IAI Accepts, published online ahead of print on 29 December 2014 Infect. Immun. doi:10.1128/IAI.02232-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
New aspects of RpoE in Uropathogenic Proteus mirabilis
2
Ming-Che Liu,a Kuan-Ting Kuo,b# Hsiung-Fei Chien,c# Yi-Lin Tsai,a Shwu-Jen
3
Liawa,d*
4
Department and Graduate Institute of Clinical Laboratory Sciences and Medical
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Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan,
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Republic of Chinaa; Department of Pathology, National Taiwan University Hospital,
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Taipei, Taiwan, Republic of Chinab; Department of Surgery, National Taiwan
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University Hospital, Taipei, Taiwan, Republic of Chinac; Department of Laboratory
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Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of Chinad
10
Keywords: RpoE, uropathogenic, Proteus mirabilis
11
Running title: RpoE in uropathogenic Proteus mirabilis
12
*Corresponding author. Mailing address: Department and Graduate Institute of
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Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine,
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National Taiwan University, 10016, No. 1, Chang-Te Street, Taipei, Taiwan, R.O.C.
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Phone: +886-02-23123456 ext 66911. Fax: +886-02-23711574.
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E-mail:
[email protected]
17
#
These authors contributed equally to this work.
18
1
19
ABSTRACT
20
Proteus mirabilis is a common human pathogen causing recurrent or persistent
21
urinary tract infections (UTIs). The underlying mechanisms for P. mirabilis to
22
establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma
23
factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria
24
expression, survival in macrophages, cell invasion and colonization in mice but
25
increased IL-8 expression of urothelial cells and swarming motility. This is the first
26
study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulating
27
mrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria
28
promoter. By real-time RT-PCR, we found the IL-8 mRNA amount of urothelial cells
29
was induced significantly by lipopolysaccharides extracted from rpoE mutant but not
30
the wild-type. These RpoE-associated virulence factors should be coordinately
31
expressed to enhance the fitness of P. mirabilis in the host including avoidance of
32
immune attacks. Accordingly, rpoE mutant-infected mice displayed more immune
33
cell infiltration in bladders and kidneys during early stages of infection and rpoE
34
mutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy
35
that urea (the major component in urine) and polymyxin B (a cationic antimicrobial
36
peptide) can induce expression of rpoE by the reporter assay, suggesting that RpoE
37
might be activated in the urinary tract. Altogether, our results indicate that RpoE is
38
important in sensing environmental cues of the urinary tract and subsequently
39
triggering expression of virulence factors, associated with the fitness of P. mirabilis,
40
to build up a UTI.
41
INTRODUCTION
42
Proteus mirabilis, a common uropathogen, is an infectious agent of pyelonephritis
43
and catheter-associated urinary tract infections (UTIs) (1, 2). P. mirabilis has many
44
virulence factors that contribute to UTIs (1-3). These factors include 2
45
fimbriae-mediated adherence to host urothelial cells and the catheters (2, 4), flagella
46
mediated motility (swarming and swimming) (1, 5), hemolysin (6), invasion of host
47
tissues and immune evasion (1, 7). Flagella-dependent swarm cell formation
48
contributes to establishing infections by migrating along the catheter (5). Haemolysin
49
is also thought to facilitate bacterial spread within the kidney and development of
50
pyelonephritis by damaging host tissues (6). Bacteria must successfully evade
51
immune responses to persist within the host. P. mirabilis uses several strategies to
52
avoid immune attacks in the urinary tract. One is to vary the antigenic structures, such
53
as flagellin by flagellar gene rearrangement (8), and fimbriae by fimbrial gene
54
diversity or phase variation to prevent antibody recognition (1, 3, 9). Other
55
immunoavoidance factors for P. mirabilis include capsules (2), IgA proteases (ZapA)
56
(10), and lipopolysaccharides (LPS) (1, 2). Capsules are effective at hiding many
57
bacterial surfaces and preventing opsonization (2). P. mirabilis is an antigenically
58
heterogeneous species due to structural differences of LPS (2). Modified LPS
59
promotes bacterial survival by increasing resistance to cationic antimicrobial peptides
60
and by altering host recognition by Toll-like receptors (TLRs) (11). Moreover,
61
capability of invading urothelial cells to survive intracellularly probably represents
62
another mechanism for immune evasion and persistence (1, 3, 7).
63
Many studies have reported that the presence of mannose-resistant Proteus-like
64
(MR/P) fimbriae of P. mirabilis is important in UTIs (12, 13). MR/P fimbriae
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facilitate colonization of the urinary tract and deficiency of the MR/P fimbriae
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decreased bacterial loads in the mouse model of UTIs (14, 15). The mrp gene cluster
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contains two transcripts, mrpABCDEFGHJ (designated as the mrp operon) and mrpI
68
(12). The promoter for the mrp operon, which contains all the genes required for
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MR/P fimbrial biogenesis, resides on a 251 bp invertible element (IE) (12). The gene
70
mrpI, transcribed divergently from the mrp operon and independent of the mrp 3
71
promoter, encodes a recombinase capable of switching the IE from either ON to
72
OFF or OFF to ON to control MR/P fimbria expression (12).
73
Less is known about the host response to uropathogenic P. mirabilis but
74
aspects of the host defense might be similar to uropathogenic E. coli (UPEC) (1-3).
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Urothelial cells secreted soluble mediators such as soluble IgA, lactoferrin, and
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bactericidal antimicrobial peptides to inhibit attachment of UPEC (16). Microbes
77
that overwhelm these early defenses contact urothelia and activate an innate
78
inflammatory response through TLRs (17). The inflammatory response consists of
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three principal steps: (a) urothelial cell activation and production of distinct
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inflammatory cytokines; (b) immune cell recruitment to the infectious site; (c)
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local destruction and elimination of the invading bacteria (16, 18).
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The bacterial envelope maintains cell homeostasis and is the site for crucial
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processes, such as metabolic energy transduction, transport of nutrients and wastes,
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signal transduction and cell-cell communication (19). RpoE, an alternative sigma
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factor, is essential for the maintenance of cell envelope integrity in Gram-negative
86
bacteria (20-22). In this regard, rpoE gene is important in pathogenesis and stress
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survival in many Gram-negative bacteria (20-22), but the function of RpoE in
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uropathogenic P. mirabilis is still not known. RseA, belonging to the rpoE operon,
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is an anti-RpoE factor under non-stressed conditions. The release of RpoE from
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RseA binding allows it to combine core RNA polymerase to transcribe
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RpoE-dependent genes (20). In this study, we characterized the roles of P.
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mirabilis RpoE in virulence by in vitro and in vivo assays. This is the first report
93
to show that P. mirabilis RpoE affected multiple traits including swarming
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motility, hemolysin activity, bacteria-mediated cytotoxicity, fimbria production,
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survival in macrophage, invasion ability, induction of cytokine expression and
96
colonization in mice. It is worth noting that P. mirabilis RpoE could not only 4
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regulate expression of MR/P fimbriae through mrpI but also modulate host immune
98
responses. We noticed that fimbria expression, survival in macrophages, invasion
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ability and colonization in mice were decreased in the rpoE mutant and induction of
100
IL-8 by rpoE mutant was higher relative to the wild-type. In addition, we found that
101
RpoE was activated by urea, a component in urine. Altogether, RpoE of P. mirabilis
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could play important roles in establishing UTIs via modulating the appropriate
103
production of virulence factors, including those associated with host immune
104
responses. To our knowledge, this is the first report describing the roles of RpoE in P.
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mirabilis.
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MATERIALS AND METHODS
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Bacterial strains, plasmids, and growth conditions. The bacterial strains and
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plasmids used in this study are listed in Table 1. Bacteria were routinely cultured at 37
109
°C in Luria-Bertani (LB) medium. The LSW- agar was used to prevent the swarming
110
motility when needed (23).
111
Construction of mutants and complementation. Sequences flanking the rpoE
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gene were amplified by PCR using the primer pairs rpoE-upF/rpoE-upR and
113
rpoE-downF/rpoE-downR (Table S1), respectively and cloned into pGEM-T Easy
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(Promega) to generate prpoEKO-up and prpoEKO-dn. The prpoEKO-up was digested
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with SalI/XbaI and the rpoE upstream sequence containing fragment was ligated to
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the SalI/XbaI-digested prpoEKO-dn to produce the prpoEKOupdn plasmid, which
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contains both upstream and downstream sequences of rpoE. A Kmr-cassette was
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inserted in the XbaI-digested prpoEKOupdn plasmid to generate the
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prpoEKOupdnKm, a plasmid containing the Kmr-cassette-disrupted combined
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upstream and downstream sequence of rpoE. The fragment containing the
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Kmr-cassette-disrupted combined upstream and downstream sequence of rpoE was
122
cleaved by SalI/SphI from prpoEKOupdnKm and ligated into SalI/SphI-cleaved 5
123
pUT/mini-Tn5(Km) to generate pUTrpoEKO. Gene inactivation mutagenesis by
124
homologous recombination and confirmation of mutants with double-crossover
125
events were performed as described previously (23). rseA mutant was obtained in
126
the same way using the primer pairs rseA-upF/rseA-upR and
127
rseA-downF/rseA-downR.
128
For complementation of rpoE mutant, the fragments containing full-length
129
rpoE and rseA were amplified by PCR using primer pairs rpoEcom-F/rpoEcom-R
130
and rseAcom-F/rseAcom-R and cloned into pGEM-T Easy (Promega),
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respectively to generate plasmids, prpoE-com and prseA-com. The prpoE-com
132
and prseA-com were transformed into the rpoE and rseA knockout mutants,
133
respectively to generate the RpoE-complemented and RseA-complemented
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strains.
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Reporter assay. The rpoE-xylE reporter plasmid (24)-transformed wild-type
136
and mutants were grown overnight in LB broth with 20 g/ml chloramphenicol,
137
diluted 100-fold in the same medium, and then the XylE activity was monitored at
138
3 h, 5 h, 7 h and overnight after incubation. For testing signals for rpoE expression,
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wild-type and mutants containing the rpoE-xylE reporter plasmid were grown
140
overnight in LB broth, diluted 100-fold, then cultured to OD600 of 0.5 at 37 °C and
141
exposed to 20 μg/ml polymyxin B (PB) or 0.5 M urea for 2 h. The XylE activity
142
was then measured as described previously (24).
143
Swarming assay. The swarming migration assays was performed as described
144
previously (24, 25). Briefly, overnight bacterial cultures (5 l) were inoculated
145
centrally onto the surface of dry LB swarming plates containing 2 % (w/v) agar,
146
which were then incubated at 37 °C. The swarming migration distance was
147
measured by monitoring the swarm fronts of the bacterial cells at 1 h intervals.
148
Measurement of the hemolysin activity. The overnight bacterial cultures 6
149
(120 μl) were inoculated onto the surface of dry LB swarming plates, which were then
150
incubated at 37 °C. The hemolysin activity was determined at 5 h and 7 h after
151
inoculation as described previously (24, 25).
152
Cytotoxicity assay. Cells (5 x 103 cells/well) of the human urothelial cell line
153
NTUB1, which was originally derived from a urinary bladder carcinoma (23), were
154
cultured in RPMI 1640 supplemented with 10 % FCS under 5.0 % CO2 for two days
155
using 96-well culture plates. Overnight bacterial cultures (120 μl) were inoculated
156
onto the surface of dry LB swarming plates and bacteria were collected and washed
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by PBS at 5 h after incubation at 37 °C. The bacterial number were adjusted to 106
158
CFU/ml by RPMI1640, 200 μl of P. mirabilis strains were applied to each well at a
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multiplicity of infection (MOI) of 20 and the plates were incubated at 37 °C for 2 h.
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The culture supernatants were collected and the lactate dehydrogenase (LDH) release
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was quantified using the CytoTox 96 nonradioactive cytotoxicity kit (Promega)
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according to the manufacturer’s instructions. Cytotoxicity was determined by
163
calculating the percentage of LDH release relative to the maximum LDH release from
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uninfected NTUB1 lysed with Triton X-100.
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The invertible element (IE) assay. The expression of P. mirabilis MR/P fimbriae,
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encoded by mrp operon, depends on the promoter orientation (9, 12). The IE ‘‘ON’’
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means the promoter direction is for mrp operon expression. Genomic DNA from
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overnight and exponential cultures (OD600 of 0.5) of various P. mirabilis strains were
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prepared for amplification of the IE element using mrpP1 and mrpP2 primers (Table
170
S1). The PCR product was digested with AflII and resolved on a 2 % agarose gel.
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Real-time RT-PCR. To study the effect of RpoE gene on the mRNA levels of
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mrpA, mrpJ, mrpI, flhDC and fliC1, total RNA of the wild-type, rpoE mutant, and
173
rseA mutant from overnight LB cultures and cells grown on agar plates for 5 h after
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seeding was extracted and real-time reverse transcription (RT)-PCR was performed as 7
175
described previously (24). The levels of mRNA were normalized against gyrB
176
mRNA.
177
Expression and purification of the MrpA-His6 fusion protein and preparation
178
of mouse antiserum against MrpA. The mouse polyclonal antiserum against MrpA
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was obtained as described previously (26). A 528-bp BamHI/XhoI fragment containing
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mrpA was cloned into the BamHI/XhoI sites of pET32a (Novagen) to construct
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pET-MrpA. MrpA was expressed as a fusion protein tagged with a His6 tail from
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pET-MrpA upon induction with IPTG (0.5 mM). The MrpA-His6 fusion protein was
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purified using Ni2+-NTA resin (Invitrogen) according to the protocol provided by the
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manufacturer. The purified MrpA-His6 fusion protein was desalted on a Ultracel-10K
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centrifugal filter device (Millipore) buffered with PBS. The protein sample was
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quantified by the Bio-Rad protein assay. The predicted 40-kDa MrpA-His6 protein was
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resolved on an SDS-12.5% polyacrylamide gel and subsequently excised from the gel.
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To raise antiserum against MrpA, the excised gel containing MrpA was emulsified in
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Freund's complete adjuvant and subcutaneously injected into BALB/c mouse (50 μg
190
protein/mouse). At 2 and 4 week after the primary immunization, each mouse was
191
given a booster injection of 50 μg protein emulsified in Freund's incomplete adjuvant.
192
Serum was collected 2 weeks after the final booster. The specificity of the antiserum
193
was established using P. mirabilis hfq mutant (whose mrp operon is off) as the
194
negative control and the correct size of MrpA predicted to be about 18 kD.
195
Western blot analysis. Overnight LB broth cultures (15 ml) of various P.
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mirabilis strains were washed and the pellets were collected and resuspended in
197
PBS (1 ml). Total proteins of bacteria were extracted by sonication, quantified by
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Bio-Rad protein assay and adjusted to the same concentration. The protein
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samples were denatured in the sample buffer (100℃, 5 min), electrophoresed on
200
an SDS- 12.5% polyacrylamide gel and then transferred to a Amersham 8
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HybondTM-P membrane (GE Healthcare). The blot was incubated with mouse
202
polyclonal antiserum against MrpA described above, followed by sheep anti-mouse
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immunoglobulin G horseradish peroxidase conjugate (GE Healthcare) and then
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developed using the enhanced chemiluminescent (ECL) detection reagents (Perkin
205
Elmer).
206
Macrophage infection assay. The assay was performed as described before (27),
207
with some modifications. Briefly, 12-well plates were seeded with 105 cells (per well)
208
of THP-1 cells and THP-1 cells were differentiated using 50 ng/ml phorbol myristate
209
acetate in RPMI 1640 with 10 % FCS under 5.0 % CO2 for 24 h. The overnight
210
cultures of P. mirabilis strains were applied to each well at an MOI of 10 (106
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CFU/well). Bacteria were brought in contact with macrophages by centrifugation and
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incubated for 30 min at 37 °C. After infection, the cells were washed with PBS and
213
incubated for 1 h in RPMI 1640 with 250 g/ml streptomycin to kill extracellular
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bacteria. Immediately, cells in some wells were lysed by 1 % Triton X-100 to
215
determine the CFU of intracellular bacterial cells at t0 ; others were incubated in
216
medium containing 250 g/ml streptomycin for additional 1 h and 4 h to obtain the
217
respective CFU.
218
Cell invasion assay. The wild-type P. mirabilis and its mutants were cultured in
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LB broth at 37 °C overnight before the cell invasion assay was performed according
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to the protocol of Jiang et al. (28). Briefly, human urothelial NTUB1 cells were grown
221
and then infected with a bacterial suspension containing 107 cells (MOI=10) for 1 h.
222
Urothelial cells were then washed and incubated at 37 °C in 1 ml of RPMI 1640
223
medium containing streptomycin for another 1 h. Cells were washed again with PBS
224
and then lysed by 1 ml of lysis solution. Cell lysates were diluted serially and viable
225
bacteria were counted by plating on LSW- agar plates. Cell invasion ability was
226
determined as the percentage of viable bacteria that survived the streptomycin 9
227 228
treatment versus the total inoculum and relative invasion was expressed . Cytokine expression of NTUB1 cells. Determination of the relative levels of
229
selected human cytokines and chemokines was performed using the Human
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Cytokine Array Panel A (R&D Systems, USA) according to the manufacturer’s
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instructions. In brief, the NTUB1 cells were grown in the 12-well plate (106
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cells/well) and incubated with overnight bacterial cultures in RPMI 1640 (107
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cells/well) at an MOI of 10 for 3 h at 37 °C. The culture supernatant and NTUB1
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cells were subjected to analyses of the cytokine array and real-time RT-PCR,
235
respectively. A mixture of NTUB1 cell culture supernatant and the detection
236
antibody was added to the membrane spotted with capture antibodies. After
237
incubation, the membrane was washed and the chemiluminescence produced by
238
the sample/antibody hybrid on the membrane was detected after adding
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streptavidin-labeled horseradish peroxidase. NTUB1 cells were washed, total
240
RNA was extracted and real-time RT-PCR was performed as described (24, 29)
241
using primers listed in Table S1 to examine the mRNA expression of IL-8, MIF,
242
PAI-1 and CXCL1 against GAPDH mRNA. To study the effect of LPS on the
243
expression of IL-8 mRNA, LPS (0.1, 1 and 5 μg/ml) extracted from P. mirabilis
244
strains as described previously (24, 28) was applied to wells with NTUB1 cells,
245
the plates were incubated for 3 h and total RNA was isolated for real-time
246
RT-PCR.
247
Infection of mice. The C57BL/6 mouse model of UTIs was used as described
248
previously (29). Briefly, 6-week-old female mice were injected transurethrally
249
with a 50-μl overnight culture suspension of P. mirabilis strains at a dose of 107
250
CFU per mouse. On day 3 and day 6 after injection, mice were sacrificed, and
251
bladder and kidney samples were collected, weighed, suspended in 0.5 ml of PBS
252
and then homogenized to determine the viable bacterial count by plating on LSW10
253
agar plates. All animal experiments were performed in strict accordance to the
254
recommendation in the Guide for the Care and Use of Laboratory Animals of the
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National Laboratory Animal Center (Taiwan), and the protocol was approved by the
256
Institutional Animal Care and Use Committee of National Taiwan University College
257
of Medicine. All surgery was performed under anesthesia, and all efforts were made
258
to minimize suffering.
259
Hematoxylin and eosin staining. One and six days after transurethral inoculation
260
of PBS, wild-type, rseA mutant and rpoE mutant, respectively, C57BL/6 mice (as
261
described above) were sacrificed and for each mouse the bladder and the kidney cut
262
were collected, preserved in 10% formalin (pH 7.2), embedded in paraffin, sectioned,
263
stained with hematoxylin and eosin, and examined microscopically. The extent of
264
renal pathology was scored as follows (30): 0, no inflammation; 1, PMNs confined to
265
the peripelvic region; 2, PMN clusters detectable in the papilla or peripelvic cortex; 3,
266
widespread extension of PMNs into the cortex or outer medulla. The severity of
267
histological modifications of each bladder was scored as follows (30): 0, no
268
histological modifications; 1, occasional submucosal immune cell infiltrates; 2,
269
widespread submucosal immune cell infiltration with minimal spread to the
270
muscularis or epithelium; 3, widespread inflammation with dense perivascular cuffs,
271
transmural distribution, and intraepithelial inflammatory cells. The investigator was
272
blind to the identity of the bacterial strain.
273
Nucleotide sequence accession numbers. The nucleotide sequences of P.
274
mirabilis intergenic region between mrpA and mrpI and the region containing rpoE
275
and rseA genes have been deposited in GenBank under accession no. KJ814628 and
276
KJ814629, respectively.
277
RESULTS
278
Identification and characterization of P. mirabilis rpoE operon. To investigate the 11
279
roles of rpoE in P. mirabilis, we first searched in the released genome of P. mirabilis
280
HI4320 (accession no. AM942759) and found the rpoE operon of P. mirabilis, like
281
that of E. coli, consists of rpoE, rseA, rseB and rseC genes. Using primers annealing
282
to conserved sequences, we cloned and sequenced the fragment containing rpoE, rseA
283
and upstream of rpoE in P. mirabilis N2. The nucleotide sequences of rpoE and rseA
284
were found to be both 99% identical to those of P. mirabilis HI4320. The amino acid
285
sequences of RpoE and RseA were 87% and 57%, respectively, identical to UPEC
286
strain CFT073. Analysis of the upstream sequence of the rpoE operon revealed a
287
putative binding site for RpoE, 5'-AACttcattctattttaatgcTCtaA-3', based on the
288
conserved binding sequence (5'-AACtt-N16-TCnaA-3') (20). Capital and lower-case
289
letters stand for the relative frequency of each base at that position; the sequences in
290
more than 80% are in capital letters. Knowing RseA is an anti-RpoE factor and RpoE
291
is autoregulated (20), we hypothesized that mutation in rseA might cause RpoE
292
overexpression in P. mirabilis. Therefore, the rpoE and rseA mutants were
293
constructed and the reporter assay was performed in the wild-type, rseA mutant and
294
rpoE mutant containing the rpoE-xylE plasmid. As shown in Fig. 1, the promoter
295
activity of rpoE (XylE activity) was decreased in rpoE mutant but increased in rseA
296
mutant compared to wild-type at 3 h, 5 h, 7 h and overnight after incubation. XylE
297
activities of both wild-type and rseA mutant, but not rpoE mutant, exhibited the
298
growth phase-dependent pattern, being the highest at stationary phase. Thus, we
299
concluded RpoE was positively autoregulated and rseA mutant was an rpoE
300
overexpression strain of P. mirabilis.
301
P. mirabilis RpoE regulated swarming motility, hemolysin activity and
302
cytotoxicity to urothelial cells. The swarming phenomenon is a metabolically
303
complicated process, any factors that affect this phenomenon would likely affect
304
the fitness of the organism (1, 2). We thus tested the swarming behavior of 12
305
wild-type and mutants and found that the swarming distance was increased for rpoE
306
mutant but decreased for rseA mutant at 4 h, 5 h, 6 h, 7 h and 8 h after inoculation
307
onto the swarming plate (Fig. 2A). Both complemented strains (rseAc and rpoEc)
308
restored almost wild-type swarming. Previous studies have shown that multiple
309
virulence factors are coordinately expressed during P. mirabilis swarming migration
310
(31, 32). P. mirabilis hemolysin was a pore-forming cytotoxin, contributing to
311
damages of human renal cells (6). We then tested the hemolysin activity. As shown in
312
Fig. 2B, the hemolysin activity was decreased in the rseA mutant but increased in the
313
rpoE mutant, compared to wild-type after growing on swarming plates for 5 h. At 7 h
314
after inoculation, rpoE mutant exhibited significant hemolysin activity, contrasting to
315
other strains. Accordingly, we found rpoE mutant differentiated into more swarmer
316
cells at 7 h after incubation than other strains (data not shown). These data indicated
317
RpoE could inhibit swarming motility, swarm cell differentiation and
318
swarming-related hemolysin activity. Knowing P. mirabilis RpoE inhibited hemolysin
319
activity, we hypothesized that RpoE might influence cytotoxicity. To identify
320
bacteria-induced cytotoxicity, wild-type and mutants (rseA and rpoE) were
321
co-cultured with NTUB1 cells, respectively, and the LDH released was measured. As
322
shown in Fig. 2C, rpoE mutant and rseA mutant displayed higher and lower
323
cytotoxicity, respectively, relative to wild-type. Thus, mutation in rpoE increased
324
cytotoxicity of P. mirabilis.
325
Regulation of MR/P fimbria expression by RpoE and the effect of
326
RpoE-regulated mrpJ on expression of flhDC and fliC1. P. mirabilis MR/P
327
fimbriae have been shown to be important in UTIs (12-15). We investigated if RpoE
328
affects expression of MR/P fimbriae in DNA, mRNA and protein levels. The
329
invertible element (IE) assay using exponential phase and overnight LB broth cultures
330
was performed to determine the ON (fragments of 336 and 407 bp) or OFF (fragments 13
331
of 204 and 539 bp) position of the IE. The IE was located on an intergenic region
332
between mrpA and mrpI (Fig. 3A). Both ON and OFF orientations were found in
333
stationary phase cultures of wild-type, rseA mutant and rpoE mutant, with less
334
“ON” in rpoE mutant (Fig. 3B). Exponential phase cultures of all strains appeared
335
to favor the OFF position compared to the stationary phase and almost no “ON” in
336
rpoE mutant. Based on ImageJ software quantification, the relative proportions of
337
DNA in the ON phase of stationary wild-type, rseA mutant and rpoE mutant were
338
34%, 34% and 10.5%, respectively; while those of exponential phase cells were
339
0.5%, 3.5% and 0.06%, respectively. Real-time RT-PCR and Western blot
340
analysis were used to further confirm the results of the invertible element assay.
341
Accordingly, mrpA and mrpJ mRNA levels of rpoE mutant were lower but those
342
of rseA mutant were significantly higher than the wild-type (Fig. 3C). Fig. 3D
343
indicated MrpA protein level of rpoE mutant was also lower than wild-type and
344
rseA mutant. These results indicated RpoE regulated expression of mrp operon.
345
Therefore, we searched the consensus sequence of RpoE binding site in the
346
intergenic region between mrpA and mrpI and found the putative binding
347
sequence, 5'-AACttgtttaaaattaacgagTaaaA-3', only in the upstream of mrpI (Fig.
348
3A). It has been shown that switching to ON orientation of IE occurred to a
349
greater extent when expression of MrpI was induced by IPTG (9). To demonstrate
350
if the expression of mrpI gene was regulated by RpoE, which further influences
351
the ON/OFF of the mrp operon, we tested for the expression of mrpI in wild-type,
352
rseA mutant and rpoE mutant. As shown in Fig. 3C, the mRNA amount of mrpI
353
was decreased in rpoE mutant but increased in rseA mutant. In summary, the data
354
revealed P. mirabilis RpoE regulated mrpI expression and suggested RpoE
355
affected mrp expression through regulating mrpI. In addition, we found excess
356
MrpI does not contribute more to turn on mrp operon, based on the observation 14
357
that the ON percentage of wild-type and rseA mutant was almost the same (Fig. 3B
358
and 3C).
359
Given that P. mirabilis mrp operon is off in agar-grown cells (9, 12), MrpJ is a
360
repressor of flhDC (encoding the flagellar transcriptional regulator) (26, 33) and RpoE
361
regulated expression of the mrp operon (Fig. 3B and 3D), we compared the mRNA
362
expression of mrpJ, flhDC and fliC1 (encoding flagellin) in wild-type, rseA mutant
363
and rpoE mutant cultured in LB broth and on plates to clarify the effect of
364
rpoE-mediated mrpJ expression on expression flhDC and fliC1. We found mrpJ was
365
positively regulated by RpoE in broth cultures but alterations of RpoE had no effect
366
on expression of mrpJ in plate cultures, a phase-off condition for mrp (Fig. 3C and
367
3E). In addition, flhDC and fliC1mRNA levels were decreased in rseA mutant and
368
increased in rpoE mutant from both broth and plate cultures (Fig. 3C and 3E). In this
369
regard, we found rseA mutant displayed a significant reduction in swimming (Fig. S1)
370
and swarming (Fig. 2A) compared to the wild-type. These results indicate
371
RpoE-mediated repression of swarming and expression of flhDC or fliC1 in cells from
372
plate cultures is not MrpJ-dependent but MrpJ contributes to RpoE-mediated
373
reduction of swimming motility and fliC1 expression in those from broth cultures.
374
The role of P. mirabilis RpoE in survival in macrophages, invasion ability and
375
induction of cytokine expression. To understand if RpoE is involved in the innate
376
response of macrophages (34) to eliminate P. mirabilis, we challenged THP-1 cells
377
with wild-type and mutants, killed external bacteria with streptomycin and assessed the
378
survival of internalized P. mirabilis. We found there was no significant difference
379
between the intra-macrophage survival of wild type and mutant cells at the time
380
immediately after streptomycin treatment. However, CFU of intra-macrophage rpoE
381
mutant was reduced significantly after further incubation for 1 h and 4 h compared to
382
wild-type (Fig. 4A), indicating loss of rpoE impaired survival of P. mirabilis in
383
macrophages. The rseA mutant exhibited the survival pattern similar to the wild-type.
384 385
Given that UPEC evaded the host defense mechanism by invading into the epithelium (2, 18, 35) and P. mirabilis also can invade into urothelial cells (7, 28), we 15
386
then assess the role of RpoE in invasion into NTUB1 cells. As shown in Fig. 4B,
387
the rpoE mutant had a significantly lower ability of cell invasion than the
388
wild-type.
389
Production of cytokines and chemokines from epithelial cells initiates the
390
innate immune responses in UTIs and the immune cells like neutrophils would be
391
attracted to the infection site to eliminate the pathogens (16). Therefore, we next
392
examined the role of RpoE in inducing expression of cytokines and chemokines.
393
To investigate a variety of cytokines and chemokines produced by NTUB1 cells
394
after co-culturing with P. mirabilis wild-type or rpoE mutant, the Human
395
Cytokine Array was used. We found the spots of IL-8, CXCL1, MIF and PAI-1
396
appeared more intense from supernatants of NTUB1 cells treated with the rpoE
397
mutant (data not shown). To confirm the expression of IL-8, CXCL1, MIF and
398
PAI-1, real-time RT-PCR was performed using NTUB1 cells treated with various
399
P. mirabilis strains. Both wild-type and rpoE mutant can induce IL-8 mRNA,
400
significantly higher by rpoE mutant than by wild-type (Fig. 4C). rseA mutant
401
(RpoE overexpression)-treated cells expressed IL-8 mRNA similar to the
402
untreated control (Fig. 4C). In addition, rpoE mutant induced a significantly
403
higher level of MIF mRNA than wild-type, and rseA mutant inhibited mRNA
404
level of PAI-1 (a cytokine supporting IL-8-mediated neutrophil migration) relative
405
to wild-type (Fig. 4C). It has been known the innate immune response relies on
406
recognition of pathogen-associated molecular patterns (PAMPs) (17). LPS is a
407
prominent feature of Gram-negative bacteria, being one of the most potent PAMPs
408
known and responsible for the inflammatory response (17). Moreover, RpoE has
409
been known to regulate biogenesis and modification of LPS in Gram-negative
410
bacteria (20, 36), so we hypothesized that RpoE-regulated LPS might be one of
411
the bacterial components to affect IL-8 expression of NTUB1 cells. To this end, 16
412
we treated NTUB1 cells with LPS (0.1, 1 and 5 g/ml) extracted from wild type, rseA
413
mutant and rpoE mutant, respectively and IL-8 mRNA level was determined. As
414
shown in Fig. 4D, the IL-8 mRNA amount of NTUB1 cells was induced about 5 fold
415
by 5 μg/ml LPS from rpoE mutant only. Altogether, RpoE-mediated LPS changes
416
could explain the difference of IL-8 expression induced by wild-type P. mirabilis and
417
rpoE mutant.
418
Colonization of the urinary tract was attenuated in P. mirabilis rpoE mutant.
419
Knowing P. mirabilis RpoE controlled expression of diverse virulence factors
420
aforementioned, including swarming, hemolysin, fimbriae, invasion and modulating
421
the host immune responses, we established the C57BL/6 mouse model for UTIs (29)
422
to determine the effect of inactivation of either rpoE or rseA on the colonization
423
ability of P. mirabilis. As shown in Fig. 5, the rpoE mutant had a severely impaired
424
ability to colonize within the bladders and kidneys relative to the wild-type. The
425
colonization of the RpoE-complemented strain showed no significant difference from
426
the wild-type and rseA mutant did so except on day 3 from bladder samples.
427
Significant difference in the bacterial load was observed between the wild type and
428
rpoE mutant on day 3 and day 6 after transurethral inoculation. This result indicated
429
RpoE is required for colonization and survival of P. mirabilis in the mouse urinary
430
tract.
431
Histopathologic evaluation of mouse bladders and kidneys. Knowing loss of
432
rpoE led to higher mRNA levels of IL-8 and MIF (Fig. 4C), which could attract the
433
immune cells to confine the bacterial loads in vivo, we also evaluated the capacity of
434
wild-type and mutants to induce immune cell infiltration in mouse bladders and
435
kidneys. The histopathology of the bladder and kidneys was evaluated according to
436
the criteria described by Alamuri et al. (30) and was expressed as a semiquantitative
437
score. On day 1 post inoculation, light microscopic evaluation revealed a significant 17
438
increase of immune cell infiltrates in bladders of the wild-type or rpoE-treated
439
mice compared to PBS control, while that of rseA mutant was similar to PBS
440
control (Fig. 6A). Only kidneys of rpoE-treated mice exhibited widespread
441
extension of immune cells into the cortex or outer medulla, scored as 3, one day
442
after inoculation (Fig. 6B, Table 2). On day 6 post inoculation, colonization of the
443
bladders by wild-type or rseA mutant was accompanied by occasional submucosal
444
inflammatory cell infiltrates (scored as 1) and those of rpoE mutant showed
445
almost no sign of inflammatory cell infiltration (Fig. 6A, Table 2). Six days after
446
inoculation, kidneys of rseA or rpoE mutant-inoculated mice displayed almost no
447
sign of immune cell infiltration, in contrast to those of the wild-type with PMN
448
clusters detectable in the papilla or the peripelvic cortex (Fig. 6B, Table 2).
449
Altogether, rpoE mutant could recruit immune cells in the bladder and kidneys
450
during the early stage of infection (Fig. 6). The wild-type induced immune cell
451
infiltration in the bladder but not kidneys on day 1 after inoculation and both the
452
bladder and kidneys on day 6 (Fig. 6). Interestingly, rseA mutant could not recruit
453
immune cells in kidneys (Fig. 6B).
454
Urea and polymyxin B induced the promoter activity of rpoE gene in P.
455
mirabilis. Based on that P. mirabilis RpoE plays such an important role in
456
virulence factor expression and colonization in mouse, it was tempting to know
457
what the signals are to activate expression of RpoE in the urinary tract, an
458
environment containing antimicrobial peptides and a lot of urea. Therefore, the
459
XylE activities of rpoE-xylE reporter in the wild-type and mutants were
460
determined in the presence of urea or polymyxin B, a cationic antimicrobial
461
peptide. The promoter activity of rpoE gene was induced by 20 g/ml PB or 500
462
mM urea after incubation for 2 h in the wild-type but not in rseA and rpoE
463
mutants (Fig. 7). The results suggested that P. mirabilis RpoE could be activated 18
464
in the host urinary tract during infection.
465
DISCUSSION
466
RpoE is involved in virulence, surface stress responses and modulation of the
467
inflammatory response during infections (20-22, 37). RpoE of uropathogenic E. coli
468
plays roles in motility, biofilm formation and sensitivity to polymyxin B (38). In this
469
study, we demonstrated the role of RpoE in regulation of virulence in uropathogenic P.
470
mirabilis. Swarming motility, hemolysin activity and cytotoxicity were increased but
471
fimbria expression, cell invasion and survival in macrophages were decreased in rpoE
472
mutant (Fig. 2, 3 and 4). rpoE mutant induced higher mRNA levels of IL-8 and MIF
473
than wild-type but RpoE overexpression (rseA mutant) decreased mRNA levels of
474
IL-8 and PAI-1 (Fig. 4C). Moreover, rpoE mutant had a significantly impaired ability
475
of colonization in mice (Fig. 5).
476
Many observations have indicated that MR/P fimbria is an important virulence
477
factor of uropathogenic P. mirabilis. In a murine model, UTIs with P. mirabilis
478
elicited a strong immune response to MrpA, indicating that MR/P fimbriae were
479
expressed in vivo (39). Increased expression of MR/P fimbriae correlates with higher
480
levels of colonization in the mouse bladder (14, 15). Vaccine trials in mice using
481
MR/P fimbriae reduce bacterial burdens after challenge (40). Transcriptome analysis
482
also revealed that genes encoding MR/P fimbriae were highly upregulated in vivo
483
(13). The phase variation of MR/P fimbriae, mediated by MrpI, has been suggested as
484
a virulence factor of uropathogenic P. mirabilis (12). Given MrpI switches the IE in
485
both directions, why can expression of mrp operon be almost off (on agar plates) or
486
on (during UTIs in mice) (12)? Selection pressures or environmental signaling in the
487
urinary tract may determine the outcome while regulation of MrpI expression and
488
epigenetic modification of IE may be involved. This is the first study to demonstrate
489
that RpoE increased fimbria (mrp operon) expression by regulating mrpI. Several lines of 19
490
evidence support the notion. First, an RpoE binding site exists upstream of mrpI
491
(Fig. 3A). Second, more OFF positions of IE was observed in rpoE mutant than
492
the wild-type and rseA mutant (Fig. 3B). Third, RpoE positively regulated mRNA
493
levels of mrpI and two genes belonging to mrp operon, mrpA and mrpJ (Fig. 3C).
494
Fourth, rpoE mutant had less MrpA protein relative to the wild-type (Fig. 3D). On
495
swarming agar plates, the IE of wild-type, rseA mutant and rpoE mutant was in
496
the OFF position (data not shown) and both ON and OFF appeared on
497
sub-culturing in broth, being more “ON” in the stationary phase when RpoE
498
expression was increased (Fig. 3B and Fig. 1). On the contrary, Lane et al. suggest
499
MrpI preferentially switches the IE from ON to OFF (41). They found that there
500
was more ON in low O2 condition than in atmospheric O2, a condition mrpI
501
expression is higher than in low O2 (41). It is possible that mrpI may be subjected
502
to different regulation in atmospheric O2 and low O2 conditions. In this regard, an
503
E. coli two-component regulator, RcsB, influences the piliation state by
504
controlling transcription of fimB and fimE recombinases in response to
505
environmental cues (42).
506
We found alterations of RpoE did affect expression of mrpJ in cells from broth
507
cultures but not those from plate cultures, a phase-off condition for mrp (Fig. 3C and
508
3E). It is reasonable to infer that the agar surface may be sensed by P. mirabilis to
509
swarm, a condition in favor of downregulating fimbriae and thus repressing
510
expression of MrpJ. It is notable that flhDC and fliC1mRNA levels were decreased in
511
rseA mutant from both broth and plate cultures (Fig. 3C and 3E). Moreover, rseA
512
mutant displayed a significant reduction in motility of swimming and swarming (Fig.
513
S1, Fig. 2A). These results suggest RpoE-mediated swarming repression on agar
514
plates is not MrpJ-dependent but RpoE-regulated MrpJ is involved in reduction of
515
swimming motility. Overexpression of RpoE (rseA mutant) could upregulate MrpI
516
expression, MrpI then switches IE to the ON position to express mrp operon and then
517
production of MrpJ inhibits flhDC and fliC1 expression when P. mirabilis was
518
sub-cultured from the LB agar plate into LB broth. The reciprocal regulation of MR/P 20
519
fimbriae and flagella involving RpoE highlights the role of RpoE in establishing a
520
UTI and the necessity for investigating the dynamics of RpoE expression during
521
infection. Mobley et al. also reported elevated expression of MrpJ in P. mirabilis
522
resulted in reduction of FliC1 protein and swimming motility using overnight broth
523
cultures (26). They found the phase ON mutant has a lower level of FlaA than the
524
OFF mutant and wild-type (26). Obviously, other factors, not MrpJ, regulated by
525
RpoE account for the repression of swarming and expression of flhDC or fliC1 in cells
526
from plate cultures. It is also possible that rpoE mutation may have other effects,
527
except low expression of MrpJ, to result in higher expression of FliC1 than wild-type
528
in broth cultures. In this view, De Lay et al. found several small RNAs inhibited
529
motility of E. coli (43). Among them, we found an ArcZ homologue in P. mirabilis
530
and a putative RpoE binding site in its promoter region. The work to characterize the
531
role of P. mirabilis ArcZ in motility has been undertaken.
532
Several results of this study suggested P. mirabilis RpoE could modulate immune
533
responses. First, rpoE mutant induced higher levels of IL-8 and MIF (both protein and
534
mRNA) and rseA mutant decreased IL-8 and PAI-1 mRNA relative to wild-type (Fig.
535
4C). Second, LPS of rpoE mutant induced high level of IL-8 mRNA (Fig. 4D). Third,
536
rpoE mutant exhibited lower survival inside macrophages than wild-type. Fourth,
537
rpoE mutant caused more immune cell infiltration both in bladders and kidneys on
538
day 1 after inoculation (Table 2, Fig. 6), which corresponds with the increased
539
pro-inflammatory cytokine expression (Fig. 4C and 4D). This is the first study to
540
demonstrate that P. mirabilis RpoE affected pro-inflammatory cytokine expression of
541
urothelial cells. All IL-8, MIF and PAI-1 could attract the migration of immune cells
542
to the infection site (16, 44-47). IL-8 has been shown to be the main neutrophil
543
attractant in humans and is secreted by both bladder and kidney cell lines in response to
544
uropathogenic E. coli (44). Our finding also supported IL-8 could be important during
545
UTIs caused by P. mirabilis.
546
The intracellular environment of a phagocyte may protect the bacteria during the
547
early stages of infection or until they develop a full complement of virulence factors. 21
548
It has been indicated that the deficiency of bactericidal activity of macrophages from
549
cystic fibrosis patients against Pseudomonas aeruginosa is correlated with the
550
increased susceptibility to bacterial infections in these patients (34). Our results that
551
surviving bacteria of rpoE mutant decreased rapidly inside macrophages indicated
552
RpoE may improve the fitness of P. mirabilis during the early stage of infection.
553
Bacterial LPS are capable of inducing bladder inflammation characterized by an
554
increase in the release of pro-inflammatory cytokines and recruitment of the immune
555
cells (17, 48). Many Gram-negative pathogens modify LPS to alter TLR4 responses
556
(11, 49). For example, Salmonella PhoP/Q (a two-component system) can sense host
557
environments, regulating genes involved in LPS modification (11). Salmonella
558
modified LPS are up to 100-fold less active for TLR4-driven NFB activation and the
559
PhoP-null mutant is immune-stimulatory in vivo (11, 50). We found that LPS up to 5
560
g/ml from rpoE mutant but not wild-type induced a significant level of IL-8 mRNA
561
(Fig. 4D), suggesting P. mirabilis RpoE may participate in LPS modification and
562
absence of RpoE-mediated LPS modification results in TLR4 activation and increased
563
IL-8 expression. In this view, RpoE in E. coli has been shown to be involved in LPS
564
modification (20, 36). LPS profile analysis also revealed difference of LPS between P.
565
mirabilis rpoE mutant and wild-type (data not shown). Moreover, knowing LPS
566
modification influences PB resistance in P. mirabilis (23, 24, 28), the finding that PB
567
MIC of wild-type was higher than rpoE mutant (over 50000 vs 12500 μg/ml) also
568
supports the notion.
569
Different authors have proposed that the induction of an innate response could
570
prevent the progress of infection and that TLR4-mediated recognition of LPS was
571
thought to activate the host defense against Gram-negative bacterial pathogens (17).
572
For example, intranasal immunization of mice with MR/P fimbriae protects P.
573
mirabilis from UTIs (40) and activating TLR4 by LPS impairs colonization of 22
574
Bordetella pertussis (51). Accordingly, we found rpoE mutant increased the
575
pro-inflammatory cytokines and the immune cell recruitment and had an impaired
576
ability of colonization.
577
P. mirabilis could cause persistent UTIs despite antibiotic treatment and catheter
578
changes (1, 2, 7). Variation of MR/P fimbriae (9) and flagellar antigens (8), secretion
579
of ZapA protease (10) and the ability of cell invasion are known mechanisms which
580
were utilized by P. mirabilis to contribute to immune evasion during infection (1, 3).
581
Formation of biofilms and urinary stones are also thought to limit bacterial exposure
582
to antibiotics and antibodies (1, 2, 7). We found loss of RpoE did not affect biofilm
583
formation and by reporter assay, RpoE did not regulate zapA and ureC (mutation of
584
the urease gene, ureC, prevents stone formation (1)) in P. mirabilis (data not shown).
585
This indicates RpoE might participate in immune evasion and persistence of P.
586
mirabilis during UTIs by facilitating invasion into urothelial cells and modulating
587
expression of fimbria and flagella and immune responses.
588
The ability to perceive changes in different environment and modify the gene
589
activity is important for bacteria. The activation signals of RpoE could be heat/cold
590
shock, oxidative stresses, osmotic stresses, stationary phase and growth in vivo in
591
Gram-negative bacteria (20). In this study, we demonstrated three signals, PB, urea
592
and the stationary phase (Fig. 1), for RpoE activation in P. mirabilis. The finding that
593
urea, a component in urine, and PB (a cationic antimicrobial peptide) can serve as
594
signals to induce expression of RpoE, reinforcing the role of P. mirabilis RpoE in
595
establishing UTIs. In 2011, Pearson et al. detected gene expression of P. mirabilis
596
from infected mice by microarray analysis (Microarray data accession number:
597
GSE25977) (13). They found genes encoding MR/P fimbriae were upregulated and
598
those for flagella were downregulated compared to in vitro overnight broth culture.
599
Accordingly, we observed that RpoE positively regulated mrp operon (fimbria 23
600
expression) and negatively regulated fliC1 (flagellin) in this study. We suggested
601
that urea could maintain the appropriate level of activated RpoE in vivo.
602
In this study, we characterized the significance of P. mirabilis RpoE in
603
building up UTIs. RpoE could sense the component of urine to regulate multiple
604
virulence factors including fimbria expression, swarming, cell invasion, survival
605
in macrophages, colonization in mice and modulation of host immune responses.
606
Given the diverse regulons of RpoE, it is not surprising for such global effects of
607
RpoE. Altogether, RpoE could help P. mirabilis adapt to its unique environmental
608
niches for building up a UTI. Our discovery adds RpoE into the complex network
609
of pathogenesis in uropathogenic P. mirabilis and deepens understanding of the
610
factors affecting the infectious process. The information is expected to contribute
611
to the designing of new strategies to improve the control of UTIs caused by P.
612
mirabilis.
613
ACKNOWLEDGMENTS
614
This work was supported by the grant NSC-100-2320-B-002-075 from the National
615
Science Council.
616
We would like to thank Dr. Yeong-Shiau Pu (National Taiwan University Hospital)
617
for providing the NTUB1 cell line, as well as Dr. Betty A. Wu-Hsieh and Dr.
618
Li-Chung Hsu for providing the THP-1 cell and technique supports in the macrophage
619
infection assay.
620
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FIGURE LEGENDS
786
FIG 1 The promoter activity of rpoE in wild-type P. mirabilis, rseA mutant and rpoE
787
mutant. The activities of XylE in the rpoE-xylE reporter plasmid-transformed
788
wild-type, rseA mutant and rpoE mutant were determined at 3 h, 5 h, 7 h and
789
overnight after incubation by the reporter assay. The XylE activity of wild-type at 3 h
790
was set at 1 and other data were relative to this value. The data are the averages and
791
standard deviations of three independent experiments. Significant difference was
792
observed by Student’s t-test analysis (*, P
