and the bladders
aseptically harvested at the indicated time
points. Horizontal bars indicate the geometric
mean titre. UTI89-tetR bladder colonization
levels ranged from 105 to 106 cfu per bladder
for up to 2 weeks post infection whereas
UTI89-tetR/Ptet fim was significantly lower at
all time points examined (*P < 0.0001;
Mann–Whitney U-test). Data represent two
independent experiments combined.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2230–2241
Type 1 pili-mediated IBC development during acute cystitis 2235
Fig. 5. wt IBCs stain robustly for type 1 pili.
Immunohistochemistry analysis of (A) UTI89
and (B) UTI89-tetR IBCs. Bacteria (green)
were stained using an anti-FimH primary
antibody. Superficial umbrella cell location
was visualized by staining with an uroplakin III
(red) primary antibody. Host cell nuclei (blue)
were visualized using Hoescht dye. Like wt
UTI89 IBCs, UTI89-tetR IBCs stain robustly
for FimH, a marker of type 1 pili.
infected bladders confirmed the IBC formation deficit seen
in LacZ staining experiments. In confocal microscopy
experiments, UTI89-tetR produced a range of 18–105
IBCs per bladder (n = 4 mice; mean = 56; median = 51)
whereas UTI89-tetR/Ptet fim produced 0–2 IBCs per
bladder (n = 4 mice; mean = 0.75; median = 0.5) (Fig. 6).
Type 1 pili are essential for intracellular fitness
Although UTI89-tetR/Ptet fim was successful at initial
invasion events, it was defective in its ability to form
IBCs and subsequently, colonized the bladder less efficiently. We analysed the IBC defect of UTI89-tetR/Ptet
fim using confocal microscopy of immunostained, whole
mount bladders at 6 and 24 h post infection. Infected
bladders were dually stained with anti-E. coli and antiFimH antibodies to visualize IBC morphology and
monitor the production of type 1 pili in vivo for UTI89tetR and UTI89-tetR/Ptet fim. UTI89-tetR produced
numerous compact, dense, IBCs containing coccoid
bacteria at 6 (n = 18–105 IBCs/mouse; n = 4 mice;
Fig. 7B–D) and 24 h (n > 30 IBCs/mouse; n = 4 mice;
data not shown) after infection while UTI89-tetR/Ptet fim
produced only a few IBC-like bacterial clusters at 6 h
after infection (n = 3 IBCs/5 mice). The UTI89-tetR/Ptet
fim intracellular bacterial clusters observed contained
bacteria which had not carried out the molecular switch
from rod to coccoid morphology (Fig. 7E–H). Further-
more, the rod-shaped UTI89-tetR/Ptet fim bacteria
remained loosely dispersed throughout the cell
(Fig. 7E–H) and stained poorly with FimH antibodies
(Fig. 7G). The lack of staining with anti-FimH antibodies
confirmed the repression and/or dilution of type 1 piliation of UTI89-tetR/Ptet fim in vivo subsequent to invasion
into the superficial umbrella cells. By 24 h, no intracellular clusters of bacteria were detected for the conditional fim strain. These results argue that type 1 pili are
necessary for the intracellular events in the IBC pathway
that lead to bacterial clustering into a tight biofilm-like
community of coccoid-shaped bacteria. The inability of
UTI89-tetR/Ptet fim to transition from rod to coccoid morphology, as typically seen for UTI89 (Justice et al., 2004;
2006) during these time points, suggested that type 1
piliation may be related to a developmental program
necessary for IBC maturation and pathway progression.
Discussion
Uropathogenic E. coli use adhesive pili, assembled by the
chaperone/usher pathway, to mediate complex host–
pathogen interactions within the urinary tract (Wright and
Hultgren, 2006). Type 1 pili, an essential virulence determinant in the pathogenesis of cystitis (Hultgren et al.,
1985; Langermann et al., 1997; Bahrani-Mougeot et al.,
2002; Hung et al., 2002; Snyder et al., 2004; 2006), bind
urothelium via the pilus adhesin, FimH (Abraham et al.,
Fig. 6. IBC enumeration by confocal
microscopy. UTI89-tetR or UTI89-tetR/Ptet fim
transurethrally infected bladders from
C3H/HeN female mice were harvested at 6 h
post infection, processed as described in
Experimental procedures, and IBCs
enumerated by laser scanning confocal
microscopy. UTI89-tetR/Ptet fim produced
either no IBCs or significantly fewer IBCs per
bladder (Range = 0–2 IBCs per bladder; N = 4
mice). Figure 6 is a representative experiment
from at least three independent experiments.
The conditional fim strain, UTI89-tetR/Ptet fim,
is significantly deficient in forming IBCs
(P = 0.0496; Mann–Whitney U-test).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2230–2241
2236 K. J. Wright, P. C. Seed and S. J. Hultgren
Fig. 7. Type 1 pili are necessary for IBC pathway initiation and IBC maturation. UTI89-tetR or UTI89-tetR/Ptet fim transurethrally infected
bladders from C3H/HeN female mice were harvested at 6 h post infection, bisected, splayed, fixed and stained with TO-PRO-3 iodide (red; A,
E) or blocked, sectioned, dually immunostained with E. coli (green; B, F) and FimH (red; C, G) antibodies, and imaged by laser scanning
confocal microscopy. Panels (D) and (H) are the merged images. Solid arrow indicates nuclei (A, B); open arrow indicates infiltrating
neutrophils (A); scale bar is equal to 10 mm. White dashed line delineates outline of the superficial umbrella cell. UTI89-tetR forms compact,
dense IBCs containing coccoid, FimH-positive bacteria whereas UTI89-tetR/Ptet fim bacteria are rod-shaped, dispersed throughout the cell, and
stain poorly for FimH.
1988). FimH–uroplakin interactions stimulate invasion
into the intracellular milieu of superficial umbrella cells
ultimately activating a complex genetic pathway which
leads to the formation of IBCs that undergo a defined
maturation and differentiation program (Anderson et al.,
2003; Justice et al., 2004). Bacteria within the IBC find a
safe haven where they are protected from clearance by
antibiotic treatment and innate host defences such as
polymorphonuclear cell attack (Justice et al., 2004). In this
study, we discovered type 1 pili to have an unexpected
function necessary for intracellular fitness and IBC initiation and maturation. These data provide the first evidence
of an adhesive pilus functioning beyond the historically
defined and accepted role in extracellular receptor binding
and bacterial colonization.
The intracellular function of type 1 pili following FimHmediated invasion was directly investigated using a conditional fim expression strain, UTI89-tetR/Ptet fim, in a
well-characterized murine model of cystitis (Mulvey et al.,
1998). Striking IBC defects were observed in the absence
of type 1 piliation following in vivo urothelium invasion,
most notable of which was the severe attenuation of IBC
formation and persistence in the bladder. UTI89-tetR/Ptet
fim was unable to form IBCs suggesting that type 1 pili are
critical for intracellular functions necessary for IBC formation subsequent to invasion. Intracellular collections of the
conditional fim strain identified by microscopy revealed
that the UPEC remained diffusely distributed throughout
the superficial umbrella cells and did not immunostain
with anti-FimH antibodies indicating the absence of type 1
pili. Additionally, these characteristics were accompanied
by the observation that UTI89-tetR/Ptet fim failed to transition from a rod to coccoid morphology, a hallmark of
UPEC within IBCs at this point in the IBC developmental
pathway (Justice et al., 2004). Thus, we have discovered
that type 1 pili may act as an intracellular aggregative
factor during IBC initiation and maturation. Bacterial interactions mediated by type 1 pili in the biofilm matrix may
serve as a developmental cue necessary for the bacteria
to undergo morphological changes key for communal
intracellular growth within IBCs.
In a murine model of UTI, the cyclic nature of the IBC
pathway is thought to contribute greatly to the severity of
acute cystitis. During the last stages of the IBC pathway,
UPEC flux out of the superficial umbrella cells to colonize and invade neighbouring cells. Thus, if type 1 pili
are important during events subsequent to fluxing, the
loss of type 1 piliation following initial invasion events
would be predicted to severely attenuate the ability of
UPEC to persist in the bladder. Relative to UTI89-tetR,
UTI89-tetR/Ptet fim bladder colonization levels rapidly
decreased ~225-fold and ~15 000-fold by six and 48 h
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2230–2241
Type 1 pili-mediated IBC development during acute cystitis 2237
after infection, respectively. However, unlike mutant fim
variants of UTI89, UTI89-tetR/Ptet fim was recovered
from the bladder at 2 weeks after infection, a result
which may suggest that the ability to carry out a FimHmediated early invasion event may provide a survival
advantage over bacteria limited to strictly extracellular,
luminal environments, even in the absence of IBC formation. In support of this hypothesis are the data that
demonstrate that UTI89 DfimH is unable to significantly
colonize extracellular niches over time. UTI89 DfimH
extracellular colonization levels ranged from 0 to 102
colony-forming units per bladder and were sterile by 6
and 48 h after infection (data not shown), respectively,
whereas wt UTI89 levels ranged from 105 to 106 by 6
and 48 h after infection, respectively (data not shown).
Taken together, these data argue that type 1 piliation is
necessary for the aggregation of the bacteria into the
IBC biofilm-like mass. The inability of UPEC to carry out
these functions greatly attenuated virulence. Thus, we
propose that the intracellular functions of type 1 pili in
the IBC pathway are important for the establishment and
severity of acute and persistent cystitis. An intriguing
possibility is that UPEC type 1 pili may function during
events immediately after invasion to facilitate biofilm formation in the cytoplasm of the superficial umbrella cells.
The UPEC biofilms facilitated by type 1 pili presumably
make them less susceptible to innate host defences and
antibiotics.
Among E. coli and UPEC isolates in particular, type 1 pili
are phylogenetically conserved with only minor variations
occurring in the adhesin, FimH (Hung et al., 2002). In this
study, we did not delineate differential function of the pilus
rod versus the FimH adhesin as the entire type 1 pilus fibre
was genetically repressed in all of our mutant constructs. It
is possible that the pilus rod either functions to present the
adhesin beyond the polysaccharide capsule for receptor
interactions (Schembri et al., 2004) and/or that the pilus
rod functions in adhesin-rod, rod-rod, or other rod–
hydrophobic interactions. Studies dissecting the individual
contributions of the pilus rod are currently underway to
address these questions. Also unknown at this time is the
source of and the molecular nature of the matrix within the
in vivo IBC. fim mutants have been shown to be unable to
produce biofilms in vitro (Pratt and Kolter, 1998; Wolfe
et al., 2003). Those studies in combination with the present
study support the hypothesis that type 1 pili are important
in both in vitro and in vivo biofilm formation. In support of
this hypothesis, we showed using a previously described
biofilm assay (Pratt and Kolter, 1998), that UTI89 Dfim,
UTI89 DfimH and UTI89-tetR/Ptet fim in the absence of AHT
failed to form biofilms whereas UTI89 and UTI89-tetR/Ptet
fim treated with AHT formed robust biofilms (data not
shown). Additionally, the inclusion of a-methyl-Dmannopyranoside at the time of inoculation or by addition
to existing 48 h biofilms inhibited formation of (Pratt and
Kolter, 1998; and data not shown) and disaggregated
biofilms, respectively (data not shown). Thus, in vitro biofilms are likely supported by a mannosylated receptor of
bacterial origin. Ongoing studies are currently in process to
identify the in vivo intracellular receptors responsible for
partnering with type 1 pili to aggregate UPEC within the
IBC. Furthermore, we suggest that UPEC unable to form
IBCs would be theoretically more susceptible to innate host
defenses such as infiltrating neutrophils (Schilling et al.,
2001; Justice et al., 2004) and antibiotic treatment (Fux
et al., 2005) because bacteria within biofilms exhibit
greater antibiotic resistance (Costerton et al., 1995;
Stewart and Costerton, 2001).
In conclusion, we have discovered an unexpected role
for type 1 pili in UTI separate from their known traditional
functions of mediating extracellular binding and invasion.
We discovered that type 1 pili have critical intracellular
functions. When type 1 pili are not produced intracellularly,
UPEC fail to transition from rod to coccoid morphology and
fail to form IBCs. These defects were associated with a
severe attenuation of virulence. Thus, our observations
emphasize the importance of previous studies in developing type 1 pili as a therapeutic target (Langermann et al.,
1997; 2000; Langermann and Ballou, 2001; Svensson
et al., 2001; Ohlsson et al., 2002). Finally, this novel role
may represent a paradigm for other pathogenic organisms
such as Pseudomonas aeruginosa which produce pili and
have been observed to form IBC-like communities within
tracheal epithelial cells (Oh et al., 2005).
Experimental procedures
Reagents
Difco® microbiological media was purchased from BectonDickinson (Sparks, MD). TO-PRO®-3 iodide, Alexa Fluor®conjugated antibodies and ProLong® Antifade were purchased
from Molecular Probes (Eugene, OR). All other chemicals, antibiotics and reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO). Restriction endonucleases were
purchased from Invitrogen (Carlsbad, CA).
Bacterial strains and culture conditions
The strains used in this study are shown in Table 1. The prototypic cystitis strain, UTI89, was obtained from an adult patient
with cystitis and has been previously described (Mulvey et al.,
2001). Bacterial strains were grown using standard techniques.
For all in vitro and in vivo studies with UTI89, UTI89 Dfim and
UTI89 DfimH, overnight (typically 16 h), aerated cultures were
diluted 1:250 into fresh Luria–Bertani (LB) broth and grown statically at 37°C for 20–24 h to induce type 1 piliation. For all in vitro
and in vivo studies with UTI89-tetR and UTI89-tetR/Ptet fim, overnight (typically 16 h), aerated cultures were diluted 1:250 into
fresh LB broth, grown to mid-log phase, induced with AHT
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2230–2241
2238 K. J. Wright, P. C. Seed and S. J. Hultgren
(1 mg ml-1), and grown statically at 37°C for two, serial 20–24 h
periods. Before all experiments, type 1 pili production was
confirmed by mannose-sensitive guinea pig erythrocyte
agglutination.
UTI89 fim mutant construction
UTI89 DfimH was constructed according to the method of Datsenko and Wanner (Datsenko and Wanner, 2000) using primers
FimHKO1 and FimHKO2 (Supplementary material Table S1)
and the template pKD13 (Datsenko and Wanner, 2000) to delete
the fimH gene in MG1655 (Schwan et al., 1992). Gene deletion
was confirmed with the fimH-flanking primers FimH1 and FimH2
(Supplementary material Table S1). UTI89 was transduced with
a P1 lysate derived from MG1655 DfimH, followed by excision of
the kanamycin cassette by introduction of the Flp recombinaseexpressing vector pCP20 (Datsenko and Wanner, 2000). UTI89
Dfim was constructed according to the method of Murphy and
Campellone (Murphy and Campellone, 2003) using primers
FimAKO1 and FimAKO2 and the template pKD4 (Datsenko and
Wanner, 2000). Gene deletion was confirmed with the fimAflanking primers FimA1 and FimA2 (Supplementary material
Table S1), followed by excision of the kanamycin cassette as
described for UTI89 DfimH (Datsenko and Wanner, 2000). Complete or partial in-frame deletion of fimA, as described above,
produced phase locked off mutant strains; thus, UTI89 DfimA
was used as a fim null strain (UTI89 Dfim). fim promoter phase
orientation was determined by PCR analysis as previously
described (Schwan et al., 1992). Unlike UTI89 Dfim and previous studies which suggested that FimH is required for the initiation of pilus assembly (Dodson et al., 1993; Saulino et al.,
1998; 2000), UTI89 DfimH produces fim encoded rods lacking
an adhesin (type1+/FimH–) as determined by FimH immunoblot
analysis, electron microscopy and N-terminal sequencing of the
major pilin subunit derived from UTI89 DfimH pili preparations
(data not shown).
tively, by electroporation to create the complemented UTI89 Dfim/
pfim and UTI89 DfimH/pfimH strains.
UTI89-tetR and conditional fim strain,
UTI89-tetR/Ptet fim, construction
A TetR-producing, isogenic derivative of UTI89 was derived as
follows. UTI89 was transduced with a P1 lysate of DH5aPRO
(Clontech) and selected on LB/spectinomycin plates (50 mg ml-1)
to create UTI89-tetR. UTI89-tetR/Ptet fim was constructed by
replacing the invertible promoter region of the type 1 pili operon
with the tetracycline promoter and operator sequences (tet O/P)
(Lutz and Bujard, 1997) as follows. First, a knockout/replacement
template was constructed. The tet O/P region was PCR-amplified
(Accuprime™ polymerase; Invitrogen) from pPROtetE.1 (Clontech) using tet O/P primers 1 and 2. An FRT-flanked kanamycin
resistance cassette was amplified from pKD4 (Datsenko and
Wanner, 2000) using the primers pKD4 1 and 2. Each reaction
was processed with the Qiagen PCR Cleanup kit to remove the
primers. A portion of each cleaned reaction was combined in a
third amplification mixture and the primers KD4 1 and tetO/P 2.
This reaction produced a linear knockout/replacement product
(KD4-tet O/P) with the kanamycin resistance gene and tet O/P in
tandem. Using KD4-tet O/P as a template, the type 1 pili
promoter-specific linear knockout product was amplified with the
primers Fim-KD4 1 and 2, yielding a product with ~50 bp flanking
regions homologous to the target sequences flanking the type 1
(fim) promoter region. The linear knockout product was introduced into UTI89-tetR/pKM208 expressing the red recombinase
machinery as previously published (Datsenko and Wanner, 2000;
Murphy and Campellone, 2003). Mutants were selected on
LB/kanamycin (50 mg ml-1), and the promoter replacement confirmed by PCR with the flanking primers PHASE 1 and fim 16.
The kanamycin cassette was removed by Flp recombinase
expressed from pCP20 as described earlier (Datsenko and
Wanner, 2000).
Haemagglutination assay for type 1 piliation
pfim and pfimH construction
pfim, a natively regulated fim operon complementation plasmid,
was constructed as follows. fimBEAICDFGH was amplified from
UTI89 genomic DNA (Accuprime™ polymerase; Invitrogen) using
primers FimOP1 and FimOP2 (Supplementary material
Table S1), ligated into pCR®-Blunt II-TOPO® (Clontech) as per
the manufacturer’s instructions, and confirmed by restriction
endonuclease digestion and DNA sequencing. pfimH, an IPTGinducible fimH complementation plasmid was constructed as
follows. fimH was PCR-amplified from pfim using primers
R1fimH5′-and BamHI fimH3′, restricted with EcoRI and BamHI,
and inserted by ligation into the polylinker of similarly restricted
pTRYC. pTRYC is a chimeric vector plasmid containing the LacIq
and polylinker sequences from pTRC99A (Amann et al., 1988)
and the chloramphenicol resistance gene and origin region from
pACYC184 (New England Biolabs). During amplification for construction of pTRYC, the EcoRI recognition sequence within the
chloramphenicol resistance gene was disrupted by introducing a
synonomous coding mutation to retain the coded amino acid
identity but disrupt the EcoRI recognition site, hence making the
EcoRI in the polylinker a unique site. pfim and pfimH were individually transformed into UTI89 Dfim and UTI89 DfimH, respec-
Strains were grown statically at 37°C described above to induce
type 1 pili production. Haemagglutination assays with guinea pig
erythrocytes (Colorado Veterinary Products) were performed following published protocols (Hultgren et al., 1986). Results represent three independent experiments.
Western blot
For immunoblot analysis of bacterial strains, gels were prepared
as previously described (Wright et al., 2005). Results represent at
least three independent experiments.
Electron microscopy
Strains were cultured as described in the ‘bacterial strains and
culture conditions’ section and prepared for EM as previously
described (Wright et al., 2005).
Mouse infections
The murine cystitis model has been described in detail (Mulvey
et al., 1998). Briefly, 7- to 8-week-old, wild-type C3H/HeN female
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 2230–2241
Type 1 pili-mediated IBC development during acute cystitis 2239
mice (in experimental groups of 4–6 mice) were obtained from
Harlan Sprague Dawley. Bacterial strains were pelleted by centrifugation at ~3000 g for 15 min and resuspended in sterile
phosphate-buffered saline (PBS) to a concentration of
~2 ¥ 108 cfu ml-1. Mice were anaesthetized by inhalation of isofluorane and infected via transurethral catheterization with 50 ml of
the bacterial suspension (~1–2 ¥ 107 cfu). At the indicated times
post infection, mice were sacrificed by cervical dislocation under
anaesthesia, and the bladders immediately aseptically harvested
and either processed for microscopy or bacterial titre determination as described below. Animal studies were approved and performed in accordance with Committee for Animal Studies at
Washington University School of Medicine.
Ex vivo IBC enumeration and imaging
For ex vivo enumeration of IBCs, infected bladders were harvested at the indicated times, bisected, splayed by pinning under
sterile PBS, gently washed with PBS, fixed with 3% paraformaldehyde (PFA; EM grade; EMS)/PBS for 45–60 min at room temperature protected from light, washed with PBS, and LacZ
stained (Justice et al., 2006) or stained with TO-PRO®-3 iodide to
image by laser scanning confocal microscopy. Whole mount IBC
immunofluorescence analysis was performed adapting previously described procedures (Mysorekar et al., 2002) with the
following modifications. Bisected, splayed and fixed bladders
were incubated with blocking buffer (PBS, containing 0.3% Triton
X-100 and 1% bovine serum albumin), sequentially stained with
primary E. coli (MedImmune) and FimH (MedImmune) antibodies
and secondary Alexa Fluor® 633-conjugated rabbit and Alexa
Fluor® 594-conjugated mouse antibodies, respectively, and
imaged by confocal microscopy as described below.
Confocal microscopy
Prepared bladders were mounted in a large drop of antifade
(Prolong®; Molecular Probes) and a coverslip placed on top.
Microscopy was performed on a Zeiss LSM 510 Meta Laser
Scanning inverted confocal microscope (Thornwood, NY) using a
63 ¥ oil immersion objective. Images were acquired using accompanying Zeiss software. TO-PRO®-3 Iodide and Alexa Fluor®
633-rabbit IgG were imaged at 633 nm excitation with emission
filters collecting between wavelengths 650–690 nm. Alexa Fluor®
594-mouse IgG was imaged at 543 nm excitation/585 nm long
pass filter emission.
Immunohistochemistry
Bladders from UTI89 or UTI89-tetR mice were prepared for
immunohistochemical analysis as previously described
(Mysorekar et al., 2002). Briefly, bladders were harvested 6 h
after inoculation, formalin fixed, embedded in paraffin, and serial
5-nm thick sections were prepared. Slides were deparaffinized
with serial washes in xylene and isopropyl alcohol, rinsed, and
rehydrated in PBS. Slides were then blocked (1% bovine serum
albumin, 0.3% Triton X-100 in PBS) and stained with a mouse
monoclonal antibody (mAb) to uroplakin III (Research Diagnostics) and rabbit antibodies to purified E. coli FimH adhesin
(Langermann et al., 1997). Antigen-antibody complexes were
visualized with Alexa Fluor® 594-conjugated donkey anti-mouse
Ig or Alexa Fluor® 488 conjugated-donkey anti-rabbit Ig (Jackson
ImmunoResearch). Nuclei were visualized using Hoescht dye.
Tissue bacterial titre determinations
To enumerate bacteria present, bladders and kidneys were aseptically harvested at the indicated times post infection, homogenized in PBS containing 0.025% Triton X-100, serially diluted,
and plated onto LB agar plates. Colony-forming units were enumerated after 24 h growth at 37°C. Luminal and intracellular
bacteria were enumerated using a gentamicin protection assay
as previously described (Justice et al., 2006).
Statistical analysis
Values of cfu per bladder were analysed for significance using the
non-parametric, Mann–Whitney U-test (InStat®; GraphPad software) to compare bladder colonization levels between wt UTI89,
UTI89-tetR and the indicated fim mutants.
Acknowledgements
We are grateful to Karen Dodson, Molly Ingersoll and Kim Kline
for critical review of this manuscript and Indira Mysorekar for her
technical expertise with the immunofluorescence analysis. EM
and confocal analyses were carried out in the Washington University Molecular Microbiology Imaging Facility. We are also
grateful for the microscopy expertise of Wandy Beatty, Ph.D and
Darcy Gill, Ph.D. This work was supported by NIH Grants
R01DK51406 and ORWH SCOR P50DK64540 (with the FDA) (to
S.J.H) and NIH Grant K12HD00850 (to P.C.S.). K.J.W. received
support as a Lucille P. Markey Special Emphasis Pathways
Fellow.
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Supplementary material
The following supplementary material is available for this article
online:
Table S1. PCR primers used in this study.
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/abs/10.1111/j.1462-5822.
2007.00952.x
Please note: Blackwell Publishing are not responsible for the
content or functionality of any supplementary materials supplied
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be directed to the corresponding author for the article.
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