INFECTION AND IMMUNITY, Nov. 2006, p. 6124–6134 Vol. 74, No. 110019-9567/06/$08.00�0 doi:10.1128/IAI.01086-06Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Protease Activity, Secretion, Cell Entry, Cytotoxicity, and CellularTargets of Secreted Autotransporter Toxin of Uropathogenic

Escherichia coli�

Nathalie M. Maroncle,1 Kelsey E. Sivick,1 Rebecca Brady,2 Faye-Ellen Stokes,3 and Harry L. T. Mobley1*Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, Michigan 481041;

Department of Biomedical Sciences, University of Maryland Dental School, Baltimore, Maryland 212012;and Society for General Microbiology, Reading, United Kingdom3

Received 11 July 2006/Returned for modification 10 August 2006/Accepted 25 August 2006

The secreted autotransporter toxin (Sat), found predominantly in uropathogenic Escherichia coli, is amember of the SPATE (serine protease autotransporters of Enterobacteriaceae) family and, as such, has serineprotease activity and causes cytopathic effects on various cell types. To assess the contribution of the serineprotease active site to the mechanism of action of Sat, mutations were made in the first (S256I), in the second(S258A), or in both (S256I/S258A) serine residues within the active site motif. Mutations in the first or bothserines reduced protease activity to background levels (P < 0.001); a single mutation in the second serinereduced activity by 60% compared to wild type (P < 0.001). After reversion of the S256I mutation to wild type(I256S), we confirmed S256 as the catalytically active serine. None of these mutations affected secretion of themature passenger domain or release into the supernatant. The S256I mutation, however, abrogated thecytotoxicity of Sat on human bladder (UM-UC-3) and kidney (HEK 293) epithelial cells, characterized byrounding and elongation, respectively, and a high level of cell detachment. Moreover, S256 is essential for Satto mediate cytoskeletal contraction and actin loss in host cells as well as to degrade specific membrane/cytoskeletal (fodrin and leukocyte function-associated molecule 1) and nuclear [microtubule-associated pro-teins, LIM domain-only protein 7, Rap GTPase-activating protein, poly(ADP-ribose) polymerase] proteins invitro. Lastly, Sat was internalized by host cells and localized to the cytoskeletal fraction where membrane/cytoskeletal target proteins reside.

Urinary tract infection (UTI) is a common extraintestinalinfection (33), and Escherichia coli is by far the most commoncausative organism (30). UTI involves the urethra and thebladder, producing cystitis, or the kidneys, producing acutepyelonephritis. Uropathogenic E. coli strains that cause theseinfections display specific phenotypic traits. These include spe-cific adhesins (P and type 1 fimbriae, necessary for attachmentto uroepithelial cells, and other fimbriae including S and F1C,commonly produced by uropathogenic strains), aerobactin, cy-totoxic necrotizing factor 1, and the pore-forming hemolysin(3, 14, 27, 53). Such isolates also typically carry large blocks ofgenes, called pathogenicity-associated islands (PAIs) (18), aswell as numerous smaller inserts (52) not found in fecal iso-lates.

Uropathogens may also damage host epithelium by the ex-port of autotransporter proteins. Studies of a number of auto-transporters (reviewed in reference 23) have demonstratedthat translocation across the inner membrane occurs via thesec-dependent pathway. Differing mechanisms for movementof the passenger domain across the outer membrane have beenreported; however, all involve the �-barrel porin structureformed by the C-terminal autotransporter domain (23, 37, 49).

Once transported to the bacterial cell surface, the passengerdomain may remain attached to the outer membrane or bereleased by proteolytic cleavage (23).

Our laboratory previously identified a 107-kDa secreted pro-tein, designated Sat (secreted autotransporter toxin), that isexpressed significantly more often by E. coli strains associatedwith the clinical symptoms of acute pyelonephritis (68% ofstrains) than by fecal strains (14% of strains) (16). Thepolypeptide, isolated from E. coli CFT073, shares highest sim-ilarity to the subcategory of autotransporters termed SPATE(serine protease autotransporters of Enterobacteriaceae) pro-teins, which are produced by diarrheagenic E. coli and Shigellaspecies isolates (23). The native Sat protein (142 kDa) includesthe three characteristic domains of SPATE proteins: an un-usually long N-terminal signal sequence, a secreted passengerdomain (the mature protein) to which the phenotype of eachprotein is attributed, and a C-terminal autotransporter do-main. The mature Sat protein (107 kDa) was shown to have acytopathic effect on various cell lines (16, 17) and to elicitglomerular damage and a vigorous antibody response in micetransurethrally infected with E. coli CFT073 (16). In addition,the sat gene was shown to reside within PAI I of E. coliCFT073, implicating Sat as another possible PAI-encoded vir-ulence determinant.

The activity of the serine protease motif of autotransportershas been characterized for a number of these proteins. Serineprotease activity can catalyze autoproteolysis of the matureprotein from the autotransporter domain at the bacterial sur-face (24) or have no such role (6, 21, 36, 46). Mutation of the

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, University of Michigan Medical School, 5641Medical Science Bldg II, 1150 West Medical Center Dr., Ann Arbor,MI 48109-0620. Phone: (734) 764-1466. Fax: (734) 763-7163. E-mail:hmobley@umich.edu.

� Published ahead of print on 5 September 2006.

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serine protease motif can result in a loss of proteolytic ormucinase activity (21), cytotoxicity on target cells (36), or en-terotoxic effects on rat jejunal tissue mounted on an Ussingchamber (36).

In this report, we demonstrate that the serine protease activesite of Sat is necessary for protease and cytotoxic activities,contraction of the cytoskeleton, and loss of actin in culturedbladder and kidney cells but not for the processing or therelease of the toxin from the bacterial surface. We show thatwild-type Sat, but not its first-serine mutant, is able to degradespecific membrane/cytoskeletal and nucleus-associated pro-teins. Lastly, we demonstrate that Sat enters human bladderand kidney epithelial cells and localizes specifically to the cy-toskeletal fraction where proposed protein targets reside.

MATERIALS AND METHODS

Bacterial strains and plasmids. E. coli HB101(pDG4) is a wild-type Sat-expressing clone (16); E. coli HB101 pBluescript SK(�) (pBS SK�) is an emptyvector control strain. Bacteria were cultured either in Luria-Bertani (LB) brothat 37°C with aeration in a shaking incubator (200 rpm) or on LB agar platescontaining 1.5% agar at 37°C. Media were supplemented with ampicillin (100�g/ml).

Site-directed mutagenesis. Site-directed mutagenesis of the serine proteaseactive site (GDS256GS258G) of Sat was performed using PCR overlap extension(25). In the first round of amplification for this two-step reaction, two DNAfragments were generated with overlapping ends which anneal during the secondround of amplification, facilitating their use as a template. Point mutations wereintroduced into the overlapping regions using oligonucleotide primers B1 (5�-AGAGCCGATGTCTCC-3�) and C1 (5�-GGAGACATCGGCTCT-3�) to mutatethe first serine to nonpolar isoleucine (S256I), B2 (5�-TAAGTATGCTCCAGCGCCG-3�) and C2 (5�-GGCGCTGGAGCATACTTA-3�) to mutate the secondserine to nonpolar alanine (S258A) and to create a double-serine mutant (S256I/S258A), and B3 (5�-ATTCGGAGACAGTGGCTCTGGAGC-3�) and C3 (5�-GCTCCAGAGCCACTGTCTCCGAAT-3�) to make isoleucine revert to serine(I256S). Flanking primers A (5�-CCAGTCACGACGTTGTA-3�) and D (5�-AGTCCGTTCCACAAAGA-3�) hybridize upstream and downstream of the serineprotease active site, respectively. By using pDG4, a plasmid expressing Sat S256Iwas generated (pDG4S256I) by A/B1 and D/C1 amplification. Second-serine anddouble-serine mutations were created by A/B2 and D/C2 amplification frompDG4 and pDG4S256I, respectively. Reversion mutation was produced by A/B3and D/C3 amplification from pDG4S256I. All final mutated PCR products weregenerated by A/D amplification.

Cloning and sequencing of PCR products. PCR products were ligated intopCR-BluntII-TOPO (Invitrogen, Carlsbad, California). Inserts were excised us-ing the appropriate restriction enzymes (either HindIII-SphI or BamHI-NotI)and separated by agarose gel electrophoresis. Inserts were excised from the geland purified using a QIAquick gel extraction kit (QIAGEN, Valencia, Califor-nia) and ligated into similarly treated pDG4. Plasmids were introduced into thelaboratory strain E. coli HB101 by electroporation (44). Sequencing of the first-,second-, and double-serine Sat mutant regions was done at the Biopolymer CoreFacility at the University of Maryland, Baltimore, using an Applied Biosystemsmodel 373A automated DNA sequencer using the Big Dye Terminator Cyclesequencing kit while the reverted mutant region was sequenced at the DNASequencing Core at the University of Michigan, Ann Arbor, according to pro-tocols for Applied Biosystems DNA sequencers.

Mass spectrometry analysis. All protein samples were digested overnight withtrypsin, including alkylation and reduction. Peptides were extracted and analyzedby matrix-assisted laser desorption ionization–time of flight mass spectrometry atthe Protein Structure Facility at the University of Michigan, Ann Arbor. Athree-pointed external calibration was performed, yielding a mass accuracy of0.1%. Results were searched in the Protein Prospector database in both NCBIand Swiss-Prot.

Sat supernatant preparations. Bacteria from overnight LB cultures of E. coliHB101 transformed with pDG4, pDG4S256I, pDG4S258A, pDG4S256I/S258A,or pDG4I256S were pelleted, and supernatants were filter sterilized. Superna-tants were then concentrated 500-fold using 100,000-molecular-weight-cutoff(MWCO) Centricon Plus-80 filters (Millipore, Billerica, Massachusetts). Proteinconcentration was determined using a bicinchoninic acid assay (Pierce, Rock-ford, Illinois).

SDS-PAGE and Western blot analysis. Samples were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteinswere either stained with Coomassie blue or electrotransferred onto a nitrocel-lulose membrane (Millipore) as described by Towbin et al. (48). Unless other-wise indicated, membranes were then incubated with primary rabbit anti-Satserum (1:5,000 dilution) followed by incubation with secondary anti-rabbit–alkaline phosphatase conjugate (1:2,000 dilution).

Serine protease and protein substrate cleavage assays. Protease activity wasdetermined using the p-nitroanilide substrate assay (7). Concentrated superna-tant containing wild-type Sat or mutant derivatives (20 �g) was added to 1 mMmethoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Calbiochem, La Jolla, Califor-nia) in a buffer containing 0.1 M morpholinepropanesulfonic acid (MOPS), pH7.3, 0.2 M NaCl, and 0.01 mM ZnSO4. Samples were incubated at 37°C for 24 h,and substrate hydrolysis was monitored at 405 nm. All absorbance measurementswere normalized to wild-type Sat values. Samples were also tested for serineprotease activity after preincubation for 30 min at room temperature with 1 mMphenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, Missouri).Reactions were conducted in quadruplicate, and the mean � standard deviationwas calculated. For protein substrate assays, spectrin (3 �g) (Sigma-Aldrich) andhuman coagulation factor V (2.5 �g) (Calbiochem) were combined with 5 �g ofeach concentrated supernatant in 20 �l of MOPS buffer (125 mM MOPS, 12.5�M ZnSO4, 250 mM NaCl, pH 7.5) and incubated overnight at 37°C. Reactionproducts were separated by SDS-6% PAGE (31).

Tissue culture assays. Cells were maintained in humidified 5% CO2-93% airat 37°C. UM-UC-3 human bladder epithelial cells (ATCC CRL-1749) and HEK-293 human kidney epithelial cells (ATCC CRL-1573) were cultured in Dulbecco’smodified Eagle’s medium (Gibco, Carlsbad, California) supplemented with 10%fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), penicillin (100 U/ml),and streptomycin (100 �g/ml) (Mediatech, Herndon, Virginia). Subconfluentcells were resuspended with trypsin-EDTA (Gibco), washed, and plated in eight-well Lab-Tek chamber slides (NNI, Naperville, IL). To perform the cytotoxicityassay, concentrated supernatants (100 �g/ml) containing wild-type and mutantderivatives of Sat were added directly to 80% confluent host cells in 200 �l ofmedium. Cells were incubated for 2 h at 37°C, washed twice with phosphate-buffered saline (PBS), and fixed with 3.7% (vol/vol) formaldehyde in PBS. Cellseither were stained with Giemsa stain (Sigma-Aldrich) and visualized by lightmicroscopy or were permeabilized by adding 0.1% Triton X-100 in PBS andstained with fluorescein isothiocyanate-phalloidin (0.5 �g/ml). Slides weremounted in Vectashield mounting medium with DAPI (4�,6�-diamidino-2-phe-nylindole; Vector Laboratories, Burlingame, California) and examined by fluo-rescence microscopy.

Isolation and cleavage of fodrin-enrichment and cellular fractions. Fodrin-enrichment fractions were prepared according to the method of Villaseca et al.(51). Briefly, bladder and kidney cells were washed and lysed in phosphate buffer.Lysed cells were centrifuged, and the resulting pellet was washed three times inhypotonic phosphate buffer to obtain bladder and kidney cell membranes, whichrepresent fodrin-enrichment fractions. Cellular fractions (cytosol, membrane,nuclear, and cytoskeleton) were prepared from bladder and kidney cells usingthe FractionPREP cell fractionation system (BioVision, Mountain View, Cali-fornia) and as directed by the manufacturer. Cytoskeletal fractions were solubi-lized by sonication in 0.2% SDS, 10 mM dithiothreitol. The same quantity offodrin-enrichment or cellular fractions was incubated overnight at 37°C witheach concentrated supernatant (5 �g) in MOPS buffer. Reaction products wereseparated by SDS-6% PAGE (31).

Purification of Sat. Bacteria from overnight LB cultures of E. coliHB101(pDG4) were harvested by centrifugation (5,000 � g, 12 min, 4°C), andsupernatants were filter sterilized. Sterile supernatants were concentrated 20-fold using a 30,000-MWCO Pellicon XL filter unit (Millipore) and then another100-fold using a 100,000-MWCO Centricon Plus-80 filter (Millipore). This crudeconcentrate was subjected to ammonium sulfate precipitation as described else-where (54). Each cut was resuspended in anion-exchange buffer (0.025 M NaCl,0.025 M Tris-HCl, pH 7.5). The appropriate cut (�40% ammonium sulfatesaturation) was dialyzed overnight against anion-exchange buffer. Dialyzed sam-ple was applied over a 5-ml Econo-Pac High Q Cartridge (Bio-Rad, Hercules,California) which had been washed with 5 column volumes of anion-exchangebuffer. Elution was carried out in 0.025 M Tris-HCl, pH 7.5, with a salt gradientfrom 25 to 500 mM NaCl at a flow rate of 2 ml/min. Fractions enriched for Sat(�25% NaCl) were pooled, concentrated, and dialyzed overnight against gelfiltration buffer (0.150 M NaCl, 0.025 M Tris-HCl, pH 7.5). Dialyzed sample wasapplied to a HiPrep 16/60 Sephacryl S-200 high-resolution column (GE Health-care, Piscataway, New Jersey) previously equilibrated with gel filtration bufferaccording to the manufacturer’s protocol. Fractions were collected using gelfiltration buffer at a flow rate of 0.5 ml/min. Protein eluting in fractions consistent

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with the predicted 107-kDa-molecular-size Sat was pooled and concentrated. Allcolumn fractionation steps were conducted using the Biologic LP chromatogra-phy system and the BioFrac fraction collector (Bio-Rad).

Sat localization assay. Confluent bladder and kidney epithelial cells wereharvested by gentle rocking with glass beads in PBS. Cells were washed threetimes in PBS and resuspended in 1 ml supplemented Dulbecco’s modifiedEagle’s medium (Gibco) without fetal bovine serum. Cells were intoxicated withpurified Sat (25 �g/ml) or the corresponding volume of gel filtration buffer andincubated for 2 h at 37°C, 5% CO2. Viability was determined by trypan blue(Mediatech) staining. Supernatants from the intoxications were collected, andcells were washed three times in cold PBS. Cellular fractions were obtained asdescribed above. Fraction proteins (200 �g) were resolved by SDS-6% PAGE.Immunoblotting was performed as described above with the following excep-tions. Nitrocellulose membranes were incubated with primary rabbit anti-Satserum (diluted 1:500). After analysis of the fractions for Sat, nitrocellulosemembranes were stripped and immunoblotted with a cytoskeletal control pri-mary antibody against vimentin (diluted 1:500) (Abcam, Cambridge, Massachu-setts). Both primary incubations were followed by incubation with secondaryanti-rabbit immunoglobulin G peroxidase conjugate (diluted 1:2,500) (Sigma-Aldrich).

RESULTS

S256 is required for Sat serine protease activity and Sat-mediated degradation of human coagulation factor V andspectrin. The serine protease active site of the passenger do-main of Sat contains two serine residues (16). By using E. coliHB101 expressing wild-type Sat, single-nucleotide mutationswere made that led to predicted amino acid changes in the first(Sat S256I), the second (Sat S258A), or both (Sat S256I/S258A) serine residues. The serine protease active site mutantswere assessed for hydrolysis of methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (7), a colorimetric substrate optimal for Sat(12). Concentrated culture supernatants containing wild-typeor mutant versions of Sat were incubated with the syntheticsubstrate, and protease activity was monitored by absorbance(Fig. 1A). Sat S256I and Sat S256I/S258A had negligible serineprotease activity comparable to the vector control (P 0.001),

while Sat S258A had a 60% reduction in serine protease ac-tivity compared to wild-type Sat supernatant (P 0.001). Toconfirm the significance of the first serine residue, we mutatedthe nonpolar isoleucine back to a serine (Sat I256S). The singlemutation resulted in a distinct nucleotide sequence that nev-ertheless encoded the same amino acid sequence as wild type.The serine protease activity of Sat I256S was restored to wild-type levels. All enzymatically active proteins were completelyinhibited by 30 min of preincubation with 1 mM PMSF, apotent serine protease inhibitor (Fig. 1A, open bars).

In addition to synthetic substrates, Sat has been reported tocleave human coagulation factor V and spectrin (12). To eval-uate whether the serine protease activity of Sat is responsiblefor degradation of these substrates, concentrated culture su-pernatants containing wild-type Sat or the serine mutant de-rivatives of Sat were incubated with these proteins. Only wild-type and revertant Sat I256S supernatants were able todegrade both chains of human coagulation factor V (Fig. 1B)and the heterodimeric subunits of spectrin (Fig. 1C). Thesedata suggest that the first serine residue, S256, is the catalyti-cally active serine of Sat.

Sat serine protease activity is not required for export andrelease of Sat. To study secretion of wild-type and mutatedversions of Sat, concentrated culture supernatants were pre-pared from E. coli HB101 expressing the wild-type Sat, SatS256I, Sat S258A, Sat S256I/S258A, and revertant Sat I256Sand analyzed by SDS-10% PAGE (Fig. 2A) and Western blot-ting using anti-Sat serum (Fig. 2B). Sat (107 kDa) was presentin every sample, indicating that none of the mutations made inthe serine protease active site nor the reversion affected thetranslocation of the mature passenger domain and release ofSat into the culture supernatant.

Sat serine protease activity is required for cytotoxicity ofSat. To determine if the S256I mutation abrogates the ability

FIG. 1. Serine protease activity and substrate specificity of wild-type and mutant versions of Sat. (A) Concentrated culture supernatants (20 �gprotein) from E. coli HB101 transformed with empty vector or plasmids expressing wild-type Sat, Sat S256I, Sat S258A, Sat S256I/S258A, orrevertant Sat I256S were incubated with 1 mM methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide at 37°C for 24 h, with (open bars) or without (filledbars) pretreatment with 1 mM PMSF. Absorbance was read at 405 nm. All absorbance measurements were normalized to those obtained forwild-type Sat. Asterisks indicate absorbance values significantly different from wild-type Sat (P 0.001). (B and C) Purified human coagulationfactor V (B) or purified spectrin (C) was incubated with supernatants containing wild-type or mutant derivatives of Sat. Reaction products wereseparated by SDS-6% PAGE. With the exception of the first lane containing untreated substrate, for each pair of lanes, the left lane containssupernatants alone while the right lane contains substrate protein incubated with each indicated supernatant.

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of Sat to cause cytopathic effects on urinary cells, concentratedsupernatants from E. coli HB101 expressing wild-type Sat, SatS256I, and the reverted mutant Sat I256S were applied tomonolayers of human bladder and kidney epithelial cells (Fig.3). Both cell lines treated with concentrated supernatants ofwild-type Sat and Sat I256S showed a release of cellular focalcontacts from the slide substratum, generating a high level ofcell detachment from the epithelium (data not shown). Inaddition, host cells showed an elevated level of morphologicalalterations characterized by rounding (Fig. 3B and 3D, arrows)and membrane ruffling (Fig. 3B and 3D, filled arrowheads) ofbladder cells and elongation of kidney cells (Fig. 3F and 3H,open arrowheads). In contrast, bladder and kidney cellstreated with Sat S256I supernatant did not show morphologicalalterations and maintained their normal structure (Fig. 3C and3G), similarly to cells treated with supernatant from the neg-ative-control E. coli HB101 clone (Fig. 3A and 3E). We con-clude that mutation of residue S256 abrogates the ability of Satto cause cytopathic effects on urinary cells despite the fact thatthe same mutation has no effect on secretion and release of Satinto the supernatant.

Sat serine protease activity is required for morphologicalalterations induced in the actin cytoskeleton of bladder andkidney cells. The rounding and membrane ruffling of bladdercells and elongation of kidney cells suggest that Sat serineprotease activity disrupts the cytoskeleton or cytoskeleton-as-sociated proteins. To further characterize these effects, kidneyand bladder cells, incubated with Sat, were stained with fluo-rescein-labeled phalloidin and observed by fluorescence mi-croscopy (Fig. 4). Bladder cells treated with negative-controlsupernatant revealed classic cytoskeletal actin structures, withlinear F-actin stress fibers (Fig. 4A, arrows). After 2 h ofexposure to wild-type Sat or to revertant mutant Sat I256Ssupernatants, the bladder cells revealed contraction of thecytoskeleton (Fig. 4B and 4D, filled arrowheads) and a loss ofactin stress fibers (Fig. 4B and 4D, asterisks). However, super-natant containing the first-serine mutant Sat S256I did notproduce any of these cytoskeletal effects (Fig. 4C), as those

cells appeared similar to the control. Similarly, kidney cellstreated with wild-type Sat and Sat I256S supernatants revealeda different actin structure than did kidney cells treated withnegative-control supernatant (Fig. 4E). Indeed, the actin pro-trusions observed at the surface of the control cells were nolonger present and were replaced by the formation of surfaceblebs (Fig. 4F and 4H, open arrows). Moreover, intoxicatedkidney cells had a globular appearance and a loss of actinfilaments normally present at the edge of control and Sat S256Isupernatant-treated cells (Fig. 4E and 4G, open arrowheads).These data reveal that, in addition to serine protease activityand cytopathic effects, residue S256 is also necessary to causecharacteristic cytoskeletal effects seen in bladder and kidneycells treated with Sat.

Identification of potential protein targets of Sat in the mem-brane fraction of bladder and kidney cells. To identify poten-tial membrane targets of Sat, isolated bladder and kidney cellmembranes were incubated with each Sat supernatant andprotein profiles were resolved by SDS-6% PAGE. The resultsshowed degradation of a protein greater than 250 kDa in bothbladder (Fig. 5A) and kidney (Fig. 5B) membrane fractionstreated with wild-type Sat and revertant mutant Sat I256S. Incontrast, membrane profiles treated with the first-serine pro-tease Sat S256I appeared similar to untreated membranes andmembranes treated with negative-control supernatant. A pro-tein with homology to nonerythroid �-spectrin was identifiedby mass spectrometry (Table 1). Fodrin is nonerythroid spec-trin, a heterodimeric cytoskeletal protein which serves to linkactin filaments with other cytoskeletal proteins and the plasmamembrane (1). To determine whether Sat cleaves fodrin aspreviously observed with erythrocyte spectrin (12), a Westernblot assay of kidney cell fodrin-enrichment membrane frac-tions was performed using anti--fodrin chain antibodies (Fig.5C). Degradation products were detected in samples incubatedwith wild-type Sat or Sat I256S. Similar results were seen withfodrin-enrichment fractions obtained from bladder cells (datanot shown). The results confirmed that -fodrin is degraded bywild-type Sat and revertant Sat I256S but not by active-sitemutant Sat S256I.

Interestingly, we found another target protein, specific to thekidney membrane, degraded by wild-type Sat but not by SatS256I (Fig. 5B). The mass spectrometry results for this protein(Table 1) revealed homology with leukocyte function-associ-ated molecule 1 (LFA-1), which is a member of the �2-integrinfamily of cell surface receptors. Integrins are receptors forextracellular matrix (ECM) molecules and are counterrecep-tors for surface proteins of apposed cells (26). These data showthat Sat degrades target proteins in both bladder and kidneymembranes and that S256 is necessary for Sat-mediated degra-dation of these putative host cell target proteins.

Identification of potential protein targets of Sat in the cy-tosol and nuclear fractions of bladder and kidney cells. As weidentified target proteins of Sat in the membrane fraction ofhost cells, we hypothesized that Sat may have protein targets inother cellular fractions. Thus, cytosolic and nuclear compart-ments of fractionated bladder and kidney cells were incubatedwith each Sat supernatant and the protein profiles were ana-lyzed by SDS-6% PAGE (Fig. 6). No bladder and kidney cellcytosolic proteins were found degraded by Sat (data notshown). In contrast, we found susceptible substrate proteins

FIG. 2. Secretion of wild-type and mutant versions of Sat. (A) Coo-massie blue-stained SDS-10% polyacrylamide gel of concentrated cul-ture supernatants from E. coli HB101 expressing wild-type or mutantversions of Sat. Molecular masses (kDa) are shown on the left. (B) De-tection of Sat by Western blotting using anti-Sat serum. Lanes are thesame as indicated in panel A.

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degraded by wild-type Sat and revertant Sat I256S superna-tants, including three in the nuclear fraction of bladder cells(Fig. 6A) and two in the nuclear fraction of kidney cells (Fig.6B). Mass spectrometry analysis of these proteins (Table 1)

revealed that the high-molecular-mass (�250-kDa) proteinsdegraded in both bladder and kidney nuclear fractions werehomologous to microtubule-associated proteins (MAPs), whichare involved in nuclear and cell division, organization of intra-

FIG. 3. Cytotoxic activity of Sat and its first-serine mutant for bladder and kidney cells. Bladder (A to D) and kidney (E to H) cells wereincubated with concentrated culture supernatants of E. coli HB101 expressing empty vector (A and E), wild-type Sat (B and F), Sat S256I (C andG), and Sat I256S (D and H) for 2 h at 37°C. Cells were fixed and stained with Giemsa stain. Arrows indicate rounding of bladder cells, filledarrowheads point to membrane blebs on bladder cells, and open arrowheads point to the elongation of kidney cells. Bars, 100 �m for left panels(magnification, �400) and 50 �m for right panels (magnification, �1,000).

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FIG. 4. Effect of Sat and its first-serine mutant on the urinary epithelial cell cytoskeleton. Bladder (A to D) and kidney (E to H) cells wereincubated with 100 �g protein/ml of concentrated supernatant from E. coli HB101 expressing the empty vector (A and E), wild-type Sat (B andF), Sat S256I (C and G), and revertant Sat I256S (D and H) for 2 h at 37°C. Actin was stained green while the nuclei were stained blue. For bladdercells, linear F-actin stress fibers are indicated by arrows, contraction of the cytoskeleton is indicated by filled arrowheads, and loss of actin stressfibers is shown with asterisks. For the kidney cells, actin protrusions are indicated by open arrowheads and formation of surface blebs in place ofactin protrusions is indicated by open arrows. Left panels depict enlarged portions of images taken at a magnification of 400� while right panelsshow images taken at a magnification of 1,000�. Bar, 50 �m.

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cellular structure, and intracellular transport (2). The twoother proteins degraded in the bladder nuclear fraction werefound to be homologous to LIM domain-only protein 7 (28)and to signal-induced proliferation-associated 1-like protein 1,which is a GAP (GTPase-activating protein) of the Rap familyof small GTPases (8). The second protein degraded in thenuclear fraction of kidney cells was found to be homologous toa poly(ADP-ribose) polymerase (PARP), an enzyme of centralimportance in a wide variety of biological processes includingmaintenance of genomic stability, DNA repair, transcriptionalregulation, centromere function, modulation of telomerelength, and regulation of protein degradation, endosomal ves-

icle trafficking, and apoptosis (11). These results show that inaddition to membrane targets, Sat also degrades nuclear pro-teins with vital cellular roles. The relevance of these in vitrosubstrates depends, of course, on the ability of Sat to enter hostcells and localize accordingly.

Sat localizes to the cytoskeletal fraction of intoxicated blad-der and kidney cells. To successfully cleave putative targetproteins, Sat must be internalized by host cells and reachspecific intracellular locations. To assess this potential, bladdercells were intoxicated with biochemically purified Sat (25 �g/ml) for 2 h. It was determined that �80% of the bladder cellswere viable after intoxication, as assessed by trypan blue stain-ing. Cells were then washed and fractionated into cytosolic,membrane, nuclear, and cytoskeletal cellular components.Fractions were analyzed for the presence of Sat by Westernblotting with anti-Sat serum (Fig. 7). As expected, mature Sat(107 kDa) was detected in the sample supernatant of intoxi-cated bladder cells (Fig. 7, lane 5). More interestingly, Sat wasalso detected in the cytoskeletal fraction of intoxicated blad-der cells (Fig. 7, lane 17) but not in the cytoskeletal fractionof either untreated cells (Fig. 7, lane 15) or buffer-treatedcells (Fig. 7, lane 16). Sat was not detected in cytosolic,membrane, or nuclear fractions of Sat-treated samples (Fig.7, lanes 8, 11, and 14, respectively). Similar results werefound with kidney epithelial cells (data not shown). Alto-gether, these data indicated that upon intoxication, Sat en-ters host cells by an unknown mechanism and localizes tothe cytoskeletal fraction, where it can cleave target proteinssuch as spectrin and integrin.

DISCUSSION

Autotransporters comprise a family of virulence factors (22,32) characterized by a unique ability to promote their ownsecretion across the outer membrane of the bacterial envelope(20, 23, 45, 47). Sat is a representative member of the auto-transporter family of secreted proteins and belongs to a sub-family termed SPATE featuring a conserved serine proteasemotif (GDSGSP) (16). This motif resides in an analogousposition within Sat and other SPATE proteins (6, 9, 13, 19, 21,38, 40, 41, 46). Sat was previously shown by our laboratory toelicit a vigorous antibody response in mice transurethrally in-fected with the pyelonephritogenic parent strain E. coliCFT073 (16) and to cause cytopathic effects on human bladderand kidney epithelial cells (17). In this study, we demonstratedthat S256 is necessary for protease and cytopathic activity ofwild-type Sat but is not involved in processing or release of Satfrom the bacterial surface. Additionally, Sat is internalized byrelevant host cells and localizes to the cytoskeletal fractionwhere cleavable target proteins reside.

The serine protease motif was observed to be solely respon-sible for the protease activity of mature Sat, and more preciselyit was shown that S256 is the catalytically active residue withinthis motif. From the p-nitroanilide substrate data (Fig. 1A) it isclear that a single-nucleotide mutation that changes the firstserine within the active site to an isoleucine (S256I) abolisheswild-type protease activity. To demonstrate that Sat S256I didnot also carry a secondary mutation that was responsible forreduced protease activity, we made the isoleucine revert to aserine (I256S) and demonstrated for the first time a complete

FIG. 5. Effect of Sat and its first-serine mutant on bladder andkidney membrane proteins. The same quantity of bladder (A) andkidney (B and C) membrane proteins was incubated overnight at 37°Cwith 5 �g protein of each concentrated supernatant. Reaction productswere separated by SDS-6% PAGE. Molecular masses (kDa) are indi-cated on the left. The closest matches of proteins degraded by wild-type Sat and revertant Sat, but not by Sat S256I, identified by massspectrometry are shown by the arrows. (C) Western blot of fodrin-enrichment fractions from kidney cells incubated with wild-type andmutant derivatives of Sat. Lanes for panels B and C are as listed forpanel A.

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restoration of the wild-type serine protease activity of Sat. Asingle mutation in the second serine (S258A) reduced theprotein activity by more than half. These results were notunexpected, as analogous mutants of Pet (36), Pic (21, 38), andEatA (39) lost their respective proteolytic activities.

Controversy exists over how cleavage of the passenger do-main from the �-barrel occurs for many autotransporters, es-pecially whether cleavage is a result of membrane-bound pro-tease or an autoproteolytic event. Some members of theautotransporter family, such as immunoglobulin A1 proteaseand Hap from Haemophilus influenzae, rely on autoproteolysisinvolving the serine protease active site for processing andrelease of the passenger domain from the outer membrane (23,24). In this study we clearly demonstrate that Sat is not auto-processed by its serine protease active site, since mutants thatlack serine protease activity are still secreted from E. coli inmature form (Fig. 2). This was also observed for Pet (36),SepA (6), EspC (46), Pic of enteroaggregative E. coli (21), Tsh

(29), and EatA (39). These results suggest that another pro-tease is required for Sat release. Navarro-Garcia et al. (34)have shown that normal processing of the Pet precursor occursin the absence of DegP, OmpP, and OmpT proteases or DsbAisomerase. Due to the high degree of homology between Satand Pet, we hypothesize that other endogenous membrane-associated enzymes are involved in release of the toxin fromthe bacterial surface.

Cytopathic activity of wild-type Sat on urinary cell lines isdependent on proteolytic activity. None of the cytopathic ef-fects observed with supernatant containing wild-type Sat or SatI256S were seen in cells treated with Sat S256I supernatant,demonstrating that the cytotoxic activity is conferred by theserine protease active site of Sat (Fig. 3). Our results appearsimilar to those previously reported for active site mutants ofPet (36). Additionally, the cell damage caused by EspC wascharacterized by cell contraction and cell detachment and wasalso due to disruption of the actin cytoskeleton, specifically

TABLE 1. Host cell membrane and nuclear proteins, identified by mass spectrometry, that are susceptible to proteolysis by Sat

Band submitted(molecular mass �kDa ) Database Mass match

(%) Tolerance Molecular mass(kDa) Identified protein(s)

Bladder membrane (�250) Swiss-Prot 83 0.789 372 Bullous pimphigoid antigen 1 (BPAG1);contains 30 beta-spectrin repeats (4)

83 0.952 288 Nonerythroid spectrin beta IVNCBI 83 0.789 372 Bullous pimphigoid antigen 1 (BPAG1);

contains 30 beta-spectrin repeats (4)83 0.952 289 Nonerythroid spectrin beta IV

Kidney membrane1 (�250) Swiss-Prot 100 1.07 416 Spectrin, nonerythroid beta-chain 4

80 0.875 288 Spectrin, nonerythroid beta-chain 3NCBI 100 1.45 372 Bullous pimphigoid antigen 1 (BPAG1);

contains 30 beta-spectrin repeats (4)100 1.07 416 Spectrin, nonerythroid beta-chain 5

2 (100–150) Swiss-Prot 66 1.78 128 Leukocyte-function associated molecule 1NCBI 66 1.78 128 Leukocyte-function associated molecule 1

Bladder nucleus1 (�250) Swiss-Prot 77 0.944 409 Abnormal spindle-like microencephaly-

associated protein � MAP77 1.37 453 AKAP450 (centrosome- and Golgi complex-

localized PKN-associated protein) � MAPNCBI 77 1.37 452 AKAP450 (centrosome- and Golgi complex-

localized PKN-associated protein) � MAP

2 (100–150) Swiss-Prot 66 1.33 192 LIM domain-only protein 7NCBI 66 1.33 192 LIM domain-only protein 7

3 (100–150) Swiss-Prot 66 1.20 199 Signal-induced proliferation-associated 1-likeprotein 1

NCBI 66 1.20 199 Signal-induced proliferation-associated 1-likeprotein 1

Kidney nucleus1 (�250) Swiss-Prot 80 0.976 306 MAP 1A (proliferation-related protein P80)

NCBI 80 0.976 306 MAP 1A (proliferation-related protein P80)80 0.976 202 MAP 1A (proliferation-related protein P80)

2 (100–150) Swiss-Prot 85 0.818 113 PARPNCBI 85 0.818 113 PARP

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perinuclear contraction of F actin and loss of stress fibers (35).These data combined with the morphological changes anddetachment of Sat-treated bladder and kidney cells make ittempting to speculate that the cytopathic effects caused by Satare associated with damage to the actin cytoskeleton or cy-

toskeleton-associated proteins, which ultimately may contrib-ute to epithelial damage in urinary tract infections. Our resultsshow contraction of the cytoskeleton and loss of actin stressfibers as early as 2 h after addition of wild-type and revertantSat supernatants to bladder and kidney monolayers (Fig. 4). Asexpected, these alterations were absent from cells treated withSat S256I, confirming the significance of this residue in thecytopathic activity of Sat.

The cytoskeletal effects mediated by Sat on urinary epi-thelial cells are likely associated with the degradation offodrin (nonerythrocyte spectrin). Fodrin/spectrin is involvedin stabilizing membrane structures, maintaining cell shape,and linking actin filaments with the plasma membrane (5,10, 50). Our results affirmed that Sat is able to degrade both- and �-spectrin chains (Fig. 1C) as previously demon-strated by Dutta et al. (12) and similarly shown for Pet andEspC. In addition, similarly to its closest homolog Pet (51),Sat was shown to cleave -fodrin (Fig. 5C). Proteolytic at-tack on fodrin, thereby altering the cytoskeleton, mayexplain the rounding, elongation, membrane ruffling, anddetachment observed when urinary cells are treated withwild-type and revertant Sat (Fig. 3).

Another protein target degraded by Sat was identified in themembrane of kidney epithelial cells. Mass spectrometry deter-mination of this protein revealed homology with leukocytefunction-associated molecule 1 (LFA-1), which is a member ofthe �2-integrin family of cell surface receptors. When cellscome in contact with ECM, they extend filopodia to sample theterrain. Integrins at the tip of filopodia bind to the ECM andinitiate formation of focal adhesion. Actin-rich lamellipodiaare then generated, often between filopodia, as the cell spreadson the ECM (15). Extracellular ligand-bound integrins trans-duce a variety of signals which induce dramatic changes in theorganization of the cytoskeleton (43). This could explain thelack of organized actin protrusions and formation of surfaceblebs seen in cells treated with wild-type and revertant Satsupernatants (Fig. 4). Moreover, these data might explain pre-vious observations from Guyer et al. (17), who showed that Satelicits significant morphological changes specific to the kidney,including dissolution of the glomerular membrane and

FIG. 6. Effect of Sat and its first-serine mutant on bladder andkidney nuclear proteins. The same quantity of bladder (A) and kidney(B) nuclear proteins was incubated overnight at 37°C with 5 �g proteinof each concentrated supernatant. Reaction products were separatedby SDS-6% PAGE. Molecular masses (kDa) are indicated on the left.The proteins degraded by wild-type Sat but not by Sat S256I wereidentified by mass spectrometry. The closest matches are shown by thearrows. Lanes for panel B are as listed for panel A.

FIG. 7. Localization of Sat to the cytoskeletal fraction of intoxicated bladder epithelial cells. Lanes 1 and 2 contain 100 ng purified Sat proteinor an equal volume of gel filtration buffer, respectively. Bladder cells were untreated (lanes 3, 6, 9, 12, and 15), treated with gel filtration buffer(lanes 4, 7, 10, 13, and 16), or treated with 25 �g/ml purified Sat (lanes 5, 8, 11, 14, and 17) and incubated at 37°C with 5% CO2 for 2 h. Afterfractionation, each intoxication supernatant (1 �g) and each cellular fraction (200 �g) were analyzed by Western blotting using anti-Sat serum.Vimentin (56 kDa), the major subunit of intermediate filaments of mesenchymal cells, served as a fractionation and loading control for thecytoskeletal fraction (bottom panel, lanes 15 to 17).

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destruction of tubular epithelial cells following an experimen-tal UTI of CBA mice infected with wild-type E. coli CFT073 oran isogenic sat::pGP704 mutant.

In addition to membrane targets, in vitro analyses showedthat wild-type and revertant Sat I256S degrade nuclear pro-teins that play vital roles in eukaryotic cells (Fig. 6). Proteinshomologous to LIM domain proteins and Rap GAPs werefound degraded in the nuclear fraction of bladder cells whereasa protein homologous to a PARP was degraded in nuclearkidney cell fractions. Proteins identified as MAPs were foundto be degraded in both cell types. Although it is interesting tospeculate on a role for the degradation of nuclear proteins inthe pathogenesis of UTI, our localization data reveal that suchtargets may not be physiologically relevant and likely do notplay a role in the cytopathic effects seen in urinary epithelialcells.

As mentioned above, proteins homologous to nonerythro-cyte spectrin and �2-integrin were degraded in the membranefraction of bladder and kidney cells in vitro. Although degra-dation was detected in the membrane fraction of urinary epi-thelial cells, these proteins are important for cytoskeletal in-tegrity and function. Due to solubility issues with thecytoskeletal fraction in early experiments, distinct degradationof protein bands could not be detected as clearly as in themembrane fraction (Fig. 5 and data not shown). A later ex-periment showed that during intoxication, Sat is internalizedand localizes specifically to the cytoskeletal fraction of bladderand kidney epithelial cells (Fig. 7 and data not shown). Local-ization of Sat to the cytoskeleton during intoxication of hostcells combined with previous in vitro data showing host cellprotein degradation indicates that, upon entry into bladder andkidney host cells, Sat likely cleaves proteins associated with thecytoskeleton. Future studies will focus on characterizing theproposed interaction between Sat and candidate cytoskeletalprotein substrates.

Numerous bacterial toxins recognize and target the actincytoskeleton. Actin-ADP-ribosylating toxins and the Vibriocholerae RTX toxin directly affect structural proteins of thecytoskeleton. Others toxins alter the function of regulatoryelements in control of the cytoskeleton. These toxins includeRho GTPases, proteins that belong to the superfamily ofRas proteins (Rho-activating and inactivating toxins), andanother group that mimics eukaryotic master regulators(bacterial GAPs and guanine nucleotide exchange factors)(reviewed in reference 1). It was shown that the bacterialtoxins acting on the cytoskeleton dramatically disturb cellmorphology, intercellular junctions, the cell barrier perme-ability, and host cell processes dependent on actin (42).Despite differences in its structure and mode of action, Satappears to attack identical or similar eukaryotic targets asother bacterial toxins. Alteration of the cytoskeleton ap-pears to be a major mechanism for the host cell cytopathiceffects caused by Sat. By virtue of cytopathic effects causedin urinary epithelial cells, Sat is proposed to play an impor-tant role in virulence during infection of the urinary tract byE. coli and may function to aid bacteria in breaching theprotective epithelial cell barrier and invading the blood-stream, causing bacteremia in afflicted individuals.

ACKNOWLEDGMENT

This work was supported in part by Public Health Service grantAI43363 from the National Institutes of Health.

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Editor: D. L. Burns

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