The Single Substitution I259T, Conserved in the ... · Microtus Pla was more efficient than wild-type Pla in 2 ... the bacterium to lungs leads to pneumonic plague, ... metastasis

JOURNAL OF BACTERIOLOGY, Aug. 2009, p. 4758–4766 Vol. 191, No. 150021-9193/09/$08.00�0 doi:10.1128/JB.00489-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Single Substitution I259T, Conserved in the Plasminogen ActivatorPla of Pandemic Yersinia pestis Branches, Enhances

Fibrinolytic Activity�

Johanna Haiko,1 Maini Kukkonen,1 Janne J. Ravantti,2Benita Westerlund-Wikstrom,1 and Timo K. Korhonen1*

General Microbiology, Faculty of Biosciences, FI-00014 University of Helsinki, Finland,1 and Institute of Biotechnology,Faculty of Biosciences, FI-00014 University of Helsinki, Finland2

Received 9 April 2009/Accepted 13 May 2009

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. Theprimary structure of the Pla �-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis,which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtusand Angola carries the single amino acid change T259I located in surface loop 5 of the �-barrel. RecombinantY. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogenactivation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasminenzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen moleculemore unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different fromthat of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in �2-antiplasmininactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, andtheir comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiproteaseinactivation. The substitution 259IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, alteredproteolysis in favor of plasmin formation, whereas the reverse substitution 259TIDKN/IIDKT in Pla alteredproteolysis in favor of �2-antiplasmin inactivation. The results suggest that Microtus Pla represents anancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestislineages.

Since the year 540, plague has killed some 200 million hu-mans in three pandemics, i.e., the Justinian plague, the BlackDeath, and the modern plague (36). Genomic studies haveestimated that the etiological agent, Yersinia pestis, evolvedfrom the oral-fecal pathogen Yersinia pseudotuberculosis sero-type O1b only shortly before the first pandemic, i.e., 5,000 to20,000 years ago (1, 2, 46), which has made the bacterium aparadigm of the rapid evolution of a severe bacterial pathogen(57). At least four biovars of Y. pestis have been identifiedthrough metabolic and genomic studies; of these biovars, An-tiqua, Medievalis, and Orientalis may be associated with thethree plague pandemics, whereas the fourth biovar, Microtus,is associated with human-attenuated Y. pestis strains from twogeographically distant infection foci in China (36, 59–61). Arecent molecular analysis indicated that the biovars are notmonophyletic and proposed the subdivision of Y. pestis intoeight molecular groupings, which represent different evolution-ary branches and histories and are only partially compatiblewith the biovars (1). Y. pestis evolved from Y. pseudotubercu-losis along branch 0, which consists of “atypical” Y. pestisstrains designated Angola, Microtus, and Pestoides; these arephylogenetically ancestral to the Antiqua, Medievalis, and Ori-entalis branches (1).

As a disease, plague exhibits various pathologies. Bubonicplague is the zoonotic form of the disease, which is usuallyacquired by humans from the bite of a flea that has beeninfected through a blood meal on a diseased rodent (36). Thebacteria invade at the intradermal flea bite site and migrate tolymphatic vessels and then to regional draining lymph nodes,where they multiply and cause the development of buboes (44).Without early treatment, bubonic plague progresses to life-threatening septicemic plague, and hematogenous spread ofthe bacterium to lungs leads to pneumonic plague, a rapidlyfatal and highly contagious airborne disease. Occasional injec-tion of Y. pestis cells by the flea directly into the circulatorysystem leads to primary septicemic plague (43).

The plasminogen activator Pla is a cell surface proteaseencoded by the Y. pestis-specific plasmid pPCP1 (10, 48). Pla isessential in the pathogenesis of bubonic (43, 49) and pneumo-nic plague (28), whereas it has less of a role in primary septi-cemic plague (43, 49). The pla gene is highly transcribed inbuboes of Y. pestis-infected mice (45), and Pla specifically po-tentiates migration of the bacteria to lymphatic tissue (43). Plaseems to have a different role in pneumonic plague, where itallows Y. pestis to replicate rapidly in the lungs, causing lethalfulminant pneumonia (28). Virulent Y. pestis strains lacking thePla-encoding plasmid pPCP1 have been isolated in Asia (3),and they can be associated with primary septicemic plague(43).

Pla is an aspartic protease (22, 55) that activates humanplasminogen (Plg) to the serine protease plasmin (47) and

* Corresponding author. Mailing address: General Microbiology,Faculty of Biosciences, FI-00014 University of Helsinki, Finland.Phone: 358 9 1915 9260. Fax: 358 9 1915 8754. E-mail: [email protected].

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inactivates the plasmin inhibitor �2-antiplasmin (�2AP), thusaffecting the main control system for plasmin activity (22). Plgis an abundant circulating zymogen, and its activation is centralin the pathogenesis of plague (13, 28, 43), and plasmin is apowerful serine protease associated with cell migration anddegradation of fibrin clots (29, 32, 37). In accordance with this,Pla-mediated bacterial adherence directs uncontrolled plasminproteolysis onto basement membranes to enhance bacterialmetastasis through tissue barriers (25, 27), and fibrinolysis byPla-generated plasmin activity plays a role in the pathogenesisof bubonic plague (8).

Compared to those of other Y. pestis biovars, Microtus iso-lates have several unique genomic features that may be in-volved in their inherent inability to attack the human host, andspecific losses of genes or gene functions are thought to beresponsible for the human attenuation (59). Interestingly, theattenuation does not apply to the murine host. The predictedamino acid sequence of the Pla polypeptide is remarkablyconserved: in the branches Antiqua, Medievalis, and Orien-talis, the Pla sequences are completely identical, whereas asingle amino acid substitution, T259I, has been detected inatypical Angola and Microtus strains (6, 38, 50). A geneticanalysis of 260 isolates of Y. pestis showed that the T259Isubstitution in Pla is shared by all isolates of biovar Microtusbut absent in those of other biovars (59). Many of the Pes-toides strains lack the pPCP1 plasmid and hence also the plagene (12), and pla sequences from Pestoides are not available.

Pla is a member of the omptin family of conserved outermembrane proteases/adhesins detected in several gram-nega-tive bacterial pathogens (15, 17, 21). The omptins have thesame molecular size, a �-barrel fold of 10 transmembrane �strands, and five surface-exposed loops, L1 to L5 (Fig. 1). Thecatalytic residues and the residues interacting with lipid A inthe outer membrane are completely conserved (17, 21–23, 41,55). The omptins cleave peptide substrates at basic residues(17) but show dramatic heterogeneity in the recognition ofbiologically important polypeptides, such as Plg, the antipro-tease �2AP, gelatin, and progelatinases. Analyses of hybridproteins created between Pla and the omptins PgtE of Salmo-nella enterica and OmpT of Escherichia coli have indicated thatthe differing polypeptide substrate selectivity of omptins isdictated by sequence variation in the mobile loop structures ofthe �-barrel (22, 40). Residue T259 in Pla is located at surfaceloop 5 and oriented inward in the active-site groove of the Plabarrel, close to residue K262, where Pla is autoprocessed (22,23) (Fig. 1).

The omptin �-barrel has spread by horizontal gene trans-fer in gram-negative bacteria and adapted to the life-stylesof host bacteria (15, 17, 21, 22, 40). Overall, the omptins givean example of an evolvable, robust enzyme fold (34) thateasily acquires novel or improved functions. The fact thatthe single substitution T259I associates with ancestral Y.pestis Microtus and Angola populations suggests that Mi-crotus Pla represents a form of the protein that precededthe common Pla protein. The central role of Plg activationin the pathogenesis of plague led us to analyze whether thesingle substitution T259I affects the fibrinolytic activities ofthe Pla molecule.

MATERIALS AND METHODS

Bacteria and plasmids. The bacterial strains and plasmids used in this studyare listed in Table 1. The pla derivatives were expressed in the inducible pSE380vector in E. coli XL1-Blue MRF� essentially as described earlier (22, 23). Plasmidconstructs pPlaT259I and pMRK3.51 were made by recombinant PCR as de-scribed earlier for the mutagenesis of pla (22) and pgtE (23), and the nucleotidesequences were verified by sequencing. The recombinant E. coli strains werecultivated overnight at 37°C in Luria broth (10 ml) supplemented with glucose(0.2% wt/vol), ampicillin (100 �g/ml), and tetracycline (12.5 �g/ml). For Plaprotein expression, the culture was pelleted, suspended in 150 �l of phosphate-

FIG. 1. Model of Pla structure (23) and location of residue Thr259.Side (top drawing) and top (bottom drawing) views of the transmem-brane �-barrel are shown. L1 to L5 are the surface loops. Catalyticresidues Asp84, Asp86, Asp206, and His208 are indicated in green,Thr259 is in red, and the autoprocessing site Lys262 is in yellow. OMis the outer membrane. (C) Amino acid sequence of residues 254 to273 at L5 and the termini of �-strands 9 and 10 in Pla, Microtus Pla,and PgtE are shown.

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buffered saline (PBS, pH 7.1), and plated on Luria plates containing 5 �Misopropyl-�-D-thiogalactopyranoside (IPTG) and antibiotics as described above.Recombinant Y. pestis strains were cultivated over 2 nights at 37°C in 10 ml ofbrain heart infusion broth supplemented with glucose (0.2% wt/vol), hemin (40�g/ml), and ampicillin (100 �g/ml), and for expression of Pla, the bacteria werecollected as described above and plated on brain heart infusion plates containing5 �M IPTG, hemin (40 �g/ml), and ampicillin (100 �g/ml) and grown over 2nights. For the assays, bacteria were collected in PBS, pelleted, and adjusted toan optical density at 600 nm of 1.2 (corresponding to ca. 109 cells/ml) or 2.0 (2 �109 cells/ml).

Measurement of Pla protein expression. Expression levels and isoforms of thePla protein were assessed from cell envelope preparations (E. coli) (22) or fromwhole-cell samples (Y. pestis). The cell envelopes were prepared by sonicating 3ml of the cell suspension (109 cells/ml) in PBS containing 2.5 mM EDTA fourtimes for 30 s (17% amplitude, 3-s pulse, 1-s break) over crushed ice. The cellswere pelleted by centrifugation (10 min, 3,000 rpm), and the supernatant wasfurther centrifuged for 2 min at 6,000 rpm. The cell envelopes in the supernatantwere pelleted (10 min, 13,000 rpm, 4°C) and suspended in PBS and then insodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loadingbuffer. Half of the sample volume was boiled for 10 min. Whole-cell sampleswere prepared by mixing bacteria (109 cells/ml) with half of the volume ofSDS-PAGE loading buffer and boiling them for 10 min. Samples were loadedinto a 12% (wt/vol) SDS-PAGE gel, electrophoresed, and transferred onto anitrocellulose membrane for detection with anti-His6-Pla antiserum (22), alka-line-phosphatase-conjugated anti-rabbit immunoglobulin G (IgG; Dako), andphosphatase substrate.

Fibrinolysis. The fibrinolysis plate assay was performed basically as describedpreviously (4), with minor modifications. Human fibrinogen (1.73 mg/ml; de-pleted of Plg, von Willebrand factor, and fibronectin; Kordia Life Sciences) wassuspended in 2 ml water containing 5 �g/ml human Glu-Plg (American Diag-nostica); 100 �l of bovine thrombin (5 NIH units; MP Biomedicals) in 100 mMsodium borate buffer (pH 7.74) was then added by gentle mixing, and thesolution was immediately poured onto a six-well plate (Nunc). Five-microlitersamples of bacterial suspensions (107 bacteria) in PBS were applied to thesolidified plate, which was then incubated at 37°C for 20 h (E. coli) or 48 h (Y.pestis). Fibrinolysis was visually assessed as a clear zone around the bacteria.

Activation and cleavage of plasminogen and cleavage of �2AP. For activitymeasurements, 4 or 0.4 �g human Glu-Plg was mixed with 8 � 107 bacteria inPBS in a total volume of 174 �l in microtiter plate (Nunc) wells. For cumulativeanalysis of plasmin formation, 30 �l of the chromogenic plasmin substrate Val-Leu-Lys-p-nitroaniline dihydrochloride (S-2251, 2.5 mg/ml; Chromogenix/Dia-Pharma) was immediately added and plasmin formation at 37°C was measuredevery 15 or 30 min for several hours in a microtiter plate reader at 405 nm (22).To analyze the time course of plasmin formation, 4 �g human Glu-Plg was mixedwith bacteria at 37°C and the plasmin substrate was added at time points of 0, 1,2, 5, 10, and 22 h; plasmin activity was then measured after 15 min. In Plgcleavage assays (22), 2.5 �g of human Glu-Plg and 4 � 107 bacteria (in PBS) wereincubated in a total volume of 25 �l while shaking for 7 h at 37°C. After 2 and

7 h, the cells were pelleted, half the volume of SDS-PAGE loading buffer wasadded and the supernatant samples were boiled for 10 min. Ten-microlitersamples were subjected to 12% (wt/vol) SDS-PAGE and transferred onto apolyvinylidene difluoride membrane (GE Healthcare), and Plg polypeptideswere detected by Western blotting with polyclonal anti-human Plg antibody (1mg/ml, diluted 1:1,000; American Diagnostica), peroxidase-conjugated anti-rab-bit IgG (1:2,500; GE Healthcare), and enhanced chemiluminescence detectionreagents (GE Healthcare) according to the manufacturer’s instructions. Themembrane was exposed to X-ray film (Agfa) for 15 s. In assays where Plgcleavage was detected with monoclonal anti-human Plg catalytic domain anti-body (R&D Systems), the following minor modifications were made. Threemicrograms of Glu-Plg and 4.8 � 107 bacteria were incubated for 2 h in a totalvolume of 30 �l. For comparison, Plg cleavage by urokinase (uPA) at a concen-tration of 2.5 �g/ml was assessed. After incubation, supernatant samples of 15 �lwere electrophoresed and detection was done with the monoclonal antibody (500�g/ml, diluted 1:500) and peroxidase-conjugated anti-mouse IgG (GE Health-care; 1:1,000). The exposure time was 2 min. Cleavage of human �2AP (0.8 �g;Calbiochem) was tested as detailed earlier (22, 26), by using 3 � 107 bacteria andincubation for 2 h under shaking (1,100 rpm) at 37°C.

Inactivation of plasmin and �2AP. Inactivation of plasmin was measured byincubating 0.5 �g human plasmin (Sigma) with bacteria (8 � 107) in 170 �l ofPBS for 30 min at 37°C in a microtiter plate. The chromogenic plasmin substrate(30 �l) was then added, and plasmin activity was measured at 405 nm after 90min of incubation at 37°C. Inactivation of �2AP was measured basically asdescribed earlier (26). Human �2AP (5.75 �g/ml) was incubated with bacteria(8 � 107) in PBS for 2 h at 37°C in a microtiter plate. Human plasmin (0.5 �g)was added, and the plate was incubated for 30 min at 37°C. Thirty microliters ofthe chromogenic plasmin substrate was added to make the final volume 200 �l,and plasmin activity was measured at 405 nm after 90 min of incubation at 37°C.

Sequence alignment and Pla structure model. Y. pestis Pla amino acid se-quences were obtained from the NCBI GenBank protein database and alignedby using the ClustalW program (7). Pairwise alignment of Pla (31) and PgtE (14)sequences was done with the needle program from the EMBOSS package (42).The Pla model was built with the MODELLER program (30) by using theexisting OmpT structure (Protein Data Bank identifier: 1i78) as the template.Visualization was done with the VMD program (18).

RESULTS

Autoprocessed �-Pla is absent in Pla T259I. To assess func-tional properties of the Microtus Pla variant, the substitutionT259I was created in the Pla molecule originating from biovarMedievalis, and Pla and Pla T259I were expressed in Y. pestisKIM D34, which lacks the pPCP1 plasmid. Western blottingwith polyclonal anti-Pla antiserum indicated that Pla and PlaT259I were expressed in similar amounts in KIM D34 cells(Fig. 2A). In SDS-PAGE, denatured Pla exhibits four isoforms(pre-Pla, �-Pla, �-Pla, and �-Pla) (24, 47, 48), of which �-Plaand �-Pla are different conformations of the mature Pla pro-tein and �-Pla is the autoprocessed form (20, 22). Pla and PlaT259I showed different isoform patterns in the KIM D34 host;all isoforms were detected with Pla, whereas Pla T259I lackedthe autoprocessed isoform �-Pla (Fig. 2A).

PgtE of S. enterica belongs to the same omptin subfamily andshares 75% sequence identity with Pla (15, 17, 21); in the L5region, the sequences differ in 6/20 residues (Fig. 1C). Weincluded in the assays the Pla derivative carrying the substitu-tion 259TIDKN/IIDKT, which was available from previouswork (40) and at L5 mimics the sequence of PgtE. To analyzethe Pla derivatives in more detail and to compare Pla and PgtE(see below), we expressed the omptin variants in recombinantE. coli XL1-Blue, which has been successfully used to charac-terize the functions of both Pla and PgtE (22–24, 27, 40, 47).Pla and the Pla variants were expressed in similar amounts incell envelope preparations from recombinant E. coli, as de-tected by Western blotting with anti-Pla antiserum (Fig. 2B).

TABLE 1. Bacterial strains and plasmids used in this study

Bacterial strain orplasmid Description Reference(s)

StrainsE. coli XL1 Blue

MRF��(mcrA)183 �(mcrCB-hsdSMR-

mrr)173 endA1 supE44 thi-1recA1 gyrA96 relA1 lac F�proAB lacIqZ�M15 Tn10(Tetr)

Stratagene

Y. pestis KIMD34

pPCP1� �pgm pYV� derivativeof Y. pestis KIM-10

11, 54

PlasmidspSE380 Expression vector; trc promoter

lacO operator lacI blaInvitrogen

pMRK1 pla in pSE380 22pPlaT259I Microtus pla in pSE380 This studypMRK1.51 pla, 259TIDKN3IIDKT 40pMRK3 pgtE in pSE380 23pMRK3.51 pgtE, 259IIDKT3TIDKN This study

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Differences in the Pla isoforms were detected in boiled samplesof the three Pla variants also in the E. coli XL1 host. Wild-typePla had all four forms, whereas Pla T259I and Pla 259TIDKN/IIDKT completely lacked the �-Pla form. In unboiled cellenvelope samples, where the native-like folding of the �-barrelis preserved (33), wild-type Pla was detected almost completelyin the �-Pla form whereas the substituted Pla molecules alsoexhibited the pre-Pla and �-Pla forms of the molecule. More-over, the �-Pla form of Pla T259I and Pla 259TIDKN/IIDKTmigrated faster than did wild-type Pla (Fig. 2B).

Microtus Pla is reduced in fibrinolysis. Formation of fibrinclots prevents bacterial migration in infected tissues (5), andthe plasmin-mediated degradation of fibrin is a possiblepathogenetic mechanism of Pla (8, 15, 28). Strain KIMD34(pPlaT259I) was less efficient than KIM D34(pMRK1) ina fibrinolysis plate assay (Fig. 3A). The same result was ob-tained when the two plasmids were expressed in E. coli XL1(Fig. 3B). Fibrinolysis was observed only when Plg was addedto the plates.

E. coli XL1(pMRK3) with PgtE exhibited weak fibrino-lytic activity, and E. coli XL1(pMRK1.51) expressing Pla259TIDKN/IIDKT, which mimics L5 of PgtE, was less effec-tive than the bacteria with the common Pla protein(Fig. 3B). The 259IIDKT/TIDKN substitution in PgtE(pMRK3.51) was therefore constructed to compare it to thereverse substitution in Pla; this construct showed slightlyhigher fibrinolytic activity than did PgtE (Fig. 3B). We ob-served no differences in isoform formation between PgtEand PgtE 259IIDKT/TIDKN (data not shown).

Formation of plasmin is reduced in recombinant Y. pestisand E. coli expressing Microtus Pla. The findings above indi-cated that the T259I substitution in Pla affects Plg activation.In measurements of cumulative plasmin formation in Y. pestis,

strains KIM D34(pMRK1), with the common Pla protein, andKIM D34(pPlaT259I) initiated plasmin formation equally ef-fectively at a high Plg concentration (20 �g/ml), whereas re-duced plasmin formation by KIM D34(pPlaT259I) was evidentin assays with a lower concentration of Plg (2 �g/ml; Fig. 4A).Similarly, slower Plg activation by Pla T259I and by Pla259TIDKN/IIDKT was observed at the lower Plg concentrationin the E. coli XL1 background (Fig. 4B). In contrast, E. coliXL1(pMRK3) with PgtE was poor in a cumulative Plg activa-tion assay, and its activity was improved by the 259IIDKT/TIDKN substitution in pMRK3.51.

We next measured plasmin activity during a 22-h incubationof bacteria with Plg. With Y. pestis KIM D34(pMRK1), theplasmin activity was highest after 1 to 2 h of incubation andthen declined, whereas with KIM D34(pPlaT259I), the plasminactivity remained lower and was more transient (Fig. 4C).Plasmin activity formed by E. coli XL1(pMRK1) with wild-typePla increased for up to 10 h but was decreased after a 22-hincubation, whereas the plasmin activity formed by E. coli withPla T259I or Pla 259TIDKN/IIDKT was already decreased af-ter 1 h of incubation and close to the background level at 22 h(Fig. 4D). A clear difference between Pla and PgtE was seen inthe time course of plasmin formation: plasmin activity did notrise after 1 h of incubation of Plg with E. coli(pMRK3) (Fig.4D). Also, in incubations for 1 to 10 h, E. coli XL1 with PgtE259IIDKT/TIDKN showed an increase in the level of PgtE-mediated plasmin formation.

We also tested the proteinases in the Y. pseudotuberculosisbackground by using as a host strain Y. pseudotuberculosis PB1�wb, which represents the probable ancestor of Y. pestis (46).The T259I substitution in Pla destabilized the formation ofplasmin activity also in this strain (data not shown).

Microtus Pla inactivates plasmin. The reduction in plasminlevels observed after a longer incubation of Plg with PgtE-, PlaT259I-, or Pla 259IIDKT/TIDKN-expressing bacteria suggestedthat these omptin variants degrade the Plg substrate into pro-teolytically inactive fragments and into plasmin, which is thenfurther degraded by the proteases. We therefore assessedwhether the omptin proteases indeed inactivate plasmin; thiswas done by incubating human plasmin with the E. coli deriv-

FIG. 2. Expression and isoform patterns of Pla and the Pla deriv-atives in recombinant bacteria. (A) Pla and Pla T259I were expressedin Y. pestis KIMD34, and the Western blot assay shows the reactivity ofwhole-cell samples with anti-Pla antiserum. (B) Expression and iso-forms of the Pla proteins in cell envelope preparations from recombi-nant E. coli. The protein constructs are indicated above the lanes, andthe migration distances of the �, �, and � isoforms of the mature Plamolecule and of nonprocessed pre-Pla are indicated on the left. Theplus sign denotes samples boiled for 10 min before electrophoresis,and the minus sign denotes unboiled samples.

FIG. 3. Fibrinolysis by recombinant Y. pestis KIM D34 and E. coliXL1. Bacteria (107 cells) in buffer were pipetted onto fibrin platescontaining 5 �g/ml Plg, and dissolution of the clot was assessed afterincubation for 20 h (E. coli) or 48 h (Y. pestis) at 37°C. (A) Bacterialnumbering: 1, Y. pestis KIM D34(pSE380); 2, Y. pestis KIMD34(pMRK1); 3, Y. pestis KIM D34(pPlaT259I). (B) Bacterial num-bering: 1, E. coli XL1(pSE380); 2, E. coli XL1(pMRK1); 3, E. coliXL1(pPlaT259I); 4, E. coli XL1(pMRK1.51); 5, E. coli XL1(pMRK3);6, E. coli XL1(pMRK3.51).

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atives and then analyzing the remaining levels of plasmin ac-tivity (Fig. 4E). The level of plasmin activity remained similarwith the vector strain E. coli(pSE380) and with E. coli-(pMRK1), indicating that Pla did not inactivate plasmin duringincubation. In contrast, the activity of plasmin was loweredafter incubation with E. coli(pPlaT259I) and E. coli-(pMRK1.51). Plasmin activity was also diminished by E. coli-(pMRK3) with PgtE, whereas E. coli(pMRK3.51) with PgtE259IIDKT/TIDKN caused only a partial reduction (Fig. 4E).

The I259T substitution is critical for generation of the cat-alytic plasmin light chain. We assessed Plg cleavage by therecombinant E. coli strains by Western blotting with polyclonalanti-Plg antibodies which mainly recognize the noncatalytic,lysine-binding kringle domains in Plg and with a monoclonalanti-Plg catalytic domain antibody which recognizes the 25-kDa light chain of plasmin (PlnL) that contains the proteasedomain. The fragmentation of Plg by the bacteria was com-pared to Plg fragments obtained in incubation with the humanPlg activator uPA. Western blotting with the anti-Plg antibodyshowed similar patterns of Plg cleavage by Pla, Pla T259I, andPla 259TIDKN/IIDKT expressed in recombinant E. coli XL1(Fig. 5A). The Pla constructs completely cleaved the Plg mol-ecule within 7 h into peptides similar in size to the plasminheavy chain (PlnH) and angiostatins. The latter are composedof kringle domains of Plg and lack the protease domain, andthey are also contained in the PlnH chains (35). Formation ofPlnH- and angiostatin-like fragments was detectable in uPA-mediated cleavage of Plg as well (Fig. 5A).

Recombinant E. coli XL1(pMRK3) with PgtE cleaved thePlg molecule as efficiently as did Pla-expressing E. coliXL1(pMRK1), whereas E. coli with the mutated PgtE259IIDKT/TIDKN protein exhibited slower cleavage (Fig. 5A).

Immunoblotting with the anti-Plg catalytic domain antibodyrevealed the presence of PlnL in Plg preparations incubatedfor 2 h with E. coli XL1 Pla or with uPA (Fig. 5B). A peptidecorresponding to PlnL was weakly detectable with PgtE259IIDKT/TIDKN, whereas we failed to detect the PlnL chainin samples treated with XL1(pPlaT259I), XL1(pMRK1.51),XL1(pMRK3), or the vector strain (Fig. 5B). PlnL contains thecatalytic domain of plasmin (51). An immunoreactive peptide(here named PlgX) was observed in the degradation of Plg bythe omptins Pla, Pla 259TIDKN/IIDKT, and PgtE 259IIDKT/TIDKN; notably, this peptide was lacking in uPA-treated Plgsamples (Fig. 5B).

Microtus Pla is efficient in �2AP inactivation. Pla and PgtEare multifunctional and cleave and inactivate the major circu-

FIG. 4. Plasminogen activation by recombinant Y. pestis KIM D34and E. coli XL1 and plasmin inactivation by recombinant E. coli XL1.(A and B) Cumulative, initial plasmin formation by bacteria tested attwo Plg concentrations (20 and 2 �g/ml). The expression host, proteinconstructs, and Plg concentrations are indicated. (C and D) Plasminformation by bacteria after incubation for up to 22 h with Plg. Plasminformation was analyzed by adding a chromogenic plasmin substrate atthe indicated time points and measuring absorbance after 15 min.(E) Inhibition of plasmin activity by recombinant E. coli. Plasmin wasincubated for 30 min with the bacteria, after which the plasmin sub-strate was added and the absorbance was measured after 90 min. Theassays were repeated at least three times, and results of a representa-tive assay with duplicate samples are shown.

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lating plasmin inhibitor �2AP (22, 26, 52), which is importantfor the in vivo activity of plasmin. To assess whether the I259Tsubstitution affects the activity of omptins toward another bi-ologically important polypeptide substrate, we compared themutated constructs for cleavage of the antiprotease (Fig. 6A).The Pla derivatives and PgtE cleaved �2AP, whereas E. coliXL1(pMRK3.51) with PgtE 259IIDKT/TIDKN exhibitedslower proteolysis than did PgtE-expressing cells. In inhibitionassays, E. coli strains expressing PgtE, Pla T259I, or Pla259TIDKN/IIDKT abolished the antiprotease activity of �2AP.In contrast, E. coli with Pla or PgtE 259IIDKT/TIDKN onlypartially affected the antiproteolytic activity (Fig. 6B), indicat-ing that although these two proteases cleaved the antiprotease,they were less effective in its inactivation.

DISCUSSION

Horizontal gene transfer, loss of gene functions, genomicrearrangements, and single-nucleotide polymorphism havebeen important in the evolution of Y. pestis (1, 57). Single-nucleotide polymorphisms have served as useful markers in theconstruction of phylogenetic patterns, but their biological ef-fects have been only partially characterized. Here we describea nonsynonymous nucleotide change that increases the fibrino-lytic potential of a major bacterial virulence factor. Pla is es-sential for the virulence and epidemic spread of Y. pestis (28,

43, 45), and the pla gene in plasmid pPCP1 has been acquiredby horizontal gene transfer after Y. pestis and Y. pseudotuber-culosis diverged. The single substitution I259T present in thePla protein of the pandemic or “typical” lineages of Y. pestisdirects the proteolytic activity of Pla toward higher fibrinolysisand plasmin formation but lower antiprotease inactivation.The reverse substrate selectivity was seen with the close or-tholog PgtE of the intracellular pathogen S. enterica, which hasan ancestral form in common with Pla (15, 47). Plg activationby Pla is essential in plague (28, 43, 49), and the I259T substi-tution in Pla has likely been important in the evolution ofplague pathogenesis. Plasmin and plasminogen bound to fibrinare resistant to regulation by �2AP (29), and the change in theselectivity of the common Pla protein toward plasmin genera-tion is in accord with the observed importance of fibrinolysis inplague (8, 28).

The eukaryotic Plg activators uPA and tissue type plasmin-ogen activator activate Plg through a single cleavage of thepeptide bond between Arg560 and Val561, which is alsocleaved by Pla (49). In addition to generating the PlnL chain,which carries the catalytic domain of plasmin, the Pla-express-ing bacteria also generated an immunoreactive Plg fragment,here termed PlgX, which was not detected with uPA-cleavedPlg. This indicates that Pla is less specific than uPA in Plgactivation and cleavage. With Pla T259I-expressing bacteria,Plg activation was slower than with Pla-expressing bacteria andwe did not detect generation of the PlnL chain by Westernblotting. As an enzyme detection method, immunoblotting is

FIG. 5. Degradation of plasminogen by Pla and PgtE variants ex-pressed in recombinant E. coli XL1. Plg degradation by the bacteriawas analyzed by Western blotting with anti-Plg antibody (A) and anti-Plg catalytic domain antibody (B). The migration distances of Plg,plasmin heavy chain (PlnH, apparent size of 70 kDa), plasmin lightchain (PlnL, 25 kDa), and angiostatins (Ang) are shown on the left.PlgX indicates a fragment of Plg produced by the bacteria. The Pla andPgtE constructs expressed in E. coli XL1 and uPA-treated Plg areindicated above the lanes, incubation times in panel A are indicatedbelow the lanes. In panel B, the incubation time was 2 h.

FIG. 6. Degradation and inactivation of �2AP by recombinant E.coli XL1. (A) Degradation of �2AP after a 2-h incubation with bacte-ria, as analyzed by Western blotting. The protein constructs expressedin E. coli XL1 are indicated above the lanes. The migration distance of�2AP is shown on the left, and the arrow indicates a cleavage productof �2AP. (B) Inactivation of �2AP by bacteria. The Pla and PgtEconstructs expressed in E. coli XL1 are indicated. Bacteria were incu-bated with �2AP for 2 h, after which plasmin and its chromogenicsubstrate were added and plasmin activity was measured after 15 min.The assay was repeated three times, and results of a representativeassay with duplicate samples are shown.

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less sensitive than measurement of plasmin enzymatic activityand may thus fail to detect small amounts of plasmin or PlnL.The bacteria expressing Microtus Pla also inactivated plasminmore efficiently than did those expressing the common Plaprotein. Taken together, the results strongly suggest that thelow plasmin activity levels formed by Microtus Pla result fromtransient formation of active plasmin, subsequent or simulta-neous cleavage of the PlnL chain, and inactivation of theformed plasmin activity.

The omptins in enteric bacteria share ca. 50% sequenceidentity, and Pla of human-pathogenic Y. pestis is the onlyomptin protease that has Thr at position 259 (41). The substi-tution T259I in Pla lowered plasmin formation but did notgrossly change the amount of Pla peptides in the outer mem-brane, and our hypothesis is that the observed change in targetselectivity results from a conformational change in Pla surfaceloop 5. Residue T259 and the autoprocessing site K262 arespatially located near the catalytic groove of the Pla protease,and it is likely that the lack of autoprocessing in Microtus Plaresults from steric inhibition of peptide bond cleavage. Pre-vention of autoprocessing in the Microtus Pla molecule willrestrict the mobility and flexibility of L5 residues and thus mayalter interactions of Pla with polypeptide substrates. A confor-mational effect is also suggested by the finding that in unboiledE. coli cell envelope preparations, where the �-barrel fold isnot destroyed (33), Pla was nearly completely in the �-Plaform, whereas pre-Pla and �-Pla were detected in the substi-tuted Pla proteins. Also, under nondenaturing conditions, the�-Pla form migrated differently from � forms of the substitutedPla molecules. The important role of the L5 region in correctsubstrate recognition by both Pla and PgtE is underlined by thefindings that the mutated PgtE 259IIDKT/TIDKN proteinshowed an increase in plasmin formation and a decrease in�2AP inactivation and that the reverse change in substrateselectivity was observed with the reverse substitution, Pla259TIDKN/IIDKT. We have earlier found that this region inL5 is important for the gelatinase activity that is expressed byPgtE but not by Pla (40).

PgtE contributes to the virulence of S. enterica by promotingbacterial survival in mouse macrophages and bacterial spreadin infected mice (26, 40). The proteolytic activities of PgtEinclude degradation of cationic antimicrobial peptides (14) andcomplement proteins (39), highly active degradation and inac-tivation of �2AP in S. enterica cells from infected macrophages(26), and degradation of gelatin and activation of mammaliangelatinases (40). Among the omptins, the latter function ap-pears unique to PgtE (15), and it is involved in the pathogen-esis of salmonellosis (16).

In contrast to Pla, PgtE appears to cleave Plg/plasminpolypeptides mainly in an inactivating manner. Several lines ofevidence support this conclusion. First, PgtE-expressing bacte-ria were as effective as Pla-expressing bacteria in cleaving thePlg molecule but produced very low levels of plasmin activityand fibrinolysis. These results are in line with previous obser-vations showing poor plasmin formation by the PgtE omptin ofS. enterica (47, 52). Second, the low level of plasmin activity didnot increase during a 2- to 5-h incubation with Plg, whereaswith Pla-expressing bacteria the plasmin activity levels wereincreased. Third, we detected generation of the PlnH chainand kringle domains but did not detect formation of the PlnL

chain by E. coli(pMRK3). This suggests that the proteolyticallyactive PlnL chain was rapidly degraded by PgtE. In the sameassay, we detected the PlnL chain after incubation with Pla-expressing bacteria, as well as with uPA. Finally, we indeedobserved that incubation of human plasmin with PgtE-express-ing bacteria lowered the enzymatic activity of plasmin. Theopposite situation was observed when �2AP was used as asubstrate; Pla-expressing bacteria cleaved the �2AP polypep-tide into a smaller fragment similarly as did PgtE-expressingbacteria but were less effective in abolishing the antiproteolyticactivity of �2AP. Measurement of �2AP antiprotease activityrelies on determining the change in apparent plasmin activity,and it should be noted that although PgtE-expressing bacteriadirectly inactivated plasmin in 30 min of incubation, we couldobserve clear plasmin activity compared to the vector strain inthe antiprotease assay. Our results support the notion thatPgtE should be functionally classified as an antiprotease inac-tivator rather than as a Plg activator (26).

Directed evolution of enzymes has shown that while mostsingle amino acid substitutions are either neutral or deleteri-ous, some single substitutions increase the enzyme’s fitness(53). Such amino acid substitutions can be located in or bedistant from the active site, and the distant mutations affectcatalysis by slightly altering the geometry, electrostatic prop-erties, or dynamics of amino acids in the active site. The �-bar-rel is a sturdy membrane-embedded structure that allows largechanges in the surface loops without disturbing the �-barrelfolding (19), and the omptin �-barrels fulfill the criteria of anevolvable and mutationally robust protein superfamily that isundergoing adaptive molecular evolution (34). The individualomptins in enteric bacteria exhibit differing functions (15), andsubstitution of nonconserved amino acid residues at the L1 toL5 structures broadens the substrate selectivity of Pla of Y.pestis and OmpT of E. coli toward progelatinase or Plg activa-tion, respectively (22, 40), which indicates that the mobilesurface loops are important in polypeptide substrate recogni-tion by omptins. Varadarajan and coworkers (56) described anS223R amino acid substitution in OmpT that changed thecleavage bond selectivity from Arg-Arg to Ala-Arg in shortsynthetic peptides. Residue S223 is located in the intimatevicinity of the catalytic residues of OmpT.

We describe here a single residue change in Pla that hasoccurred naturally and has persevered in the evolution of pan-demic Y. pestis branches. Plg is synthesized mainly in the liverand is abundant in circulation, whereas only very low concen-trations of Plg mRNA or Plg protein have been detected inextrahepatic tissue sites, such as adipose tissue (58) and extra-cellular matrices (9), which the plague bacterium must bypassto reach lymphatic tissue. Hence, the improved Plg utilizationand enhanced fibrinolysis by the common Pla protein can becritical in the pathogenesis of plague.

ACKNOWLEDGMENTS

We thank Raili Lameranta and Johanna Heikkinen for technicalassistance.

This work was financially supported by the Research Foundation ofthe University of Helsinki, the European Union Network of ExcellenceEuroPathoGenomics program, the Viikki Graduate School in Molec-ular Biosciences, and the Academy of Finland (grants 116507 and130202).

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REFERENCES

1. Achtman, M., G. Morelli, P. Zhu, T. Wirth, I. Diehl, B. Kusecek, A. J. Vogler,D. M. Wagner, C. J. Allender, W. R. Easterday, V. Chenal-Francisque, P.Worsham, N. R. Thomson, J. Parkhill, L. E. Lindler, E. Carniel, and P.Keim. 2004. Microevolution and history of the plague bacillus, Yersinia pestis.Proc. Natl. Acad. Sci. USA 101:17837–17842.

2. Achtman, M., K. Aurth, G. Morelli, G. Torrea, A. Guiyoule, and E. Carniel.1999. Yersinia pestis, the cause of plague, is a recently emerged clone ofYersinia pseudotuberculosis. Proc. Natl. Acad. Sci. USA 96:14043–14048.

3. Anisimov, A. P., L. E. Lindler, and G. B. Pier. 2004. Intraspecific diversity ofYersinia pestis. Clin. Microbiol. Rev. 17:434–464.

4. Beesley, E. D., R. R. Brubaker, W. A. Janssen, and M. J. Surgalla. 1967.Pesticins. 3. Expression of coagulase and mechanism of fibrinolysis. J. Bac-teriol. 94:19–26.

5. Bergmann, S., and S. Hammerschmidt. 2007. Fibrinolysis and host responsein bacterial infections. Thromb. Haemost. 98:512–520.

6. Chain, P. S. G., P. Hu, S. A. Malfatti, L. Radnedge, F. Larimer, L. M. Vergez,P. Worsham, M. C. Chu, and G. L. Andersen. 2006. Complete genomesequence of Yersinia pestis strains Antiqua and Nepal516: evidence of genereduction in an emerging pathogen. J. Bacteriol. 188:4453–4463.

7. Chenna, R., H. Sugawara, T. Koike, R. Lopez, T. J. Gibson, D. G. Higgins,and J. D. Thompson. 2003. Multiple sequence alignment with the Clustalseries of programs. Nucleic Acids Res. 31:3497–3500.

8. Degen, J. L., T. H. Bugge, and J. D. Goguen. 2007. Fibrin and fibrinolysis ininfection and host defense. J. Thromb. Haemost. 5(Suppl. 1):24–31.

9. Farina, A. R., A. Tiberio, A. Tacconelli, L. Cappabianca, A. Gulino, and A. R.Mackay. 1996. Identification of plasminogen in Matrigel and its activation byreconstitution of this basement membrane extract. BioTechniques 21:904–909.

10. Ferber, D. M., and R. R. Brubaker. 1981. Plasmids in Yersinia pestis. Infect.Immun. 31:839–841.

11. Finegold, M. J., J. J. Petery, R. F. Berendt, and H. R. Adams. 1968. Studieson the pathogenesis of plague. Blood coagulation and tissue responses ofMacaca mulatta following exposure to aerosols of Pasteurella pestis. Am. J.Pathol. 53:99–114.

12. Garcia, E., P. Worsham, S. Bearden, S. Malfatti, D. Lang, F. Larimer, L.Lindler, and P. Chain. 2007. Pestoides F, an atypical Yersinia pestis strainfrom the former Soviet Union. Adv. Exp. Med. Biol. 603:17–22.

13. Goguen, J. D., T. Bugge, and J. L. Degen. 2000. Role of the pleiotropic effectsof plasminogen deficiency in infection experiments with plasminogen-defi-cient mice. Methods 21:179–183.

14. Guina, T., E. C. Yi, H. Wang, M. Hackett, and S. I. Miller. 2000. A PhoP-regulated outer membrane protease of Salmonella enterica serovar Typhi-murium promotes resistance to alpha-helical antimicrobial peptides. J. Bac-teriol. 182:4077–4086.

15. Haiko, J., M. Suomalainen, T. Ojala, K. Lahteenmaki, and T. K. Korhonen.2009. Breaking barriers—attack on innate immune defences by omptin sur-face proteases of enterobacterial pathogens. Innate Immun. 15:67–80.

16. Handley, S. A., and V. L. Miller. 2007. General and specific host responsesto bacterial infection in Peyer’s patches: a role for stromelysin-1 (matrixmetalloproteinase-3) during Salmonella enterica infection. Mol. Microbiol.64:94–110.

17. Hritonenko, V., and C. Stathopoulos. 2007. Omptin proteins: an expandingfamily of outer membrane proteases in gram-negative Enterobacteriaceae.Mol. Membr. Biol. 24:395–406.

18. Humphrey, W., A. Dalke, and K. Schulten. 1996. VMD—Visual MolecularDynamics. J. Mol. Graphics 14:33–38.

19. Koebnik, R., K. P. Locher, and P. Van Gelder. 2000. Structure and functionof bacterial outer membrane proteins: barrels in a nutshell. Mol. Microbiol.37:239–253.

20. Kramer, R. A., D. Zandwijken, M. R. Egmond, and N. Dekker. 2000. In vitrofolding, purification and characterization of Escherichia coli outer membraneprotease OmpT. Eur. J. Biochem. 267:885–893.

21. Kukkonen, M., and T. K. Korhonen. 2004. The omptin family of enterobac-terial surface proteases/adhesins: from housekeeping in Escherichia coli tosystemic spread of Yersinia pestis. Int. J. Med. Microbiol. 294:7–14.

22. Kukkonen, M., K. Lahteenmaki, M. Suomalainen, N. Kalkkinen, L. Emody,H. Lång, and T. K. Korhonen. 2001. Protein regions important for plasmin-ogen activation and inactivation of �2-antiplasmin in the surface protease Plaof Yersinia pestis. Mol. Microbiol. 40:1097–1111.

23. Kukkonen, M., M. Suomalainen, P. Kyllonen, K. Lahteenmaki, H. Lång, R.Virkola, I. M. Helander, O. Holst, and T. K. Korhonen. 2004. Lack ofO-antigen is essential for plasminogen activation by Yersinia pestis and Sal-monella enterica. Mol. Microbiol. 51:215–225.

24. Kutyrev, V., R. J. Mehigh, V. L. Motin, M. S. Pokrovskaya, G. B. Smirnov,and R. R. Brubaker. 1999. Expression of the plague plasminogen activator inYersinia pseudotuberculosis and Escherichia coli. Infect. Immun. 67:1359–1367.

25. Lahteenmaki, K., S. Edelman, and T. K. Korhonen. 2005. Bacterial metas-tasis: the host plasminogen system in bacterial invasion. Trends Microbiol.13:79–85.

26. Lahteenmaki, K., P. Kyllonen, L. Partanen, and T. K. Korhonen. 2005.Antiprotease inactivation by Salmonella enterica released from infected mac-rophages. Cell. Microbiol. 7:529–538.

27. Lahteenmaki, K., R. Virkola, A. Saren, L. Emody, and T. K. Korhonen. 1998.Expression of plasminogen activator Pla of Yersinia pestis enhances bacterialattachment to the mammalian extracellular matrix. Infect. Immun. 66:5755–5762.

28. Lathem, W. W., P. A. Price, V. L. Miller, and W. E. Goldman. 2007. Aplasminogen-activating protease specifically controls the development ofprimary pneumonic plague. Science 315:509–513.

29. Lijnen, H. R., and D. Collen. 1995. Mechanisms of physiological fibrinolysis.Baillieres Clin. Haematol. 8:277–290.

30. Martí-Renom, M. A., A. C. Stuart, A. Fiser, R. Sanchez, F. Melo, and A. Sali.2000. Comparative protein structure modeling of genes and genomes. Annu.Rev. Biophys. Biomol. Struct. 29:291–325.

31. McDonough, K. A., and S. Falkow. 1989. A Yersinia pestis-specific DNAfragment encodes temperature-dependent coagulase and fibrinolysin-asso-ciated phenotypes. Mol. Microbiol. 3:767–775.

32. Mignatti, P., and D. B. Rifkin. 1993. Biology and biochemistry of proteinasesin tumor invasion. Physiol. Rev. 73:161–195.

33. Nakamura, K., and S. Mizushima. 1976. Effects of heating in dodecyl sulfatesolution on the conformation and electrophoretic mobility of isolated majorouter membrane proteins from Escherichia coli K-12. J. Biochem. 80:1411–1422.

34. O’Loughlin, T. L., W. M. Patrick, and I. Matsumura. 2006. Natural historyas a predictor of protein evolvability. Protein Eng. Des. Sel. 19:439–442.

35. O’Reilly, M. S., L. Holmgren, Y. Shing, C. Chen, R. A. Rosenthal, M. Moses,W. S. Lane, Y. Cao, E. H. Sage, and J. Folkman. 1994. Angiostatin: a novelangiogenesis inhibitor that mediates the suppression of metastases by aLewis lung carcinoma. Cell 79:315–328.

36. Perry, R. D., and J. D. Fetherston. 1997. Yersinia pestis—etiologic agent ofplague. Clin. Microbiol. Rev. 10:35–66.

37. Plow, E. F., T. Herren, A. Redlitz, L. A. Miles, and J. L. Hoover-Plow. 1995.The cell biology of the plasminogen system. FASEB J. 9:939–945.

38. Prentice, M. B., K. D. James, J. Parkhill, S. G. Baker, K. Stevens, M. N.Simmonds, K. L. Mungall, C. Churcher, P. C. Oyston, R. W. Titball, B. W.Wren, J. Wain, D. Pickard, T. T. Hien, J. J. Farrar, and G. Dougan. 2001.Yersinia pestis pFra shows biovar-specific differences and recent commonancestry with a Salmonella enterica serovar Typhi plasmid. J. Bacteriol. 183:2586–2594.

39. Ramu, P., R. Tanskanen, M. Holmberg, K. Lahteenmaki, T. K. Korhonen,and S. Meri. 2007. The surface protease PgtE of Salmonella enterica affectscomplement activity by proteolytically cleaving C3b, C4b and C5. FEBS Lett.581:1716–1720.

40. Ramu, P., L. A. Lobo, M. Kukkonen, E. Bjur, M. Suomalainen, H. Raukola,M. Miettinen, I. Julkunen, O. Holst, M. Rhen, T. K. Korhonen, and K.Lahteenmaki. 2008. Activation of pro-matrix metalloproteinase-9 and deg-radation of gelatin by the surface protease PgtE of Salmonella entericaserovar Typhimurium. Int. J. Med. Microbiol. 298:263–278.

41. Rawlings, N. D., F. R. Morton, C. Y. Kok, J. Kong, and A. J. Barrett. 2008.MEROPS: the peptidase database. Nucleic Acids Res. 36:D320–D325.

42. Rice, P., I. Longden, and A. Bleasby. 2000. EMBOSS: the European Molec-ular Biology Open Software Suite. Trends Genet. 16:276–277.

43. Sebbane, F., C. O. Jarrett, D. Gardner, D. Long, and B. J. Hinnebusch. 2006.Role of the Yersinia pestis plasminogen activator in the incidence of distinctsepticemic and bubonic forms of flea-borne plague. Proc. Natl. Acad. Sci.USA 103:5526–5530.

44. Sebbane, F., D. Gardner, D. Long, B. B. Gowen, and B. J. Hinnebusch. 2005.Kinetics of disease progression and host response in a rat model of bubonicplague. Am. J. Pathol. 166:1427–1439.

45. Sebbane, F., N. Lemaitre, D. E. Sturdevant, R. Rebeil, K. Virtaneva, S. F.Porcella, and B. J. Hinnebusch. 2006. Adaptive response of Yersinia pestis toextracellular effectors of innate immunity during bubonic plague. Proc. Natl.Acad. Sci. USA 103:11766–11771.

46. Skurnik, M., A. Peippo, and E. Ervela. 2000. Characterization of theO-antigen gene clusters of Yersinia pseudotuberculosis and the cryptic O-antigengene cluster of Yersinia pestis shows that the plague bacillus is most closelyrelated to and has evolved from Y. pseudotuberculosis serotype O:1b. Mol.Microbiol. 37:316–330.

47. Sodeinde, O. A., and J. D. Goguen. 1989. Nucleotide sequence of the plas-minogen activator gene of Yersinia pestis: relationship to ompT of Escherichiacoli and gene E of Salmonella typhimurium. Infect. Immun. 57:1517–1523.

48. Sodeinde, O. A., and J. D. Goguen. 1988. Genetic analysis of the 9.5-kilobasevirulence plasmid of Yersinia pestis. Infect. Immun. 56:2743–2748.

49. Sodeinde, O. A., Y. V. Subrahmanyam, K. Stark, T. Quan, Y. Bao, and J. D.Goguen. 1992. A surface protease and the invasive character of plague.Science 258:1004–1007.

50. Song, Y., Z. Tong, J. Wang, L. Wang, Z. Guo, Y. Han, J. Zhang, D. Pei, D.Zhou, H. Qin, X. Pang, Y. Han, J. Zhai, M. Li, B. Cui, Z. Qi, L. Jin, R. Dai,F. Chen, S. Li, C. Ye, Z. Du, W. Lin, J. Wang, J. Yu, H. Yang, J. Wang, P.Huang, and R. Yang. 2004. Complete genome sequence of Yersinia pestisstrain 91001, an isolate avirulent to humans. DNA Res. 11:179–197.

VOL. 191, 2009 FIBRINOLYTIC ACTIVITIES OF THE Pla MOLECULE 4765

on July 15, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

51. Summaria, L., B. Hsieh, W. R. Groskopf, and K. C. Robbins. 1967. Theisolation and characterization of the S-carboxymethyl � (light) chain deriv-ative of human plasmin. The localization of the active site on the � (light)chain. J. Biol. Chem. 242:5046–5052.

52. Suomalainen, M., J. Haiko, P. Ramu, L. Lobo, M. Kukkonen, B. Westerlund-Wikstrom, R. Virkola, K. Lahteenmaki, and T. K. Korhonen. 2007. Usingevery trick in the book: the Pla surface protease of Yersinia pestis. Adv. Exp.Med. Biol. 603:268–278.

53. Tracewell, C. A., and F. H. Arnold. 2009. Directed enzyme evolution: climb-ing fitness peaks one amino acid at a time. Curr. Opin. Chem. Biol. 13:3–9.

54. Une, T., and R. R. Brubaker. 1984. In vivo comparison of avirulent Vwa�

and Pgm� or Pstr phenotypes of yersiniae. Infect. Immun. 43:895–900.55. Vandeputte-Rutten, L., R. A. Kramer, J. Kroon, N. Dekker, M. R. Egmond,

and P. Gros. 2001. Crystal structure of the outer membrane protease OmpTfrom Escherichia coli suggests a novel catalytic site. EMBO J. 20:5033–5039.

56. Varadarajan, N., J. Gam, M. J. Olsen, G. Georgiou, and B. L. Iverson. 2005.Engineering of protease variants exhibiting high catalytic activity and exquis-ite substrate selectivity. Proc. Natl. Acad. Sci. USA 102:6855–6860.

57. Wren, B. W. 2003. The yersiniae—a model genus to study the rapid evolutionof bacterial pathogens. Nat. Rev. Microbiol. 1:55–64.

58. Zhang, L., D. Seiffert, B. J. Fowler, G. R. Jenkins, T. C. Thinnes, D. J.Loskutoff, R. J. Parmer, and L. A. Miles. 2002. Plasminogen has a broadextrahepatic distribution. Thromb. Haemost. 87:493–501.

59. Zhou, D., Y. Han, Y. Song, P. Huang, and R. Yang. 2004. Comparative andevolutionary genomics of Yersinia pestis. Microbes Infect. 6:1226–1234.

60. Zhou, D., Z. Tong, Y. Song, Y. Han, D. Pei, X. Pang, J. Zhai, M. Li, B.Cui, Z. Qi, L. Jin, R. Dai, Z. Du, J. Wang, Z. Guo, J. Wang, P. Huang, andR. Yang. 2004. Genetics of metabolic variations between Yersinia pestisbiovars and the proposal of a new biovar, microtus. J. Bacteriol. 186:5147–5152.

61. Zhou, D., Y. Han, Y. Song, Z. Tong, J. Wang, Z. Guo, D. Pei, X. Pang, J.Zhai, M. Li, B. Cui, Z. Qi, L. Jin, R. Dai, Z. Du, J. Bao, X. Zhang, J. Yu, J.Wang, P. Huang, and R. Yang. 2004. DNA microarray analysis of genomedynamics in Yersinia pestis: insights into bacterial genome microevolutionand niche adaptation. J. Bacteriol. 186:5138–5146.

4766 HAIKO ET AL. J. BACTERIOL.

on July 15, 2019 by guesthttp://jb.asm

.org/D

ownloaded from