Filamentation by subverts innate defenses during urinary tract infection

Sheryl S. Justice*†, David A. Hunstad*‡, Patrick C. Seed*‡§, and Scott J. Hultgren*

Departments of *Molecular Microbiology and ‡Pediatrics, Washington University School of Medicine, St. Louis, MO 63110

Edited by M. J. Osborn, University of Connecticut Health Center, Farmington, CT, and approved November 8, 2006 (received for review July 26, 2006) To establish disease, an infecting organism must overcome a vast initial UTI. Many women use daily to reduce the risk array of host defenses. During cystitis, uropathogenic Escherichia of recurrence only to suffer recrudescence when this prophylac- coli (UPEC) subvert innate defenses by invading superficial um- tic regimen is discontinued. The use of in vitro and in vivo models brella cells and rapidly increasing in numbers to form intracellular has allowed the description of events that delineate the acute and bacterial communities (IBCs). In the late stages of the IBC pathway, chronic stages of UTI. Mannosylated uroplakin proteins coating filamentous and bacillary UPEC detach from the biofilm-like IBC, the surface of the superficial umbrella cells of the bladder serve fluxing out of this safe haven to colonize the surrounding epithe- as the receptors for binding and invasion of UPEC by means of lium and initiate subsequent generations of IBCs, and eventually the FimH adhesin at the tips of type 1 pili (3, 4). During murine they establish a quiescent intracellular reservoir. Filamentous UPEC cystitis, intracellular bacterial communities (IBCs) appear ini- are not observed during acute infection in mice lacking functional tially as rod-shaped, loosely organized colonies with a doubling Toll-like receptor 4 (TLR4), suggesting that the filamentous phe- time of 30 min (5). Between 6 and 8 h postinfection, the IBC notype arises in response to host innate immunity. We investi- matures into a tightly packed, highly organized cluster of coccoid gated SulA, a cell division inhibitor associated with the SOS with a doubling time greater than 45 min. Approxi- response, to gain insight into the role of filamentous UPEC in mately 16 h postinfection, the bacteria on the outer surface of the pathogenesis. A transcriptional reporter from PsulA revealed spatial IBC regain a rod shape, become motile, detach from the IBC, and temporal differences in expression within IBCs, and it was and eventually they escape from the superficial umbrella cell (5), active in the majority of filamentous UPEC. Although UTI89 and entering into the bladder lumen to attach to a naı¨ve epithelial cell UTI89 ⌬sulA both formed first-generation IBCs equally well, UTI89 or to leave the host through micturition. During this escape ⌬sulA was sharply attenuated in formation of second-generation (fluxing), filamentous bacteria (up to 70 ␮m in length) are IBCs and establishment of the quiescent intracellular reservoir. The of UTI89 ⌬sulA was restored in TLR4-deficient mice, observed on the luminal surface (5, 6). In the C3H mouse suggesting that filamentation facilitates the transition to addi- background, this developmental cascade occurs in two cycles, tional rounds of IBC formation by subverting innate immune with the second generation displaying much slower kinetics than responses. These findings demonstrate that transient SulA-medi- the first (5). Exfoliation of the superficial umbrella cells occurs ated inhibition of cell division is essential for UPEC virulence in the at late stages of the acute infection (6) in an effort to eliminate murine model of cystitis. the bacterial burden. After superficial umbrella cell exfoliation, UPEC establish a chronic, quiescent intracellular reservoir bacterial filamentation ͉ SOS response ͉ pathogenesis (QIR) within the bladder epithelium (5, 7). Shortly after exfo- liation, the urine is sterile on most days, but the bladder tissue maintains a bacterial burden for weeks to months after acute he interaction between bacterium and host ultimately results Tin symbiosis, disease, and/or clearance. The innate immune cystitis (7, 8). This QIR resists treatment with oral antibiotics; it response comprises a myriad of biochemical (e.g., oxidizing is apparently undetected by host immunity, and it can be the agents), cytological (e.g., phagocytic cells), and biomechanical source of a recurrent infection (7). (e.g., exfoliation of epithelial cells) tactics that prevent coloni- UPEC have developed specific strategies to bypass or over- zation and eradicate infectious microorganisms. Polymorphonu- come host defenses. The bladder epithelium produces cytokines, clear leukocytes (PMNs; neutrophils) are phagocytic cells that primarily through stimulation of host Toll-like receptor 4 represent a first line of defense, and they are recruited from the (TLR4) by bacterial LPS, that recruit PMNs to assist in the bloodstream directly to the site of invading bacteria. The primary elimination of the bacteria. UPEC actively suppress the produc- role for PMNs is the neutralization of invading organisms by tion of cytokines produced by bladder epithelial cells (9). In using degradative enzymes, reactive oxygen species, and anti- addition, the intracellular niche provides a safe haven for UPEC microbial peptides. of bacteria results in the stim- evasion from infiltrating PMNs (5). Finally, our recent data ulation of apoptosis within the PMN, modulating inflammation suggest that bacterial filamentation might confer resistance to and reducing collateral destruction of host tissues (1). Productive killing by PMNs (5). pathogens use multiple mechanisms to evade and/or cope with the stresses presented by these host defenses. Urinary tract infections (UTIs) are an attractive model to study the molecular Author contributions: S.S.J. and D.A.H. designed research; S.S.J. and D.A.H. performed research; P.C.S. contributed new reagents/analytic tools; S.S.J., D.A.H., and S.J.H. analyzed details of this delicate balance between host and pathogen data; and S.S.J., D.A.H., and S.J.H. wrote the paper. because the bladder employs diverse tactics to combat infectious The authors declare no conflict of interest. organisms. Uropathogenic Escherichia coli (UPEC) have This article is a PNAS direct submission. adapted multiple mechanisms to evade these host defenses. Abbreviations: IBC, intracellular bacterial community; MMC, mitomycin C; PMN, polymor- UTIs are among the most common infections in the United phonuclear leukocyte (neutrophil); QIR, quiescent intracellular reservoir; UPEC, uropatho- States. Each year, at least 4 million women seek treatment for genic E. coli; UTI, urinary tract infection. Ϸ UTIs, and $1.6 billion will be spent in the diagnosis and †To whom correspondence should be addressed at the present address: Center for Micro- treatment of UTIs (2). UPEC are the cause of 70–90% of all bial Pathogenesis, Columbus Children’s Research Institute, 700 Children’s Drive, W532, community-acquired UTIs (2). Clinicians are frustrated by the Columbus, OH 43205. E-mail: [email protected]. rise in resistance among organisms that cause UTIs §Present address: Department of Pediatrics, Duke University, Durham, NC 27710. and by frequent recurrences in healthy adult females after an © 2006 by The National Academy of Sciences of the USA

19884–19889 ͉ PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606329104 Downloaded by guest on September 26, 2021 Fig. 1. Microscopic examination of IBC formation and UPEC filamentation. Female 7- to 9-week old C3H/HeN (a–e) or C3H/HeJ (f) mouse strains were infected with 107 cfu of UTI89/pCOMGFP (a and c) or UTI89 ⌬sulA/pCOMGFP (b, d, and f) or UTI89 (e; red) plus UTI89 ⌬sulA/pCOMGFP (e; green) by transurethral catheterization. Bladders were harvested at6h(a and b), 16 h (e), or 24 h (c, d, and f) postinfection, and they were prepared as described in Materials and Methods for visualization by confocal laser scanning fluorescent microscopy with three-dimensional reconstruction or as an optical slice (d). (Scale bars: 30 ␮m.)

Bacteria have developed multiple strategies to cope with response in murine cystitis (5). In addition, filamentation may environmental stresses. DNA damage can result from certain play a role in human cystitis because filamentous UPEC are environmental stresses or insults (e.g., UV light, oxidative observed in the urine of UTI patients (D. Rosen and S.J.H., conditions). The SOS response is one such strategy that is unpublished data). To assess directly a role for filamentation in MICROBIOLOGY designed to inhibit cell division while damaged DNA is repaired, cystitis, the bacterial system responsible for filamentation was thereby preventing transmission of deleterious mutations to investigated. Because PMNs use oxidative molecules to eradicate daughter cells (for review, see ref. 10). On a molecular level, bacterial pathogens, the oxidative environment of the host mutations resulting in mismatched base pairs produce regions of inflammatory response could lead to mutation of bacterial single-stranded DNA. RecA, the major bacterial recombinase, DNA. DNA repair would be accomplished by induction of the binds single-stranded regions, and it is activated in the presence of nucleotide triphosphates. Activated RecA stimulates the SOS response, ultimately leading to bacterial filamentation autoproteolysis of the SOS transcriptional repressor, LexA. caused by the cell division inhibitor, SulA (17). Therefore, a LexA represses the of at least 17 genes that are kanamycin cassette disruption of sulA was transduced into our involved in DNA repair, and it has recently been implicated in prototypic cystitis UPEC isolate, UTI89. Cystitis was induced in the regulation of Ͼ30 unlinked genes (11). The LexA regulon 7- to 9-week-old female C3H/HeN mice by transurethral inoc- includes a cell division inhibitor, SulA, to prevent transmission ulation of 107 cfu of statically grown UTI89 or UTI89 ⌬sulA. The of mutant DNA to new daughter cells. SulA specifically inhibits ability of the sulA mutant to proceed through all stages of the FtsZ polymerization (12, 13), preventing FtsZ ring formation developmental pathway was evaluated by fluorescence micros- (14–16) and thereby blocking septation. Once DNA repair is copy or by recovery of cfu from the bladder. UTI89 and UTI89 complete, LexA repression of the SOS genes is restored. In ⌬sulA were transformed with pCOMGFP (18) for ease of addition, Lon, the general cytoplasmic protease, degrades SulA, visualization of intracellular bacteria by fluorescence micros- restoring cell division capacity. copy. During the first 16 h postinfection, the size and morphol- UPEC filamentation may serve as an additional mechanism to ogy of IBCs were indistinguishable between UTI89 and UTI89 evade host innate immune responses. The current study directly ⌬sulA (Fig. 1 a and b), suggesting that UTI89 ⌬sulA is competent tested a role for SulA-mediated cell division inhibition in the for first-generation IBC formation. Consistent with these mi- pathogenic lifestyle of UPEC. Inactivation of sulA in UPEC sharply croscopic results, the total numbers of bacteria recovered at 6 attenuated virulence in the murine cystitis model, but only when the and 16 h from the bladders infected with UTI89 ⌬sulA were TLR4-mediated response was intact, demonstrating that filamen- tation is a critical component of the bacterial arsenal against the indistinguishable from bladders infected with UTI89 (Fig. 2a). in vitro However, at 16 h, when filamentous UTI89 is readily observed innate immune response. Induction of the SOS response was ⌬ not sufficient to protect UPEC from PMN phagocytosis, suggesting in infected bladders (Fig. 1e), UTI89 sulA appeared only in that factors in addition to filamentous morphology are required for bacillary form, both intracellularly and on the luminal surface. evasion of innate immunity. To characterize more fully the absence of filamentation in UTI89 ⌬sulA, bladders coinfected with equivalent numbers of UTI89 Results and UTI89 ⌬sulA/pCOMGFP were studied by fluorescence SulA Mediates Filamentation in Vivo. Bacterial filamentation has microscopy at 16 h postinfection. Wild-type UTI89 displayed red been proposed as a mechanism of evasion of the innate immune fluorescence caused by the counterstaining of whole mounts

Justice et al. PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19885 Downloaded by guest on September 26, 2021 bacterial load was much lower than in wild-type infection at this time point (Fig. 2a). After the second generation of IBC formation, UPEC enter into a QIR (5, 7, 19). Bacteria were not recovered from any of the bladders infected with UTI89 ⌬sulA at 2 weeks postin- fection, whereas bladders infected with UTI89 all contained viable cfu, reflecting the presence of a QIR (Fig. 2a) (103 to 105 cfu per bladder) as observed in our earlier studies (7). The inability of the sulA mutant to initiate effectively second- generation IBC formation suggests that SulA-mediated bacterial filamentation is important for the transition between genera- tions of IBC formation. Moreover, the transition into a QIR is also preceded by the emergence of a large number of filamentous bacteria on the luminal surface (5). Thus, the pathogenesis of murine cystitis hinges on SulA-mediated filamentation, a phe- notype that might underlie reentry into naı¨ve superficial um- brella cells or confer resistance to host immune responses, including PMN engulfment and killing.

SulA Is Not Required in the Absence of Functional TLR4. PMN influx into the bladder during UTI is greatly impaired in the C3H/HeJ mouse strain (20) because of a mutation in the cytoplasmic tail of TLR4, blocking transduction of LPS-initiated signals (21, 22). Our previous studies indicated that filamentous UTI89 are not observed during the acute infection in the C3H/HeJ mouse strain (5). Therefore, we hypothesized that inactivation of sulA would have no effect on the course of cystitis in the C3H/HeJ mouse. UTI89 ⌬sulA persisted to the same extent as UTI89 after inoculation into the bladders of C3H/HeJ mice (Fig. 2b)from6h to 2 weeks. IBCs formed by UTI89 ⌬sulA in C3H/HeJ bladders were similar in number and architecture at 24 h postinfection to Fig. 2. Virulence of UTI89 ⌬sulA. Female C3H/HeN (a) or C3H/HeJ (b) mice those observed in C3H/HeJ bladders infected with UTI89 (Fig. were transurethrally inoculated with 107 UTI89 (Š), UTI89 ⌬sulA (‹), or UTI89 ⌬ ⌬ ࡗ 1f). Moreover, the ability of UTI89 sulA to establish a QIR was lon ( ). Bladders were harvested at 6 h, 16 h, 24 h, 48 h, or 2 weeks Ϫ/Ϫ postinfection and homogenized, and viable bacteria were enumerated as cfu restored in the Tlr4 mouse strain (Fig. 2b), suggesting that the per bladder. Each graph is representative of three independent experiments. requirement for SulA-mediated filamentation during IBC mat- The statistical significance was assayed by using the Mann–Whitney test (ns, uration and establishment of cystitis relates to evasion of host not significant; *, P ϭ 0.03; **, P ϭ 0.004; nd, not determined). immunity.

UPEC Virulence Requires Recovery from Filamentation. Previous with a monomeric cyanine nucleic acid dye after paraformalde- studies indicated that cell division capacity was restored in hyde fixation. Surface-associated UTI89 was observed in both filamentous UPEC during UTI (5). To determine whether bacillary and filamentous forms; however, only bacillary UTI89 restoration of cell division within the filamentous population is ⌬sulA/pCOMGFP was observed on the surface (Fig. 1e). The ⌬ essential for uropathogenesis, lon was inactivated in UTI89 to absence of filamentous UTI89 sulA during first-generation IBC prevent recovery from SulA-mediated filamentation. The cyto- formation suggests that SulA is required for filamentation in plasmic protease, Lon, degrades SulA to restore FtsZ polymer- vivo. ization and septation after repair of damaged DNA (23). UTI89 Fluxing at the end of first-generation IBC formation leads to ⌬lon displayed a defect in cfu per bladder of 5 orders of invasion of naı¨ve superficial umbrella cells and generation of a magnitude in C3H/HeN mice at 48 h (Fig. 2a), but virulence of large number of second-generation IBCs (5). In marked contrast UTI89 ⌬lon was restored in the C3H/HeJ mice (Fig. 2b), similar to infections with UTI89 (Fig. 1c), UTI89 ⌬sulA was primarily on to that observed with UTI89 ⌬sulA infection. the luminal surface at 24 h (Fig. 1d). Only four IBCs were observed in 10 bladders infected with UTI89 ⌬sulA alone at 24 h When filamentous UTI89 on the bladder surface is visualized by postinfection; typically, UTI89 forms 50 to several hundred IBCs SEM, the majority of filaments exhibit a smooth surface (5). This per bladder. In addition, at time points corresponding to the observation is consistent with previous studies reporting a smooth second round of IBC formation (24 and 48 h), there was a surface for E. coli that have undergone filamentation mediated by decrease of 5–7 orders of magnitude in cfu when bladders were SulA (5, 6). However, occasionally, a partial septum is observed on infected with UTI89 ⌬sulA compared with bladders infected the surface of filamentous UPEC by SEM. Lutkenhaus and col- with UTI89 (Fig. 2a). Thirty percent of the bladders infected leagues (24) have previously demonstrated that disassembly of with UTI89 ⌬sulA were sterile at 48 h postinfection. Sterile preformed septation complexes results in an aborted septum that bladders are rarely, if ever, observed at this time point after cannot be used in further septation events. Thus, the majority of infection with wild-type UTI89. These data suggest that SulA is daughter cells arising after Lon-mediated restoration of cell division critical for UTI89 pathogenesis after first-generation IBC for- would display the normal cell length that we observed. SulA- mation. This time frame is the same as when filamentous bacteria mediated division inhibition has been described as a transient are typically observed, suggesting that the inability of UTI89 process to prevent septation temporarily during the repair of DNA ⌬sulA to filament dramatically attenuates virulence. damage (25). Our data suggest that the filamentation that occurs At 36–48 h postinfection, when filamentous bacteria are ob- during pathogenesis must also be a transient process and that served in UTI89-infected bladders (5), filamentous UTI89 ⌬sulA septation must be restored for continued progression through the was not observed (data not shown), although the UTI89 ⌬sulA IBC cascade during acute infection.

19886 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606329104 Justice et al. Downloaded by guest on September 26, 2021 7 Fig. 3. Expression of sulA in vivo. Female C3H/HeN (a–d) or C3H/HeJ (e and f) mice were transurethrally inoculated with 10 UTI89 attB::PsulA-gfp. Bladders were harvested at4h(a),6h(b), or 16 (c–f), and they were prepared as described in Materials and Methods for visualization by laser scanning fluorescent microscopy. They were visualized with three-dimensional reconstruction (a–d) or as an optical slice (e and f). (Scale bars: a, b, e, and f,50␮m; c and d,30␮m.)

sulA Is Expressed in Vivo. To demonstrate further that SulA is control of Plac. Filamentous and bacillary UTI89 were opsonized involved in UPEC pathogenesis, the in vivo transcriptional with 50% normal human serum and challenged with purified profile of sulA was evaluated. To observe directly expression of human PMNs. There was no apparent difference in the ability of sulA during UTI pathogenesis in vivo, the sulA promoter was the PMNs to engulf and kill the filamentous and bacillary forms

used to drive expression of the gene encoding GFP from a single of UTI89 (Fig. 4 a and b), suggesting that filamentation per se is MICROBIOLOGY copy integrated at the attB site. In vitro, UTI89 attB::PsulA-gfp not sufficient for protection from PMNs. The observation that exhibited strong fluorescence only on exposure to SOS-inducing size alone is not sufficient to protect the bacteria from engulf- agents, e.g., mitomycin C (MMC; data not shown). Expression of ment is not surprising, considering that PMNs engulf the much sulA was readily observed within a minority of the individual larger hyphal form of Candida albicans (26). The ability of PMNs bacteria in early IBCs in C3H/HeN mice infected with UTI89 to engulf the SulA-induced filaments in vitro suggests that there attB::PsulA-gfp (Fig. 3 a and b). The number of bacteria within an are additional factors coregulated with the filamentous response, IBC that expressed sulA varied among IBCs depending on their needed to subvert PMNs. Such factors might represent other size and maturation state. Approximately 60% of the Ͼ800 IBCs components of the SOS regulon, or they may be produced observed within 10 murine bladders infected with UTI89 independent of the SOS response. Alternatively, the extended attB::PsulA-gfp exhibited differential sulA expression, both tem- length might provide a more secure attachment to the bladder porally and spatially, by confocal microscopy. Fluorescence epithelium, with increased number of adhesins over a greater emitted from GFP was also observed in the majority of filamen- surface area that would make removal by PMNs more difficult. tous E. coli (Fig. 3 c and d) analyzed in 15 murine bladders To test whether chemical induction of the SOS response infected with UTI89 attB::PsulA-gfp, consistent with the hypoth- provides protection from PMN phagocytosis in vitro, UTI89 was esis that expression from PsulA leads to filamentation of UPEC grown under static conditions with the addition of 300 ng/ml during cystitis. Infection of C3H/HeJ (Tlr4Ϫ/Ϫ) mice with UTI89 MMC in early log phase at 37°C. PMNs were isolated from both attB::PsulA-gfp did not lead to detectable expression of sulA humans and mice as described in ref. 27, and UTI89 was grown within Ͼ600 IBCs analyzed (Fig. 3 e and f). Thus, TLR4- in the presence or absence of MMC and opsonized with normal dependent innate responses lead to induction of sulA expression, serum or mixed directly with human or mouse PMNs. As likely reflecting induction of the SOS response caused by host visualized by microscopy, both human and mouse PMNs were production of DNA-damaging (e.g., oxidative) agents. able to engulf both opsonized (data not shown) and nonopso- nized (Fig. 4 c–f) filamentous and bacillary UTI89 readily. From SulA Overproduction Does Not Protect from PMNs in Vitro. Our data these in vitro data, it appears that a putative bacterial component suggest that SulA is critical in uropathogenesis, but they do not for protection is not induced by brief exposure to a chemical directly address the function of SulA in subverting innate DNA-damaging agent. Additionally, it is unknown whether the defenses in vivo. Previous studies have suggested that filamen- observed induction of sulA in vivo occurs as a component of the tous UTI89 evades PMN killing (5). Current methods do not SOS response. LexA-dependent induction mediated by DNA permit isolation of sufficient numbers of filamentous bacteria damage is the only known mechanism that leads to sulA expres- from the urine and/or bladders to assess directly the ability of in sion. Recently, microarray analysis of the SOS response in vivo-produced filamentous UPEC to survive PMN phagocytosis laboratory (K-12) strains of E. coli have demonstrated that ex vivo. To circumvent this problem in vitro, UTI89 was trans- additional genes are transcriptionally active on initiation of DNA formed with a plasmid (pGC165) that encodes sulA under damage (11). Given the increased size of the UTI89 genome

Justice et al. PNAS ͉ December 26, 2006 ͉ vol. 103 ͉ no. 52 ͉ 19887 Downloaded by guest on September 26, 2021 Fig. 4. PMN phagocytosis in vitro. Human and mouse PMNs were incubated with UTI89 under inducing conditions for 20 min, and they were visualized by using phase microscopy (a, b, e, and f) or fluorescent microscopy (c and d). (a) Opsonized, uninduced UTI89 with human PMNs. (b) Opsonized induced UTI89 Plac::sulA. (c and d) MMC-induced UTI89 with mouse PMNs. (e) Uninduced UTI89 with human PMNs. (f) MMC-induced UTI89 with human PMNs. (Scale bars: a–d,30␮m; e and f,50␮m.)

relative to E. coli K-12 (28), it is likely that additional genes may cells (29). In addition, the potential roles of UPEC filamentation be present in the SOS regulon within pathogenic organisms. in resistance to killing mediated by neutrophil extracellular traps However, the presence of other signals that induce sulA in vivo (30) and by antimicrobial peptides such as cathelicidin (31) cannot be eliminated. SOS induction for periods longer than 4 h warrant further investigation. may result in the expression of additional molecules that may be In summary, we have demonstrated that in response to innate protective. The putative bacterial component for PMN protec- immunity, UPEC employs filamentation as a means to evade this tion may be produced in response to the same signal that results immune response. Characterization of putative host signals and in PsulA expression, or it may occur through an alternative signal further understanding of the regulation of sulA will offer insight that occurs coincidently with the signal that induces PsulA in vivo. into the pathogenesis of diverse bacterial infections and will foster the design of novel therapeutic agents. Discussion Materials and Methods The data presented here provide strong evidence that filamen- tation, mediated by SulA, is a vital component of the UPEC Strains and Media. Bacterial strains were grown in Luria broth pathogenic cascade within an immunocompetent host. The statically at 37°C for 16 h to facilitate type 1 pilus production transition of UPEC from the first to second generation of IBC (32). UTI89 is a clinical UPEC isolate obtained from a patient formation depends on SulA-mediated division inhibition and with cystitis (8). The sulA::kan insertional mutation (33) was Lon-mediated restoration of cell division competence. This transduced into UTI89 by using P1 transduction (34), and transductants were selected by using 50 ␮g/ml kanamycin sulfate. conclusion is based on the observation of a rapid decline in cfu Transcriptional activity of sulA in vivo was assessed by introduc- per bladder 24 h postinfection, which is the time point associated tion of the sulA promoter driving expression of the gene encod- with formation of second-generation IBCs (5). The restoration ⌬ ing for green fluorescent protein (gfp) in single copy into UTI89. of virulence of UTI89 sulA in C3H/HeJ mice argues that Briefly, the sulA promoter region (Ϫ322 to ϩ51 relative to the SulA-mediated filamentation protects UPEC from components start ATG) was amplified by PCR from UTI89 genomic DNA by of host innate defenses. However, overproduction of SulA or using primers ACTGTATCGATCAACGGTCAGGCTGTA- chemical induction of the SOS response was insufficient to ACTACCAAA and ATGAACCCGGGTGCTGTGGAT- confer protection from phagocytosis in vitro. Although sulA is GAGAACGACGAATCA. The 373-bp product was restricted expressed in vivo during cystitis, the mechanism of its induction with ClaI and SmaI, cloned into the same sites of pPSSH10, is unknown. We propose that components of the innate immune sequenced for verification, and integrated into the ␭ attachment response (e.g., oxidative stresses) likely induce the SOS response site of E. coli MG1655 as described in ref. 35. The fusion was in vivo but that sulA induction in vivo might instead rely on signals moved into UTI89 by P1 phage transduction. Plasmid pGC165 independent of the SOS pathway. Our data suggest that the contains sulA under the control of the Plac promoter (36). To filamentous phenotype that is observed at the transition between induce the SOS response in vitro, saturated cultures were diluted stages of murine cystitis is more than a morphologic one and that 1:1,000 and allowed to grow with aeration or statically at 37°C filamentation may provide multiple means of protection for until they reached early log phase (A600 ϭϷ0.3). MMC (Sigma– UPEC. For example, previous reports have suggested that Aldrich, St. Louis, MO) was added to a final concentration of 300 induction of the SOS response and subsequent SulA-mediated ng/ml, and growth continued under the same conditions for division inhibition protect against antibiotics that act on dividing 2–4 h.

19888 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606329104 Justice et al. Downloaded by guest on September 26, 2021 Murine Cystitis. Female C3H/HeN (Harlan, Indianapolis, IN) or Institutional Review Board for human subjects research. Alter- C3H/HeJ (Jackson Laboratories, Bar Harbor, ME) mice, 7–9 weeks natively, female mice were treated with heparin (200 units s.c.) old, were infected with 107 cfu of E. coli transurethrally, in a volume for 10 min, then blood was harvested by cardiac puncture. Mouse of 50 ␮l as described in ref. 6. At the times indicated, bladders were and human PMNs were isolated as described in ref. 27. Where aseptically harvested and homogenized for bacterial enumeration; indicated, bacteria were opsonized with 50% normal human or they were bisected, splayed, and fixed in 3% paraformaldehyde serum in PBS for 30 min at 37°C before incubation with isolated for1hforvisualization by laser scanning confocal fluorescent PMNs. Bacteria and PMNs were coincubated in RPMI medium microscopy (Zeiss LSM510 Meta; Carl Zeiss, Inc., Thornwood, 1640 with 10 mM unbuffered Hepes for up to 30 min at 37°C. NY). Fixed bladders were treated with 0.01% Triton X-100 in PBS Samples were analyzed at specified intervals by using phase for 10 min, and then they were counterstained with a red fluores- microscopy or epifluorescent microscopy. cent monomeric cyanine nucleic acid dye (ToPro3; Molecular Probes, Eugene, OR) for an additional 10 min. Three-dimensional We thank L. Rothfield and M. J. Osborn for invaluable discussions. This image reconstruction was performed by using Volocity software work was supported by National Institutes of Health Grants F32- (Improvision, Lexington, MA). DK10168 (to S.S.J.), K08-DK067894 (to D.A.H.), K12-HD00850 (to P.C.S.), and R01-DK51406 (to S.J.H.) and Office of Research on PMN Isolation. Human blood was collected from peripheral veins Women’s Health Specialized Center of Research Grant P50-DK64540 of healthy donors in accordance with a protocol approved by the with the Food and Drug Administration (to S.J.H.).

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