the omptins of yersinia pestis and salmonella enterica cleave the

JOURNAL OF BACTERIOLOGY, Sept. 2010, p. 4553–4561 Vol. 192, No. 180021-9193/10/$12.00 doi:10.1128/JB.00458-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Omptins of Yersinia pestis and Salmonella enterica Cleave theReactive Center Loop of Plasminogen Activator Inhibitor 1�

Johanna Haiko,1 Liisa Laakkonen,1,2 Katri Juuti,1 Nisse Kalkkinen,3 and Timo K. Korhonen1*General Microbiology, Department of Biosciences, P.O. Box 56,1 Neuroscience Center, P.O. Box 56,2 and

Institute of Biotechnology, P.O. Box 65,3 University of Helsinki, FI 00014 Helsinki, Finland

Received 21 April 2010/Accepted 23 June 2010

Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule thatregulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important humanpathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhi-murium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavageof PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved,or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteasesplasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family oftransmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected withthe omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptinsubfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E.coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novelserpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypassnormal control of host proteolysis.

Plasminogen activator inhibitor 1 (PAI-1) is a key regulatorof the mammalian fibrinolytic/plasminogen system (29, 37).The fibrinolytic system comprises the serine protease zymogenplasminogen, urokinase-type plasminogen activator (uPA), tis-sue-type plasminogen activator (tPA), PAI-1, and plasmin in-hibitor �2-antiplasmin (�2AP) (for a review, see reference 52).Plasminogen is converted to plasmin, which is a broad-spec-trum serine protease that dissolves fibrin in blood clots, de-grades laminin of basement membranes, and activates matrixmetalloproteinases that degrade collagens and gelatins in tis-sue barriers. Herewith, plasmin controls physiological pro-cesses such as fibrinolysis/coagulation, cell migration and inva-sion, and tumor metastasis (29, 37). PAI-1 maintains normalhemostasis by inhibiting the function of the plasminogen acti-vators tPA and uPA, which are serine proteases and highlyspecific for cleavage of the plasminogen molecule. tPA binds tofibrin and is associated with plasmin-mediated breakdown offibrin clots, whereas uPA has low affinity for fibrin and associ-ates with cell surface proteolysis, cellular migration, and dam-age of tissue barriers (52).

The mammalian fibrinolytic and coagulation systems aretargeted by invasive bacterial pathogens during infection (re-viewed in references 6, 11, 34, and 61). In bacterial sepsis,increased production of fibrin clots at a damaged endotheliumresults from enhanced thrombin-catalyzed fibrin generationand from an increased serum level of PAI-1. Coagulation canprotect the host by activating immune systems or by physicallyrestraining the bacteria (6, 15, 25, 41). On the other hand,several invasive bacterial pathogens enhance fibrinolysis either

through direct plasminogen activation or by immobilizing plas-minogen/plasmin on the surface (6, 34, 61). Activation of theplasminogen system by bacteria enhances bacterial dissemina-tion and invasiveness through release of bacteria from fibrindeposits and through degradation of tissue barriers. Bacterialplasminogen activators and receptors have been under exten-sive structural and functional studies, but much less is knownabout interactions of bacteria with the regulatory proteins offibrinolysis.

PAI-1 is present in a large variety of tissues and is secretedby several human cells (37). In healthy individuals, the level ofPAI-1 antigen in human plasma is low (6 to 85 ng/ml), butsynthesis and secretion of PAI-1 are strongly elevated in dis-ease states and induced by, e.g., inflammatory cytokines andendotoxin of Gram-negative bacteria (37). PAI-1 is a serineprotease inhibitor (serpin), which exists in two forms. In itsactive form, PAI-1 rapidly inactivates both tPA and uPA byforming a covalent bond between the hydroxyl group of acatalytic serine residue of tPA/uPA and the carboxyl group ofthe residue R346 at the reactive center loop (RCL) of PAI-1(52). The RCL of PAI-1 is a 19-amino-acid-long flexible loopwhich inserts into the catalytic center of tPA/uPA and containsthe “bait” residues R346 and M347, which mimic the normaltarget of tPA/uPA. PAI-1 induces distortion of the active siteof tPA/uPA, which prevents completion of the catalytic cycle(70). The active form of PAI-1 is unstable, with a half-life of 2to 3 h at 37°C, and it changes spontaneously and irreversiblyinto a latent form, where the RCL is incorporated into acentral �-sheet of the PAI-1 molecule and therefore cannotreact with tPA or uPA. This conformational change takes placealso after proteolytic cleavage of PAI-1 at the R346-M347bond. The active form of PAI-1 binds with high affinity tovitronectin (Vn), and PAI-1/Vn complex formation increasesthe half-life of PAI-1 2- to 4-fold (10, 46, 69). Most circulating

* Corresponding author. Mailing address: General Microbiology,Department of Biosciences, P.O. Box 56, University of Helsinki, FI00014 Helsinki, Finland. Phone: 358 9 19159260. Fax: 358 9 19159262.E-mail: [email protected].

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PAI-1 is thought to be in a complex with Vn, and the complexserves as the reservoir of physiologically active PAI-1 (44).

Plague disease caused by Yersinia pestis is associated withimbalance of the fibrinolytic system, and decreased fibrin(o-gen) deposition has been observed in both bubonic and pneu-monic plague (11, 36). The plasminogen activator Pla, which isencoded by a Y. pestis-specific 9.5-kb virulence plasmid, pPCP1(59), does not degrade fibrin directly but mimics the action oftPA and uPA in converting plasminogen to plasmin by cleav-age at R561-V562. Pla also degrades the serpin �2AP and thuscreates uncontrolled plasmin activity (32, 60). Pla belongs tothe omptin superfamily of bacterial �-barrel outer membraneproteases (for reviews of omptins, see references 21 and 23).The omptins share molecular size and transmembrane fold butdiffer markedly in their substrate selectivities. In their catalyticcenters, omptins combine structural features of aspartic andserine proteases (66).

Increased fibrinolysis observed in plague led us to investigatewhether Y. pestis increases plasminogen activation also indi-rectly by controlling the activity of PAI-1. We compared Y.pestis to Salmonella enterica serovar Typhimurium and Yersiniapseudotuberculosis, and the study also included omptins ofother enterobacterial species.

MATERIALS AND METHODS

Bacterial strains and plasmid constructs. The bacterial strains and plasmidsare listed in Table 1. pla and pgtE and their derivatives and ompT of Escherichia

coli AAEC072 were available from previous work (32, 33). The chromosomalompT and the plasmid-encoded ompTP genes of E. coli IHE 3034, sopA ofShigella flexneri M90T, kop (Klebsiella outer membrane protease) of Klebsiellapneumoniae 342, epo (plaA) of Erwinia pyrifoliae Ep1/96, and the Yersinia chro-mosomal omptin genes ycoA of Y. pestis KIM D34 and ycoB of Y. pseudotuber-culosis IP 32953 were cloned and expressed as described earlier (32, 33). S.enterica and E. coli XL1 were cultivated as described previously (22, 48). Y. pestiswas cultivated over two nights at 37°C on brain heart infusion (BHI) plates,inoculated in BHI broth with hemin (40 �g/ml), and cultivated twice over twonights at 37°C. Y. pseudotuberculosis strains were cultivated overnight at 37°Cin Luria broth, and Y. pseudotuberculosis PB1 was grown with kanamycin (50�g/ml). E. pyrifoliae was cultivated in Luria broth overnight at 28°C. E. coliIHE 3034, K. pneumoniae, and S. flexneri were cultivated in Luria brothovernight at 37°C.

Degradation of the PAI-1/Vn complex. Recombinant active human PAI-1 (2.5�g; American Diagnostica, Stamford, CT) and human plasma Vn (5 �g; Pro-mega, Madison, WI) were incubated for 1 h at 37°C in a volume of 5 �l to forma complex (69). Bacteria (20 �l, 4 � 107 cells) in phosphate-buffered saline (PBS;pH 7.1) were incubated with the complex for 2 to 8 h at 37°C. The bacteria werepelleted, the supernatants were run in a 12% (wt/vol) SDS-PAGE gel, and PAI-1and Vn were detected by Western blotting with polyclonal anti-Vn (1:1,000;Calbiochem, Darmstadt, Germany) and anti-PAI-1 (1:5,000; Calbiochem) anti-bodies, alkaline phosphatase-conjugated anti-rabbit immunoglobulin G (Dako,Glostrup, Denmark), and phosphatase substrate.

Peptide analyses. For peptide analysis, recombinant E. coli bacteria (4 � 107

cells) expressing Pla or PgtE were incubated with PAI-1 (12 �g) for 2 h in 26 �lof PBS. The N-terminal sequence of degraded PAI-1 (PAI-1*) was obtained byEdman degradation. For matrix-assisted laser desorption ionization–time offlight mass spectrometry (MALDI-TOF MS), the PAI-1* fragment was cut fromthe gel, digested with trypsin, and exposed to MS. The peptides were analyzedwith the Mascot search engine (www.matrixscience.com). To determine the exactcleavage site of PAI-1, we incubated 24 �g PAI-1 with 40 �l bacteria (8 � 107

TABLE 1. Bacterial strains and plasmids used in this study

Bacterial strainor plasmid Description Reference(s)

or source

StrainsErwinia pyrifoliae Ep1/96 Wild type 51Escherichia coli IHE 3034 Wild type, meningitis isolate 45Escherichia coli XL1-Blue MRF� �(mcrA)183 �(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1

gyrA96 relA1 lac �F� proAB lacIqZ�M15 Tn10 (Tetr)�Stratagene

Klebsiella pneumoniae 342 Wild type 16Salmonella enterica serovar

Typhimurium 14028RRough derivative of 14028 68

Salmonella enterica serovarTyphimurium 14028R-1

�pgtE derivative of 14028R 35

Shigella flexneri M90T Wild type 55Yersinia pestis KIM D27 pPCP1 �pgm pYV derivative of Y. pestis KIM-10 14, 64Yersinia pestis KIM D34 pPCP1, otherwise identical to KIM D27 14, 64Yersinia pseudotuberculosis IP 32953 Wild type 9Yersinia pseudotuberculosis PB1

�wb(pYV)Rough derivative of PB1, with the 20-kb ddhD-wzz

chromosomal fragment deletedJ. A. Bengoechea

Yersinia pseudotuberculosis PB1�wb(pYV)

Rough derivative of PB1, with the 20-kb ddhD-wzzchromosomal fragment deleted, cured of pYV

33

PlasmidspSE380 Expression vector, trc promoter, lacO operator, lacI bla InvitrogenpMRK1 pla in pSE380 32pMRK3 pgtE in pSE380 33pMRK111 pMRK13Pla D206A 32pMRK31 pMRK33PgtE D206A 33pMRK2 ompT of E. coli AAEC072 in pSE380 32pMRK2b Chromosomal ompT of E. coli IHE 3034 in pSE380 This workpMRK2c Plasmid ompT of E. coli IHE 3034 in pSE380 This workpMRK4 epo in pSE380 This workpMRK7 sopA in pSE380 This workpMRK8 ycoA in pSE380 This workpMRK9 ycoB in pSE380 This workpMRK10 kop in pSE380 This work

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cells), isolated the cleavage products by reversed-phase chromatography, andperformed MALDI-TOF MS analysis.

PAI-1 activity. PAI-1 (0.5 �g) and Vn (1.0 �g) were pipetted on 96-wellmicrotiter plates (Nunc, Roskilde, Denmark) in 44 �l PBS and incubated for 1 hat 37°C. Bacteria (16 �l, 3.2 � 107 cells) were then added, and after incubationfor 4 h, high-molecular-weight uPA (160 ng, 12.8 IU; American Diagnostica) wasadded and the incubation was continued for 30 min. Chromogenic uPA substrateS-2444 (2.1 mg/ml; Chromogenix, Milan, Italy) was added, and A405 was mea-sured after 3 h. The averages and standard deviations were calculated from threeassays with duplicate samples; for Y. pestis, two assays were used. Significanceswere calculated with Student’s t test, and probability values of less than 0.01 wereregarded as significant differences.

Modeling of Pla with PAI-1. The complex between Pla and the RCL of PAI-1was modeled based on crystal structures of OmpT (Protein Data Bank [PDB]code 1i78) and PAI-1 (PDB code 1dvm, residues 340 to 351) with the programModeller 9v7 (54). The RCL was cut out of the overall structure and broughtmanually close to the active site residues of Pla. A water molecule was addedbetween D84 and H208, as suggested previously (66). To allow for better con-formational searching, seven central residues of the PAI-1 fragment were omit-ted from the template, and the remaining residues were mutated to alanines. Theformation of the complex was guided by distance constraints from the reactivewater molecule to D84, H208, and the carbonyl carbon of the scissile peptidebond. Several starting positions were generated. In each run, 20 structures wereproduced with the standard modeling scheme, optimized, and analyzed visuallywith the program VMD (24).

Protein structure accession numbers. NCBI accession numbers of proteinstructures used in this study are as follows: Pla, AAA27667 (58); PgtE,AAF85951 (19); Epo, YP_003208082 (M. Kube et al., unpublished); Kop,YP_002235876 (16); Enterobacter omptin, YP_001176628 (A. Copeland et al.,

unpublished); YcoA, NP_670257 (12); YcoB, YP_069801 (9); E. coli K-12(AAEC072) OmpT, CAA30008 (18); E. coli IHE 3034 OmpT, ADE89723 (40a);IHE 3034 OmpTP, HM210637; SopA, AAC45084 (13); OmpP, BAA97899 (27);Leo, YP_124757 (8); Vibrio fischeri omptin, AAW85741 (53); Desulfotalea psy-chrophila omptin, YP_066438 (47); Mesorhizobium loti omptin, NP_106686 (26);and CroP, YP_003365570 (43).

RESULTS

Y. pestis and S. enterica degrade the PAI-1/Vn complex. Inorder to investigate the interaction of Y. pestis and S. entericaserovar Typhimurium with PAI-1, the bacteria were incubatedwith the PAI-1/Vn complex and the proteins were detectedwith Western blotting. We also analyzed degradation of PAI-1without Vn (data not shown), and the results were the same asthose with the PAI-1/Vn complex. Results obtained with thePAI-1/Vn complex are shown (Fig. 1), because the complexrepresents the circulating form of PAI-1.

Pla and PgtE require bound lipopolysaccharide (LPS) fortheir proteolytic activity, but the presence of O antigen in theLPS sterically inhibits proteolysis by Pla, PgtE, and OmpT (31,33). Y. pestis has lost the genes encoding the O antigen (57),and S. enterica serovar Typhimurium modifies its LPS to arough form during infection (35). For these reasons, we per-formed the analyses with rough mutant strains S. enterica

FIG. 1. Degradation of the PAI-1/vitronectin complex by S. enterica, Y. pestis, and Y. pseudotuberculosis, detected with anti-PAI-1 (A) andanti-Vn (B) antibodies. The bacteria are indicated above the lanes; PBS indicates PAI-1/Vn incubated in buffer without bacteria. Migrationdistances of molecular mass markers (in kilodaltons) are indicated on the left.

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14028R and Y. pseudotuberculosis PB1; the latter representsthe likely evolutionary ancestor of Y. pestis (1, 57).

Y. pestis KIM D27 and S. enterica 14028R induced a limiteddegradation of PAI-1 into a smaller fragment, termed herePAI-1*, and they also cleaved Vn into smaller fragments (Fig.1). No degradation of PAI-1 or Vn was observed with Y. pestisKIM D34, which lacks the pla-encoding plasmid pPCP1, orwith S. enterica 14028R-1, from which pgtE has been deleted.We assessed the degradation of PAI-1 by Y. pseudotuberculosisPB1 �wb with or without pYV, a virulence plasmid shared byY. pestis and Y. pseudotuberculosis. Both Y. pseudotuberculosisstrains failed to cleave PAI-1 and Vn (Fig. 1).

Omptins of Y. pestis and S. enterica cleave PAI-1. The resultsdescribed above suggested that omptins of Y. pestis and S.enterica cleave PAI-1, and we next assessed the time course ofPAI-1/Vn cleavage by recombinant E. coli expressing omptingenes. We tested degradation from 2 to 8 h, as the 8-h timepoint is close to the half-life of active PAI-1 in a complex withVn. Western blotting with the empty vector strain E. coliXL1(pSE380) did not reveal degradation of PAI-1, and thedegradation pattern of PAI-1 by XL1(pMRK1) expressing Plaremained limited to the formation of PAI-1* (Fig. 2).XL1(pMRK3) with PgtE cleaved PAI-1 more rapidly, withnearly all PAI-1 degraded within 2 h, and a major fragment ofthe size of PAI-1* as well as two smaller PAI-1-derived pep-tides of about 25 kDa was detected.

We next determined the cleavage site in the PAI-1* peptideproduced by Pla and PgtE expressed in E. coli. N-terminalsequencing revealed the presence of an intact N terminus(VHHPP…) in PAI-1*. MALDI-TOF MS analysis of the re-versed-phase purified fragments gave an m/z of 38,972 Da(PgtE-generated PAI-1*) and an m/z of 38,982 Da (Pla-gener-ated PAI-1*), values which correspond well, within the massaccuracy of the analysis method, to the calculated average mass(38,983 Da) of the PAI-1 fragment V1-R346. The analyses thuslocated the cleavage site between R346 and M347 in the RCLof PAI-1. Peptide mass fingerprint analysis of trypsin-cleavedPla- or PgtE-generated PAI-1* identified peptide fragmentscovering only sequences from V1-R346, which further sup-ported the location of the cleavage site at the bait residues ofPAI-1. By Western blotting, two PAI-1*-like peptides formedby PgtE-expressing XL1 were occasionally observed (see Fig.4); MS analysis, however, revealed the presence of one PAI-1*fragment only. The 25-kDa polypeptides generated by PgtEreacted strongly with the anti-PAI-1 antibodies in Western

blotting but were present in amounts too small for N-terminalor mass spectrometric characterization.

Serpin activity of PAI-1 is decreased by Pla and PgtE. Weassessed the serpin activity of the PAI-1* fragment, i.e., itsability to inhibit uPA, the natural target of PAI-1, by incubat-ing the bacteria with the PAI-1/Vn complex, uPA, and uPAsubstrate. Control samples without bacteria (bars designatedPBS in Fig. 3) showed that the PAI-1/Vn complex completelyabolished uPA activity. PAI-1* formed by Pla- and PgtE-ex-pressing Y. pestis or S. enterica did not exhibit considerableserpin activity, whereas the serpin activity of PAI-1 incubatedwith Y. pestis KIM D34 or S. enterica 14028R-1 was high andsimilar to the activity of PAI-1 incubated without bacteria (Fig.3). The vector strain E. coli XL1(pSE380) did not abolishPAI-1 activity, whereas recombinant E. coli XL1 expressing Plaor PgtE impaired serpin activity (Fig. 3). In contrast, PAI-1incubated with E. coli XL1(pMRK111) expressing Pla D206Aor XL1(pMRK31) expressing PgtE D206A retained the capac-ity to inhibit uPA. Thus, proteolytic action of Pla and PgtE onPAI-1 resulted in loss of the serpin activity.

Cleavage of PAI-1 by other enterobacterial omptins. Wenext compared degradation of PAI-1 by several enterobacterialomptins expressed in E. coli XL1. A cladogram presentation ofthe omptin sequence alignment is shown in Fig. 5. In a 2-hincubation, PAI-1 cleavage and PAI-1* formation were de-tected with XL1 expressing Pla, PgtE, Epo (PlaA) of E. pyri-foliae, and Kop of K. pneumoniae (Fig. 4), which all belong tothe Pla subfamily of omptins (Fig. 5). E. coli XL1(pMRK111)and XL1(pMRK31) strains, expressing the catalytic residuemutations Pla D206A and PgtE D206A, respectively, werenegative for degradation, which confirms that the proteolyticactivity is needed for PAI-1 cleavage. The individual omptinsdiffered in efficacy of PAI-1 degradation, with PgtE and Kop

FIG. 2. Degradation of the PAI-1/vitronectin complex by recombi-nant E. coli incubated for 2, 4, 6, and 8 h, detected with anti-PAI-1antibody. The proteins expressed in E. coli XL1 are indicated abovethe lanes; vector indicates the host strain with blank vector. Migrationdistances of molecular mass markers (in kilodaltons) are indicated onthe left.

FIG. 3. Inactivation of PAI-1 by Y. pestis, S. enterica, and recombi-nant E. coli XL1. Bacteria were incubated with the PAI-1/Vn complexor in buffer, uPA and uPA substrates were added, and the cleavage ofthe uPA substrate was measured by absorbance at 3 h. The bacteria areindicated at the bottom. PBS denotes a control without bacteria. Av-erages and standard deviations from two (Y. pestis) or three (S. en-terica, E. coli, and PBS) assays with duplicate samples are shown. *,P � 0.01.



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degrading nearly all PAI-1 in 2 h. No degradation was detectedwith the omptins YcoA of Y. pestis and YcoB of Y. pseudotu-berculosis (Fig. 4), which form another omptin subfamily.OmpT occurs as two major variants, the chromosomally en-coded OmpT and the plasmid-encoded OmpTP, which differby 24 to 25% in mature sequences. We included chromosoma-lly encoded OmpT variants from the E. coli K-12 strainAAEC072 and from the meningitis isolate IHE 3034 and aplasmid-encoded OmpTP variant from IHE 3034. No deg-radation of PAI-1 was observed with either of the OmpT

variants or with SopA of S. flexneri, which belongs to thesame subfamily.

Bioinformatic analysis of the interaction between Pla andPAI-1. The 16 known complete omptin protein sequences werealigned and analyzed with regard to the known OmpT struc-ture (see Fig. 7 for the alignment). A majority of the fullyconserved residues (19/24) are found in the periplasmic half ofthe barrel. Interestingly, seven of them (E29, L43, W45, M82,I104, Y168, and Q170) cluster at the bottom of the barrelopening, hydrogen bonded to the active site residues, and seemto form the binding site walls for a large and basic amino acid,such as R346 in the RCL of PAI-1 (Fig. 6). Docking of thePAI-1 RCL to the Pla model shows how R346 can extend intothe catalytic, acidic groove of omptins formed by the conservedresidues.

When those omptins that are able to cleave PAI-1 werecompared as a group to the inactive ones, all differences werefound in the extracellular loops. Specifically, comparing OmpTand Pla, insertions, deletions, and major differences are foundat residues 36 and 37, 93 and 94, 151 to 156, 210 and 211, and269 to 273 of Pla (Fig. 7). Comparing these sites betweenPla/PgtE/Epo/Kop and OmpT/SopA/Yco, putatively importantamino acid differences are found at T36, L213, I260, D266, andD270.

DISCUSSION

To our knowledge, this is the first description of a specificinactivation of the serpin activity and reactive center of PAI-1by enteric bacteria. The omptins of Y. pestis and S. entericaserovar Typhimurium exhibited limited cleavage of the RCL ofthe PAI-1 molecule at the R346-M347 bond, which is thefunctional bait peptide bond of PAI-1. The resulting PAI-1*peptide was unable to inhibit enzymatic activity of uPA and

FIG. 4. Degradation of the PAI-1/vitronectin complex by recombi-nant E. coli incubated for 2 h, detected with anti-PAI-1 antibody. Theproteins expressed in E. coli XL1 are indicated above the lanes; vectorindicates the host strain with blank vector, and PBS indicates PAI-1/Vnincubated in buffer without bacteria. OmpTP denotes plasmid-encodedOmpT of IHE 3034. Omptin subfamilies are indicated. Migrationdistances of molecular mass markers (in kilodaltons) are shown on theleft.

FIG. 5. Cladogram presentation of omptin sequence alignment.Phylogenetic analyses were conducted using the MEGA4 program(63). The phylogenetic tree was linearized assuming equal evolutionaryrates in all lineages. The tree is drawn to scale, with branch lengths inthe same units as those of the evolutionary distances used to infer thephylogenetic tree. The evolutionary distances were computed using thePoisson correction method and are reported as the number of aminoacid substitutions per site.

FIG. 6. Model of the Pla–PAI-1 complex. The extracellular regionof the omptin barrel is shown as a white cartoon structure. Surface-accessible segments of the extracellular loops L1 to L5 of 20 differentPla model structures are shown as thin white C-alpha traces, to high-light their mobility. The PAI-1 RCL is bound to the active site and isshown in yellow. The catalytic residues of Pla are shown as green sticks,the conserved residues at the bottom of the binding site are in blue,and the most variable surface residues between Pla/PgtE/Epo/Kop andOmpT/SopA/Yco are in red. The water molecule attacking the peptidecarbonyl is shown as a red sphere. Nitrogens are blue and oxygens red.The loops are indicated in red letters.



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thus lacks serpin activity. The overall outcome of the inactiva-tion of PAI-1 is enhanced host-derived proteolysis, i.e., plas-min formation, during infection and inflammation. For Y. pes-tis, the role of plasmin generation and activity in bacterialinvasiveness has been well documented (11, 36, 56, 60),whereas the role in other enterobacterial infections has beenless studied. We have earlier observed that Pla and PgtE de-grade another serpin, �2AP (32, 35), and our present resultssuggest that serpinolytic activity is a common function of theproteinases in the Pla subfamily of omptins. Y. pestis evolvedfrom Y. pseudotuberculosis only 1,500 to 20,000 years ago (1),and the molecular basis of their different pathogenetic poten-tials has attracted considerable interest. We observed here thatY. pseudotuberculosis cells and the Yco omptins do not cleavePAI-1, and they neither cleave nor activate plasminogen (J.Haiko, unpublished). The Pla-mediated, wide attack on con-trol of an important hemostatic process in humans, i.e., thefibrinolysis/coagulation balance (32, 60, 71), represents a con-siderable difference in pathogenetic potential between the twobacterial species.

The omptin Epo also cleaved human PAI-1; this omptinassociates with the plant pathogen E. pyrifoliae, which causespear blight (30), and epo is carried on a mosaic plasmid,pEP36, that is closely related to the virulence plasmid pEA29of Erwinia amylovora (40). Serpins of mammals and plantsshare structural features, which include a similar, exposedRCL that is inserted into the catalytic groove of the targetproteinases (17). Searches in databanks for homologs of PAI-1did not identify any in pears but several in the rose family (49).Thus, it is plausible that a function of Epo is associated withserpinolytic activity and tissue damage in plants. Our ongoingresearch (J. Haiko, unpublished) has shown that Epo is a verypoor plasminogen activator, and hence we prefer the nameEpo (Erwinia pyrifoliae omptin) instead of PlaA, introduced byMcGhee et al. (40).

Pla and PgtE caused limited proteolysis of the PAI-1 RCL.Pla activates plasminogen through a single cut at the peptidebond R561-V562, and in both plasminogen and PAI-1 thecleavage site for Pla is located in loop regions that are flexiblebut do not share sequence similarity. Pla and PgtE also impairthe activity of �2AP through a single cut (32, 35). �2AP has an18-amino-acid-long RCL that is only 30% identical to thePAI-1 RCL in primary sequence but has Arg-Met as the baitresidues (17). The cleavage site in �2AP for Pla/PgtE has notbeen determined, but it might be that the serpinolytic mecha-nism is similar to what was observed here with PAI-1. PAI-1cleavage by PgtE-expressing bacteria was rapid and also pro-duced peptides that were not detected with Pla-expressingbacteria, indicating slightly different cleavages of PAI-1 by Plaand PgtE. Cleavage of PAI-1 by E. coli expressing Pla did notlead to complete degradation of PAI-1 in an 8-h incubation.Eight hours is approximately the half-life of PAI-1 in a com-plex with Vn, and our hypothesis is that a fraction of the PAI-1molecules spontaneously changed into the latent form duringthe incubation, and the poorly accessible RCL in latent PAI-1could not be cleaved by the bacteria. PgtE-expressing recom-binant E. coli caused a more rapid cleavage of PAI-1 to PAI-1*than did E. coli with Pla. Both omptins are expressed at similarlevels in recombinant E. coli (33), and the results thus suggestthat PAI-1 is a better substrate for PgtE than for Pla.

FIG. 7. Sequence alignment of omptins. Sixteen known omptin se-quences were aligned with ClustalW, and the image was rendered withAlscript. The profile mode alignment was guided by the secondary struc-ture of the experimentally known OmpT structure. The � strands and theextracellular loops are numbered, and the surface-accessible residues areboxed. Irregularities in the � strands are marked in red. Amino acids arecolored according to their chemical nature. Names of those omptins thatare able to cleave PAI-1 are written in green, and those of the noncleav-ing, tested omptins are in red. Residue numbers refer to Pla. The catalyticresidues are indicated by arrowheads.



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We observed that Pla expressed in Y. pestis KIM D27 fromits natural promoter in plasmid pPCP1 inactivated PAI-1 moreefficiently than did Pla expressed in the E. coli XL1 host.Acylation of LPS and substitution of lipid A phosphates withaminoarabinose have an indirect effect on the enzymatic activ-ity of Pla in Y. pestis (62). At 37°C, Y. pestis expresses tetra-acylated LPS, with a low content of aminoarabinose, and theouter membrane is more fluid (4, 5, 28), which increases theenzymatic activity of Pla (62). E. coli expresses mainly hexa-acylated LPS, and this probably explains the slower formationof PAI-1* by Pla expressed in E. coli. On the other hand, PgtEin recombinant E. coli showed more efficient inhibition ofPAI-1 than did PgtE encoded by the chromosome of S. en-terica, which possibly results from the higher copy number ofpgtE in recombinant E. coli.

Molecular modeling indicated that both the catalytic resi-dues and the substrate binding pocket are conserved inomptins, and therefore the observed difference in PAI-1 cleav-age by Pla/PgtE/Epo/Kop and OmpT/SopA/Yco cannot be ex-plained by properties of the binding pocket or by catalyticinactivity. Instead, the explanation seems to arise from theability to form the enzyme-substrate complex. We hypothesizethat amino acid residues at the sites where majority of thedifferences are found promote, or the corresponding residuesin OmpT/SopA/Yco prevent, the interaction with PAI-1. Thishypothesis is supported by mutagenesis scanning, loop swap-ping, and substitution analyses showing that the polypeptidesubstrate selectivities of Pla, OmpT, and PgtE for plasminogenand gelatin are dictated by their surface loops (22, 32, 48).

Vn serves a wealth of physiological functions in addition tostabilizing PAI-1 in circulation (46), and hence the Vn degra-dation observed here may have other biological consequencesas well. The characterization of the biological role of Vn deg-radation, however, is beyond the scope of this communication.Vn is organized into three domains, of which the N-terminalsomatomedin B domain is the binding site for PAI-1 (46). Ouranalyses have shown that Pla and PgtE cleave Vn at the C-terminal region (J. Haiko and N. Kalkkinen, unpublisheddata), which indicates that the binding epitope for PAI-1 in theVn molecule is not cleaved by Pla or PgtE.

The primary functions of PAI-1 are in physiological controlof the coagulation/fibrinolysis balance. Recent studies have,however, demonstrated that PAI-1 is important in host defenseagainst respiratory tract and systemic infections caused byGram-negative bacteria. PAI-1 levels are elevated in patientswith severe pneumonia and sepsis, and host defense againstpneumonia and sepsis caused by K. pneumoniae in mice wasimpaired by PAI-1 deficiency and improved by overexpressionof PAI-1 (20, 50). The exact mechanisms of how PAI-1 en-hances protection against Klebsiella infection in vivo remain tobe characterized, but the PAI-1 degradation by Kop of Kleb-siella indeed may enhance virulence of K. pneumoniae.

Degradation of PAI-1 has earlier been observed with theserine protease subtilisin NAT of Bacillus subtilis and the ther-molysin-like metalloproteinases aureolysin of Staphylococcusaureus and LasB of Pseudomonas aeruginosa (2, 3, 7, 65). Theseare extracellularly secreted enzymes, and their cleavage pat-terns with PAI-1 and plasminogen are different from those ofPla and PgtE. S. aureus and P. aeruginosa are severe pathogenswhich show multiple interactions with the human fibrinolytic

system (2, 6, 34, 39, 61), and LasB is an acknowledged viru-lence factor in lung and skin infections. A functional differencewith pathogenetic potential is that the aureolysin/LasB metal-loproteinases cleave plasminogen at the amino-terminal regionand at S441-V442, which results in miniplasminogen that is notenzymatically active but can be activated by tPA (2, 3), whereasPla/PgtE cleave plasminogen at R561-V562, which generatesactive plasmin. Subtilisin NAT cleaves PAI-1 at the bait resi-dues, similarly as in hydrolysis by Pla and PgtE, but does notcleave �2AP and thus appears to have a more limited serpino-lytic activity than Pla or PgtE (65).

Y. pestis and S. enterica cause life-threatening invasive infec-tions, and due to the important pathophysiological role ofPAI-1, we assume that the disturbance of the hemostatic bal-ance by Pla and PgtE plays an important role in pathogenesisof plague and salmonellosis. Whereas Y. pestis spreads mainlyextracellularly, S. enterica is an intracellular pathogen that in-vades the host in the intestine and spreads within phagocyticcells to cause systemic infection (42). S. enterica serovar Ty-phimurium rarely causes systemic infections, but other sero-vars, including severe pathogens S. Typhi and S. Paratyphi,which typically cause systemic infections, possess pgtE geneswhose predicted protein sequences are at least 98% identicalto that of PgtE of S. Typhimurium (49). PgtE is a poor plas-minogen activator (22) but promotes growth of S. Typhi-murium inside mouse macrophages and its systemic spread inmice (35, 48). Plasminogen activation has not been reported insalmonellosis in vivo, but recent findings have suggested that S.enterica serovar Typhimurium also engages the plasminogensystem to advance cell migration (35, 48). Endotoxin-activatedmacrophages show increased surface-located plasminogen ac-tivation (67), and through inactivation of PAI-1 and the sub-sequent increase in plasminogen activation, PgtE may increaseproteolysis and mobility of phagocytes as well as of bacterialcells that are released from inflammatory phagocytes (38).

ACKNOWLEDGMENTS

We thank E. Ahola-Iivarinen, S. Helenius, A. Jaatinen, R. Lamer-anta, G. Ronnholm, and H. Tossavainen for technical assistance. J.Ravantti is thanked for computer resources.

This work was financially supported by the European Union Net-work of Excellence EuroPathoGenomics program, the Viikki Gradu-ate School in Biosciences, and the Academy of Finland (grant numbers116507 and 130202).

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