Molecular Microbiology 2001) 40 5), 1097±1111

Protein regions important for plasminogen activation and inactivation of a2-antiplasmin in the surface Pla of

Maini Kukkonen,1 Kaarina LaÈhteenmaÈki,1 influenced by residue R211 in L4 as well as by Marjo Suomalainen,1 Nisse Kalkkinen,2 unidentified residues in L3. OmpT, which is not Levente EmoÈ dy,3 Hannu LaÊng1 and associated with invasive bacterial disease, was Timo K. Korhonen1* converted into a Pla-like protease by deleting resi- 1Division of General Microbiology, Department of dues D214 and P215, by substituting residue K217 for Biosciences, and 2Institute of Biotechnology, PO Box 56, R217 in L4 of OmpT and also by substituting the FIN-00014, University of Helsinki, Finland. entire L3 with that from Pla. This simple modification 3Department of Microbiology, University Medical School, of the surface loops and the substrate specificity of PeÂcs, Hungary. OmpT exemplifies the evolution of a housekeeping into a virulence factor by subtle mutations at critical protein regions. We propose that inactivation Summary of a2-antiplasmin by Pla of Y. pestis promotes The plasminogen activator, surface protease Pla, of uncontrolled proteolysis and contributes to the the plague bacterium Yersinia pestis is an important invasive character of plague. virulence factor that enables the spread of Y. pestis from subcutaneous sites into circulation. Pla-expres- sing Y. pestis and recombinant Introduction formed active plasmin in the presence of the major Plague is an acute zoonotic disease and among the most human plasmin inhibitor, a2-antiplasmin, and the severe bacterial diseases ofman. The aetiological agent bacteria were found to inactivate a2-antiplasmin. In ofthe disease is Yersinia pestis, which is usually contrast, only poor plasminogen activation and no transmitted by the bite of infected fleas and produces cleavage of a2-antiplasmin was observed with recom- the systemic and often fatal disease. Y. pestis is a highly binant bacteria expressing the homologous gene uniform species and genetically closely related to Yersinia ompT from E. coli.Ab-barrel topology model for pseudotuberculosis Bercovier et al., 1980), which is Pla and OmpT predicted 10 transmembrane b-strands transmitted via the oral route and causes intestinal disease and five surface-exposed loops L1±L5. Hybrid Pla± Brubaker, 1972). It was estimated recently that Y. pestis OmpT were created by substituting each of has evolved from Y. pseudotuberculosis serotype O:1b the loops between Pla and OmpT. Analysis of the only 1500±20 000 years ago Achtman et al., 1999; hybrid molecules suggested a critical role of L3 and Skurnik et al., 2000). Despite its recent emergence, Y. L4 in the substrate specificity of Pla towards pestis has caused three waves ofpandemic plague of plasminogen and a2-antiplasmin. Substitution analy- devastating magnitude reviewed by Perry and Fetherston, sis at 25 surface-located residues showed the 1997). The bacterium still persists endemically in many importance of the conserved residues H101, H208, countries and causes local outbreaks. D84, D86, D206 and S99 for the proteolytic activity of Given the highly virulent and invasive character of Y. Pla-expressing recombinant E. coli. The mature a-Pla pestis and its relatedness to Y. pseudotuberculosis,it of 292 amino acids was processed into b-Pla by an seems a paradox that Y. pestis lacks the inv and yadA autoprocessing cleavage at residue K262, and resi- genes that enhance the invasiveness of Y. pseudotuber- dues important for the self-recognition of Pla were culosis and Yersinia enterocolitica. The invasin gene of Y. identified. Prevention of autoprocessing of Pla, pestis is inactivated as a result ofinsertion ofan IS 200- however, had no detectable effect on plasminogen like element Simonet et al., 1996), and the yadA adhesin activation or cleavage of a2-antiplasmin. Cleavage of on account of a frameshift mutation Skurnik and Wolf- a2-antiplasmin and plasminogen activation were Watz, 1989). Y. pestis has two plasmids not shared by other species of Yersinia, the 96.2 kb plasmid pFra and Accepted 19 March, 2001. *For correspondence. E-mail timo. [email protected]; Tel. 1358) 9 1915 9260; Fax 1358) 9 1915 the 9.5 kb plasmid pPCP1, also called pPst Ferber and 9262. Brubaker, 1981; Perry and Fetherston, 1997). The

Q 2001 Blackwell Science Ltd 1098 M. Kukkonen et al. plasmid pFra has been suggested to contribute to the are included in the omptin family of serine transmission ofthe plague bacillus by the flea Hinne- Mangel et al., 1994), which lack the classical serine busch et al., 1998), and the plasmid pPCP1 has been protease consensus sequences. OmpT is the best- shown to contribute to the invasive character ofplague. characterized member ofthis protein family;it has been Loss ofpPCP1 increases the median lethal dose of Y. associated with human urinary tract infections Stumpe pestis for mice by about a million-fold, but only when the et al., 1998), but not with invasive infections. Its role in E. infection initiates subcutaneously Brubaker et al., 1965). coli infectivity still remains open, and it appears most likely The plasmid pPCP1 encodes at least three proteins: a to be a housekeeping protein functioning in the degrada- bacteriocin termed pesticin, a protein conferring immunity tion ofdenatured proteins White et al., 1995). A recent to pesticin and an outer membrane protease termed Pla study identified the active-site serine and histidine and Sodeinde and Goguen, 1988). Sodeinde et al. 1992) provided an antiparallel b-barrel topology model for the showed by mutagenesis ofthe genes in pPCP1 that the protein Kramer et al., 2000a). virulence-enhancing property is encoded by the pla gene The exact mechanisms ofhow Pla enhances invasive- and that pla is essential for the invasive character of plague. ness of Y. pestis remain to be elucidated. Pla is an Pla of Y. pestis is a 292-amino-acid outer membrane efficient proteolytic activator of plasminogen Sodeinde protease Sodeinde and Goguen, 1989) that shows a high and Goguen, 1989), and analysis ofthe virulence ofwild- degree ofsequence identity with fourother enterobacter- type and pla knock-out Y. pestis in plg1/1 and plg±/± mice ial surface proteases, PgtE of Salmonella typhimurium have stressed the importance ofplasminogen activation in Guina et al., 2000), OmpT of Escherichia coli Grodberg the virulence function of Pla Sodeinde et al., 1992; et al., 1988), OmpP of E. coli Kaufmann et al., 1994) and Goguen et al., 2000). It remains unclear how plasminogen SopA of Egile et al., 1997). The proteins activation enhances invasion by Y. pestis, and other

Table 1. Effect of mutations in Pla on plasminogen activation, inactivation of a2-antiplasmin and autoprocessing ofPla by E. coli XL1 pMRK1).

Plasminogen activationa Inactivation ofAutoprocessing b c Mutation in Pla Mean Range a2-antiplasmin into b-Pla

None 100 11 H28V 125 90±173 11 H98V 94 72±134 11 H101V 0.8 , 0.1±1.2 ± ± H108V 121 85±162 11 H203V 109 82±152 11 H208V 1.7 0.9±2.6 ± ± S77A 133 117±163 11 S99A 1.0 , 0.1±2.9 ± ± S267A 108 70±146 11 D84A 3.7 , 0.1±6.3 ± ± D86A 3.5 , 0.1±7.2 ± ± D97A 56 36±72 11 D206A 1.3 , 0.1±1.9 ± ± D212R 111 76±149 11 M210G 58 45±80 11 R211K 11 3.3±18 ± 1 L213I 123 113±134 11 F215Y 42 38±46 1 1) E217S 52 31±75 1 ± K218A 103 96±110 11 K240A 151 150±153 11 K249A 132 114±150 11 K254A 90 67±114 11 K262A 85 72±100 1 ± K280A 98 86±111 11 ::D211P212 0.8 , 0.1±2.4 ± ± a. Pla activity was assessed using 8 Â 107 recombinant bacteria, human plasminogen and the chromogenic plasmin substrate Val-Leu-Lys-p- nitroaniline hydrochloride. The rate ofplasmin formationwas estimated according to the procedure ofMangel et al. 1994) for a coupled omptin reaction, using values from the time interval 15±60 min. The results are means of four independent assays; the range gives the lowest and highest value in the assays. The plasmin formed by E. coli XL1 pMRK1) was assigned as 100% in each individual test. b. Inactivation was assessed as the inability of a2AP to prevent conversion ofplasminogen by recombinant E. coli and as the cleavage of a2AP by the strain. c. Formation of b-Pla by the mutated proteins was analysed by Western blotting ofcell envelope preparations with anti-His 6±Pla as primary antibodies. 1) Partial autoprocessing.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 Plasminogen activator Pla of Yersinia pestis 1099 functions have also been proposed for Pla. It cleaves the adherence of Y. pestis to laminin and human and murine C3 component ofcomplement and thus suppresses basement membrane LaÈhteenmaÈki et al., 1998). Y. migration of inflammatory cells to the infection site, pestis degrades laminin and subepithelial extracellular which may reduce phagocytosis ofthe invading bacterium matrix through its capacity to generate plasmin LaÈhteen- Sodeinde et al., 1992). Pla also has weak coagulase maÈki et al., 1998), which may enhance the spread ofthe activity, which is detectable only with rabbit plasma bacterium through tissue barriers. Pla-generated plasmin Jawetz and Meyer, 1944). Pla contributes to adhesion also degrades fibrin Beesley et al., 1967); this can to eukaryotic cells Kienle et al., 1992) and promotes enhance bacterial migration by preventing entrapment of bacteria in fibrin clots Sodeinde et al., 1992). The formation and activity of proteases in the human circulation are tightly controlled, and circulating, unbound plasmin is rapidly inactivated by plasma antiproteases. Approximately 10% ofthe protein mass in human plasma is composed ofproteinase inhibitors, and the main inhibitor ofplasmin in plasma is the serine protease

inhibitor serpin) a2-antiplasmin a2AP) Travis and Salvesen, 1983). a2AP inhibits plasmin by forming an equimolar complex with it. Pla of Y. pestis can activate plasminogen in human plasma Sodeinde et al., 1992), but the mechanism ofhow Pla-generated plasmin activity can overcome inhibition by a2AP has not been addressed. We report here that Pla inactivates human a2AP. This promotes uncontrolled proteolysis and can be important for the highly invasive character of Y. pestis. The b-barrel fold in bacterial outer membrane proteins is stable and tolerates changes in the surface-exposed loops Koebnik, 1999; Koebnik et al., 2000; Schulz, 2000). On the other hand, small changes in the specificity pocket of serine proteases can have dramatic effects on the substrate specificity and catalytic rates of the enzymes Craik et al., 1985; Wells et al., 1987). We substituted putative surface loops and amino acid residues between Pla of Y. pestis and OmpT of E. coli and identified regions and residues in the Pla molecule that contribute to plasminogen activation and antiprotease inactivation.

Fig. 1. Inactivation of a2AP by Pla-expressing bacteria. A. Plasmin activity generated by the pPCP1-carrying Y. pestis KIM D27, the pPCP1-negative Y. pestis KIM D34 and the recombinant strain E. coli XL1 pMRK1) expressing Pla. Bacteria and plasminogen were incubated in PBS without or with a2AP, and plasmin activity was measured with the chromogenic plasmin substrate Val-Leu-Lys-p- nitroaniline. Plasmin activity in the absence ofbacteria alone is also shown and, for comparison, activity of free exogenous plasmin in the absence or presence of a2AP right bottom). B. Cleavage ofplasminogen Plg) by Y. pestis KIM D27, Y. pestis KIM D34, E. coli XL1 pMRK1) Pla1) and the host strain E. coli XL1 pSE380) lacking Pla. Bacteria were incubated with plasminogen in the absence top) or the presence of a2AP bottom), and the cleavage was estimated by SDS±PAGE and Western blotting ofthe cell suspensions using anti-plasminogen antibodies as primary antibodies. C. Cleavage of a2AP by the bacteria. The bacteria were incubated with a2AP, and the cleavage was estimated by SDS±PAGE and Western blotting ofthe suspensions using anti- a2AP as primary antibodies.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 1100 M. Kukkonen et al. Results Protection ofplasmin frominhibition by a2AP could result from binding of plasmin to the bacterial surface, as Inactivation of a -antiplasmin by Pla-positive Y. pestis 2 receptor-bound plasmin is protected from inactivation by and recombinant E. coli a2AP Wiman et al., 1979), or from inactivation of a2AP We observed that a2AP only transiently inhibited the by Pla. The presence ofplasminogen receptors on Y. plasmin activity formed by the Pla-expressing Y. pestis pestis has not been reported, and we observed that two- strain KIM D27, whereas it permanently inactivated free thirds ofthe plasmin formedby Pla-expressing Y. pestis plasmin Fig. 1A). No plasmin activity was detected with or recombinant E. coli was soluble in the buffer data not the Y. pestis strain KIM D34 that has been cured ofthe shown). The transient nature ofprotease inhibition by plasmid pPCP1 and thus lacks pla Fig. 1A). In order to a2AP suggested that the bacteria inactivated a2AP. study the role ofPla activity more directly, we cloned the Indeed, cleavage ofthe 70 kDa a2AP was observed open reading frame ORF) of pla into the plasmid pSE380 with Pla-expressing Y. pestis KIMD27 and E. coli behind the inducible trc promoter. a2AP-resistant plasmin XL1 pMRK1), whereas no detectable cleavage of a2AP activity was also formed in the presence of the resulting was seen with the Pla-negative strains Y. pestis KIMD34 recombinant E. coli strain XL1 pMRK1) pla1) Fig. 1A), or E. coli XL1 pSE380) Fig. 1C). A 50 kDa peptide but not in the presence ofthe host strain E. coli reacting with anti-a2AP antibodies was formed rapidly in XL1 pSE380) lacking pla not shown). We also observed the presence ofPla-expressing bacteria, and it appeared that a2AP at a physiological concentration did not prevent that cleavage of a2AP did not proceed into smaller conversion ofplasminogen to plasmin by Pla-carrying Y. peptides. The 50 kDa peptide was detected in the cell- pestis or recombinant E. coli Fig. 1B). free assay supernatant only, whereas the apparently

Fig. 2. Comparison ofthe expression and proteolytic activity ofPla and OmpT in the E. coli XL1 host strain.

A. SDS±PAGE left) and Western blotting right) with anti-His6±Pla and anti-His6±OmpT polyclonal antibodies ofcell envelope preparations from recombinant E. coli. Lanes 1±3, cell envelopes from E. coli XL1 pSE380) 1), E. coli XL1 pMRK1) pla1) 2) and E. coli XL1 pMRK2) ompT1). Migration oftwo molecular weight marker proteins is shown on the left. B. Cleavage of a2AP top) and plasminogen Plg) bottom) by the recombinant E. coli. The bacteria were incubated with a2AP or Plg, and samples were collected at the indicated time points for SDS±PAGE and Western blotting with anti-a2AP or anti-Plg as primary antibodies. Lanes C1 and C2, plasmin C1) and cleavage ofthe proteins by E. coli XL1 pSE380) C2).

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 Plasminogen activator Pla of Yersinia pestis 1101

Fig. 3. Topology models and sequence alignment ofPla of Y. pestis and OmpT of E. coli. Identical residues are marked by an asterisk, the predicted surface-associated loops are indicated by L1 to L5, and the transmembrane antiparallel b-strands are marked by dashed lines above the sequences. Lines above the sequences indicate fusion sites in Pla and OmpT hybrid proteins. The symbol q indicates residues important for the proteolytic activity of Pla, and the arrow in L5 indicates the autoprocessing site in Pla. uncleaved 70 kDa form of a2AP was detected in both the larger form that most probably represented the unpro- cell-bound and the free forms data not shown). cessed pre-Pla Sodeinde and Goguen, 1988; Sodeinde et al., 1988; Kutyrev et al., 1999). We estimated that pre- Pla comprised 11% ofthe immunoreacting Pla peptides, Comparison of Pla and OmpT in recombinant E. coli a-Pla 43%, b-Pla 30% and g-Pla 16%. The anti-OmpT Another omptin, OmpT of E. coli, has also been reported antibodies reacted with OmpT of39 kDa as well as with a to activate plasminogen Leytus et al., 1981), but is not polypeptide of31 kDa, both ofwhich are close to the sizes associated with invasive disease. We therefore compared ofthe two conformationsofmature OmpT described the catalytic rates ofPla and OmpT expressed in recently Kramer et al., 2000b). No serological cross- recombinant E. coli. We performed Western blotting reactivity was detected between Pla and OmpT, and analysis with anti-His6-Pla and anti-His6-OmpT antibodies neither did the antibodies react with cell envelope proteins ofcell envelope preparations from E. coli XL1 pMRK1) from E. coli XL1 pSE380) Fig. 2A). The anti-Pla and anti- pla1), E. coli XL1 pMRK2) ompT1) and the control strain OmpT antibodies also reacted specifically with recombi- E. coli XL1 pSE380) Fig. 2A). The anti-Pla antibodies nant strains expressing Pla or OmpT in an indirect detected four forms of the Pla protein, the presumably immunofluorescence staining of bacterial cells data not processed polypeptides a-, b- and g-Pla as well as a shown).

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 1102 M. Kukkonen et al. coli XL1 pMRK2) was also inefficient in cleavage of a2AP; no degradation was observed in a 22 h incubation under conditions in which E. coli XL1 pMRK1) rapidly cleaved a2AP Fig. 2B). We estimated the cleavage activities by bacteria carrying the protein derivatives described below after 4 h and 22 h incubations in order to detect both the rapid Pla-like and the slow OmpT-like activities. Plasminogen activation by OmpT is often measured using cell envelopes from OmpT-expressing E. coli Leytus et al., 1981; Mangel et al., 1994). We found no difference between plasminogen activation by cell envel- ope preparations or by cells ofthe recombinant E. coli XL1 derivatives data not shown). In addition, use ofthe ompT lon E. coli strain BL21 pREP4) as the expression host did not change the observed activity levels, indicating that the low endogenous OmpT activity of E. coli XL1 did not affect the observed results. It was concluded that the recombinant E. coli strains expressed functional Pla or OmpT on their cell surface and that the differences observed in plasminogen activation and a2AP cleavage resulted from different substrate specificities of the two omptins.

Proteolytic activities of Pla±OmpT hybrids The overall design ofthe omptin familyofouter membrane proteases appears to be a b-barrel Kramer et al., 2000a,b), which forms a rather rigid membrane- embedded structure with more flexible surface loops Schulz, 2000). Alignment ofPla and OmpT with other omptin sequences and prediction oftransmembrane b- strands in the proteins suggested the presence offive surface-exposed loops in both Pla and OmpT. Topology models for mature Pla and OmpT are shown in Fig. 3. While this study was in progress, two essentially similar topology models for OmpT were presented Kramer et al., 2000a,b), which helped us to refine the model of Pla. The Fig. 4. Plasmin generation and cleavage of a2AP by E. coli XL1 putative surface loops L1±L5 of Pla and OmpT shared pMRK1) pla1)andE. coli XL1 pMRK2) ompT1) top) and by the between 39% and 60% identity and between 61% and E. coli XL1 derivatives expressing the Pla±OmpT hybrid proteins 77% similarity. We raised polyclonal antibodies against Hy). The bacteria were incubated with closed circles) or without open circles) plasminogen in the presence ofthe chromogenic peptides encompassing each ofthe putative loop struc- plasmin substrate Val-Leu-Lys-p-nitroaniline; triangles in the top tures in Pla; in Western blotting ofthe hybrid proteins right graph show plasmin generation by E. coli XL1 pSE380). The described below, each antiserum recognized only those surface loops L1±L5 in Pla closed boxes) and OmpT open boxes) as well as in the hybrid proteins are indicated. Inserts in each hybrids that carried the antigen sequence examples with graph show cleavage of a2AP by the E. coli XL1 derivative, and anti-L5 are shown below). The antipeptide antibodies the migration distances ofnative and cleaved a2AP are indicated. reacted in indirect immunofluorescence staining of E. coli XL1 pMRK1), but not of E. coli XL1 pMRK2) data not Western blot analyses showed that E. coli XL1 pMRK1) shown), which is in accordance with the topology model converted plasminogen into plasmin, whereas only poor for Pla. The anti-L1 and anti-L4 sera reacted less strongly conversion was seen with E. coli XL1 pMRK2) and none with E. coli XL1 pMRK1) cells than the other antipeptide with E. coli XL1 pSE380) Fig. 2B). In contrast to the Pla- sera, which means that the antigen sequences were mediated plasminogen conversion, the slow OmpT- poorly surface exposed or had a conformation in the mediated conversion to plasmin was totally inhibited by external loop that was weakly recognized by the a2AP data not shown). In line with this observation, E. antibodies raised against linear epitopes.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 Plasminogen activator Pla of Yersinia pestis 1103

A rapid activation ofplasminogen by E. coli E. coli, and the reverse substitution in OmpT Hy8) XL1 pMRK1) was seen in assays with the chromogenic exhibited direct S-2251 degradation. These results substrate S-2251, and it is noticeable that the bacteria did pointed to a critical role ofL4 in determining the not hydrolyse the substrate S-2251 in the absence of substrate specificity of Pla, and we therefore con- added plasminogen Fig. 4). In contrast, E. coli structed hybrids Hy18 and Hy17, in which residues XL1 pMRK2) hydrolysed S-2251 in the absence of 191±209 and residues 210±229 in Pla were substituted added plasminogen, and the addition ofplasminogen for the corresponding residues in OmpT. Hybrid Hy18 caused only a small increase in colour formation Fig. 4). exhibited a high level ofplasminogen activation, This indicated that OmpT hydrolysed the chromogenic whereas Hy17 was inactive Fig. 4), indicating that the substrate S-2251 directly, in addition to slowly forming C-terminal end ofL4 was important forplasminogen plasmin. Only marginal plasmin activity was detected in activation by Pla. the presence of E. coli XL1 pSE380) Fig. 4). In order to Western blotting analyses showed that bacteria expres- locate regions in Pla important for its substrate specificity, sing the hybrids Hy5, Hy10, Hy12, Hy1 and Hy18 cleaved Pla±OmpT hybrid molecules were constructed and plasminogen, whereas no conversion could be detected assessed for plasminogen activation and cleavage as with bacteria expressing the other hybrids data not well as for cleavage of a2AP. The Pla±OmpT hybrids shown), which is in accordance with the results shown were constructed by substituting individual loops between in Fig. 4. Cleavage of a2AP was detected with Pla and OmpT at the conserved transmembrane regions recombinant E. coli expressing Hy5, Hy10 and Hy18, or, in the case ofL1, at the conserved C-terminal end of but not with cells expressing the other hybrids Fig. 4). L1 see Fig. 3). The surface expression of the hybrid These data indicated the importance ofL5 substituted molecules was confirmed by indirect immunofluorescence in Hy1), L3 substituted in Hy12) and the C-terminal end ofbacterial cells data not shown) as well as by Western ofL4 substituted in Hy17) in the inactivation of a2AP blotting ofcell envelope preparations see examples by Pla. As observed with the bacteria expressing Pla, below) with the anti-His6-Pla, the anti-His6-OmpT and plasminogen cleavage by bacteria carrying the hybrids the antipeptide sera raised against the individual pre- Hy5, Hy10 and Hy18 was not inhibited by a2AP data dicted loop structures. The hybrids could be detected at not shown). the cell surface, and these analyses revealed only minor differences in the level of surface expression of the hybrid Identification of proteolytically important residues in Pla molecules. We estimated that the rate ofplasminogen activation by 8 Â 107 XL1 pMRK1) cells varied on Pla and the other omptins are considered to be serine different days between 60% and 185% of the mean. proteases that have the catalytic triad histidine±serine± Although such variation is significant, we considered it aspartate in their active centre Rawlings and Barrett, acceptable for the identification of mutations that affect 1994). In order to identify the active-site residues in Pla, a the substrate specificity of Pla. In comparing the rate of series ofsubstitutions was constructed at histidine, serine plasmin formation by the hybrids as well as by the and aspartate residues in Pla. We substituted all six mutated proteins described below, the primary data from histidine residues in Pla for valine. The substituted serines the parabolic curves shown in Fig. 4 were linearized and aspartates were chosen on the basis that they are Mangel et al., 1994), and the values at the time interval conserved in omptins and located in surface-exposed 15±60 min were used. loops, and we also searched for sequence homology to Substitution ofthe L1-containing amino-terminus, L2 or the active-site sequences in tissue-type tPA) or uroki- L3 in Pla with the corresponding protein regions from nase uPA) plasminogen activators, which cleave plas- OmpT hybrids Hy5, Hy10 and Hy12) reduced plasmin minogen at the same R560±V561 bond as does Pla formation by recombinant E. coli XL1 pMRK1) by . 70% Rijken, 1995). A total of25 substitution mutations was Fig. 4). The reverse substitutions in OmpT decreased the created in Pla; ofthese, six mutations resulted in a Pla direct hydrolysis ofS-2251 hybrids Hy4 and Hy9) or variant that exhibited 4% or less ofthe plasminogen induced a low level ofplasmin formation hybrid Hy11). activation exhibited by Pla Table 1). Substitution ofL4 in Pla hybrid Hy14) resulted in Bacteria with the substitutions H101V and H208V bacteria lacking plasminogen activation capacity but, Fig. 3) expressed only 0.8% and 1.7% ofthe plasmino- instead, expressing a low level ofOmpT-like activity in gen activation level shown by XL1 pMRK1) Table 1). The the degradation ofS-2251. The reverse substitution in H101V and H208V substitution mutants also failed to OmpT hybrid Hy13) resulted in bacteria expressing a inactivate a2AP, whereas the other histidine mutants marginal level ofplasminogen activation. Substitution of were active Table 1). It was concluded that H101 and the L5-containing C-terminus ofPla Hy1) caused a H208 are important for the proteolytic activity of Pla. The 95% decrease in plasminogen activation by recombinant substitution S99A decreased the plasminogen activation

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 1104 M. Kukkonen et al. proteolytic activity ofthe recombinant bacteria Table 1). These results also indicated that the C-terminal end ofL4 is involved in substrate specificity of Pla.

Autoprocessing of Pla

Pla occurs in three processed forms of a-Pla, b-Pla and g- Pla see Fig. 2), ofwhich a-Pla corresponds to full-size mature Pla, and b-Pla and g-Pla are thought to result from processing of a-Pla Sodeinde and Goguen, 1988; Sodeinde et al., 1988; Kutyrev et al., 1999). We observed that none ofthe Pla mutants lacking proteolytic activity formed b-Pla Table 1; see Fig. 5 for examples), which suggests that b-Pla results from an autoprocessing event. g-Pla, however, was observed in all Pla variants, Fig. 5. Western blotting ofPla, Pla substitution derivatives and regardless oftheir proteolytic activity Fig. 5). The Pla±OmpT hybrid Hy) proteins in cell envelopes from E. coli XL1. Envelope proteins were separated by SDS±PAGE, and blotting substitution E217S abolished b-Pla formation by recom- was carried out with anti-His6±Pla and anti-L5 polyclonal binant E. coli, and the substitution F215Y lead to antibodies. The position of a-Pla, b-Pla and g-Pla are indicated. For decreased b-Pla formation Fig. 5). It is notable that surface loop structures of Hy1, Hy6 and Hy8, see Figs 4 and 6. these two mutated Pla proteins supported plasminogen activation and a2AP cleavage Table 1). The N-terminal by 99% Table 1). The S99A mutant strain was also amino acid sequence of b-Pla ASSQLIPNISPDS) was inactive in a2AP cleavage. The substitutions at residues found to be identical to that of a-Pla. Pla has no cysteine D84, D86 and D206 abolished detectable plasminogen residues and disulphide bonds and, hence, it seems likely cleavage and reduced plasmin formation by . 96%. that the Pla molecule is autoprocessed at the C-terminal Recombinant bacteria with the mutations D84A, D86A end. The apparent molecular weight of b-Pla, as or D206A were also poor in a2AP cleavage. The measured from SDS±PAGE gel, suggested that it is substitutions at other histidine, serine or aspartate approximately 30 amino acids shorter than a-Pla Fig. 2). residues had less effect on plasminogen activation and Omptins cleave proteins after basic residues, and we a2AP cleavage. replaced each basic residue at or near L5 K218, K240, K249, K254, K262 and K280) with alanine. Ofthese mutated proteins, only K262A did not form b-Pla, although L4 amino acid residues affecting the substrate specificity it retained proteolytic activity Fig. 5; Table 1). The K262A of Pla substitution derivative ofPla supported high-level plasmi- Results obtained with the Pla±OmpT hybrid molecules nogen activation and inactivation of a2AP by recombinant and the single amino acid substitutions suggested E. coli Table 1). The presence ofat least partial L5 in b- important roles for L2 and L4 in the proteolytic activity of Pla was also supported by its reactivity with the anti-L5 Pla. L2 is well conserved between Pla and OmpT, peptide serum Fig. 5). The specificity of the antiserum for whereas L4 differs between Pla and OmpT Fig. 3). We L5 was indicated by the finding that it reacted with Hy8, also analysed the importance ofthe C-terminal end ofL4 which carries OmpT with L5 from Pla, but failed to react in the proteolytic activity ofPla by substituting each with Hy1, which was Pla substituted with L5 from OmpT residue at locations 210±217 that differs between the Pla Fig. 5). It is noteworthy that the Hy8 protein was not and OmpT sequences Table 1). The substitution R211K processed into a smaller form. These results suggested decreased plasminogen activation by 89%. a2AP was not that K262 is the autoprocessing site and that E217 and cleaved by the recombinant E. coli with the substitution F215 are important in self-recognition by Pla. R211K, and a2AP inhibited plasminogen conversion by this mutant strain Table 1). The other substitutions did Conversion of OmpT into a Pla-like protease not have dramatic effects on plasminogen activation, with values ranging between 42% and 123% ofthat shown by Pla and OmpT seem to possess very similar overall recombinant bacteria expressing wild-type Pla protein. topology in the bacterial outer membrane. Most ofthe Pla and OmpT also differ at this region, in that OmpT has important amino acid differences are located in the three the residues D214 and P215, which are lacking in Pla last surface loops, L3, L4 and L5. We found it appealing to Fig. 3). Insertion ofthese two residues into the corre- modify the substrate specificity of OmpT towards that sponding site in the Pla molecule decreased the ofPla by engineering small changes in key areas of

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 Plasminogen activator Pla of Yersinia pestis 1105

Fig. 6. Conversion ofthe substrate specificity ofOmpT into a Pla-like protease.A. Enhancement ofplasmin formationwas assessed by incubating recombinant E. coli XL1 derivatives with plasminogen and the chromogenic plasmin substrate Val-Leu-Lys-p- nitroaniline; the colour formation in the absence ofadded plasminogen was subtracted. The surface loop structures of mutated or hybrid Hy) proteins expressed in E. coli XL1 are shown: Pla closed boxes), OmpT open boxes), the genetically modified L4 ofOmpT hatched box). B. Cleavage ofplasminogen Plg) by recombinant E. coli in the absence top) or the presence bottom) of a2AP. The bacteria were incubated with Plg, and the cleavage was assessed by Western blotting with anti- plasminogen as primary antibodies. C. Cleavage of a2AP by the recombinant bacteria. The bacteria were incubated with a2AP, and cleavage was assessed by SDS± PAGE and Western blotting using anti-a2AP as primary antibodies. The protein construct expressed in E. coli XL1 is indicated on the right.

the protein and, in this way, also to ascertain the role coli XL1, compared with XL1 pMRK1) expressing the ofL3 and the C-terminal end ofL4 ofPla in entire Pla Fig. 6A). Plasminogen cleavage by bacteria plasminogen activation and cleavage of a2AP. We with the protein OmpT/DDP/K217R/L3 was partially first deleted residues D214 and P215 to shorten L4 in inhibited by a2AP Fig. 6B), and a low degree of OmpT; the resulting mutant protein OmpT/DDP was a2AP modification by these bacteria was evident not significantly improved in plasminogen activation or Fig. 6C). For comparison, the activities ofhybrids a2AP cleavage Fig. 6). The residue R211 in Pla was Hy8, Hy7, Hy6 and Hy5 with increasing C-terminal found to be important for Pla function Table 1), and substitutions for Pla surface loops are shown in Fig. 6. lack ofdetectable cleavage of a2AP by the bacteria A gradual increase in plasminogen activation in expressing Hy12 Fig. 4) suggested the importance of parallel with increasing numbers ofsubstitutions is L3 in a2AP inactivation. The substitution K217R in the evident Fig. 6A). Ofthese mutant or hybrid proteins, corresponding site ofOmpT/ DDP improved plasmin only Hy5 and Hy6 supported detectable a2AP formation, and further substitution of L3 of Pla into cleavage by recombinant E. coli Fig. 6C), and their this mutated protein resulted in the protein OmpT/ plasminogen cleavage was only partially inhibited by DDP/K217R/L3, which supported 8.5% activity in E. a2AP Fig. 6B).

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 1106 M. Kukkonen et al.

Discussion Kramer et al. 2000a) identified S99 and H212 as active-site residues in OmpT. These residues are located The Pla protease is an essential virulence factor of Y. in L2 and L4 ofOmpT. We observed that the correspond- pestis that enables the plague bacterium to spread through ing residues S99 and H208 in Pla are needed for tissue barriers into the circulation Sodeinde et al., 1992). activation ofplasminogen, cleavage of a2AP and autop- The homologous protein in E. coli, OmpT, is not associated rocessing. Kramer et al. 2000a) used a dipeptide with invasive disease. OmpT was originally described as a substrate to identify the active-site residues, and we plasminogen activator Leytus et al., 1981), but the tested various tripeptide chromogenic substrates mimick- biological significance of the plasminogen activator function ing the cleavage site in the single-chain plasminogen, but ofOmpT has remained questionable. The ompT gene is found none that was cleaved by Pla-expressing bacteria. present in the vast majority of E. coli isolates, but This may indicate that the specificity pocket of Pla is plasminogen activation is very rarely detected with these better suited for polypeptide substrates. The residues S99 bacteria, and the observed activity levels have been very and H208 are conserved in all omptin sequences, and it low Sodeinde and Goguen, 1988; Parkkinen et al., 1991; seems reasonable that they are part ofthe active site in Lundrigan and Webb, 1992). We observed differences in Pla as well. We observed that substitution ofthe residues the substrate specificity of these two omptins: Pla efficiently D84, D86 and D206 abolished the proteolytic activities of activated plasminogen and cleaved the important antipro- Pla. The residue D206 was chosen for mutagenesis as tease a2AP, whereas OmpT hydrolysed the small-mole- the sequence AHDND-206 resembles the sequence cular-weight substrate Val-Leu-Lys-p-nitroaniline, but AHHND-102 lining the active-site aspartate-102 in the cleaved plasminogen very slowly and did not cleave catalytic domain ofurokinase Spraggon et al., 1995). a2AP at all. We located regions in Pla that contribute to Kramer et al. 2000a) found that the substitution at the its virulence-associated substrate specificity and, using this histidine corresponding to H101 in Pla abolished the knowledge, we modified the substrate specificity of OmpT reactivity ofOmpT with a dipeptide substrate but not with of E. coli towards that ofPla of Y. pestis. a pentapeptide substrate. In the absence ofa low- Pla and OmpT both appear to be structurally similar molecular-weight substrate for Pla, we cannot settle the antiparallel b-barrel proteins localized to the outer exact roles ofD84, D86, S99, H101, D206 and H208 in membrane. In addition to omptins, this structural archi- the proteolytic activity ofPla. However, we hypothesize tecture is present in several outer membrane proteins that that S99 is the active nucleophile, H208 the general base function in solute transport or in biogenesis of surface and D206 the acidic amino acid in the active site ofPla. filaments or are enzymes for recent reviews, see Koebnik D84, D86 and H101 may be indirectly involved in et al., 2000; Schulz, 2000). The topology model for Pla proteolysis via interactions with the substrates or are fulfils the construction rules for transmembrane b-barrels needed for the correct contact of the nucleophile S99 with Koebnik et al., 2000; Schulz, 2000). While this study was the substrates. The resolution ofthis problem requires a in progress, Kramer et al. 2000a,b) reported two b-barrel three-dimensional model ofan omptin, which is not topology models for OmpT that were used to refine our presently available. The crystal structure ofone b-barrel model for Pla. Our finding that polyclonal antibodies outer membrane enzyme has been determined. Phos- against loop-located peptides recognized Pla on the pholipase A of E. coli is a 12-stranded dimer with two surface of recombinant E. coli is in accordance with the active centres at the dimer interface Snijder et al., 1999). proposed topology model ofPla. The anti-L2, anti-L3 and At present, it is not known how omptins are oligomerized anti-L5 sera bound strongly to E. coli XL1 pMRK1) cells, in the outer membrane. Purified OmpT exhibits an whereas the binding ofanti-L1 and anti-L4 antibodies was apparently pentameric form in the presence of Triton X- weaker. The latter could result from the conformational 100 Sugimura and Nishihara, 1988). nature ofthe surface-exposedloops. On the other hand, The Pla substitution mutant R211K could not inactivate we found that L4 residues are important for the proteolytic a2AP, but supported autoprocessing and weak plasmino- activity ofPla. By analogy with the active sites in serine gen activation, which suggests that this residue is proteases, such as the catalytic domain ofuPA Spraggon particularly important in the correct interaction ofPla et al., 1995), we hypothesize that the L4 residues are part with the a2AP substrate. The point mutation R211K did ofthe specificitypocket and hence inaccessible to not change the basic character ofthe residue, and we did antibodies. The b-barrel is a sturdy membrane-embedded not detect any change in the surface expression of the structure that allows large changes in the surface loops protein. L4 ofOmpT has two amino acid residues, D214 without disturbing the b-barrel folding Koebnik, 1999). and P215, that are absent from the other omptin This enabled us to exchange surface loops and residues sequences. Insertion ofthis dipeptide into the correspond- between Pla and OmpT to identify their significance for ing site in L4 ofPla greatly decreased plasminogen substrate specificity and the virulence functions of Pla. activation and abolished a2AP cleavage as well as

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 Plasminogen activator Pla of Yersinia pestis 1107 autoprocessing. This insertion most probably distorts the change the substrate specificity of OmpT into a more correct positioning ofD206 and H208 towards these `virulent' one, i.e. towards plasminogen activation. The physiological substrates and, conversely, the presence of key elements in this modification were the deletion of D214 and P215 in L4 ofOmpT may denote its substrate D214 and P215 from the L4 of OmpT as well as the specificity. substitution ofL3, which is fouramino acids longer in The mature form of Pla, a-Pla, is slowly processed into OmpT than in Pla. We also made the substitution K217R, b-Pla Sodeinde and Goguen, 1988). A third, apparently as the presence ofarginine at the corresponding position smaller form, g-Pla, is also present in cell envelopes from in Pla was important for plasminogen activation and Pla-positive E. coli Kutyrev et al., 1999). These three cleavage of a2AP by Pla. The resulting protein, OmpT/ forms of Pla were also present in E. coli XL1 pMRK1) DDP/K217R/L3, was well expressed on the bacterial envelope preparations, and we observed that the muta- surface and exhibited an activation level that was 8.5% of tions that abolished the cleavage ofplasminogen and that shown by Pla. This protein also supported a low a2AP also abolished the formation of b-Pla. This strongly degree of a2AP cleavage not seen with OmpT or the two suggested that b-Pla is formed as a result of autoproces- other OmpT substitution proteins, but the conversion of sing. On the other hand, g-Pla was formed by active-site plasminogen to plasmin by the OmpT/DDP/K217R/L3 mutant Pla derivates and also when Pla was expressed in derivative was partially inhibited at a physiological the ompT-null strain E. coli BL21 pREP4). g-Pla probably concentration of a2AP. Further substitution at the N- represents full-size Pla that has folded differently from a- terminal region in hybrid Hy5 increased the activation Pla, as was observed recently with variants ofOmpT level as well as cleavage of a2AP, indicating that the N- Kramer et al., 2000b); it may also be formed by terminal regions ofPla also modulate the specificityofthe proteolysis by another protease of E. coli. Omptins cleave active site towards plasminogen and a2AP. The modifica- target proteins at basic residues Grodberg and Dunn, tion ofOmpT into a Pla-like protease is an example ofhow 1988; Sugimura and Nishihara, 1988), but the exact a bacterial protein may evolve into a powerful virulence preferences of Pla towards its target sequences are not factor by the accumulation of subtle mutations at critical known. We found that the substitution K262A abolished sites without changing the overall architecture ofthe b-Pla formation without affecting plasminogen or a2AP protein. cleavage. Cleavage at this site results in a truncated A wealth ofevidence has suggested that Y. pestis protein with a calculated mass of29 578, which is close to evolved fairly recently from the enteric pathogen Y. the observed apparent molecular weight of b-Pla. The N- pseudotuberculosis serotype O:1b Bercovier et al., terminal amino acid sequence of b-Pla was identical to that 1980; Achtman et al., 1999; Skurnik et al., 2000). This ofmature Pla, and it also reacted with the anti-L5 antiserum evolution has involved loss offunctional inv and yadA in Western blotting. Thus, we concluded that K262 is the genes and, on the other hand, acquisition oftwo Y. pestis- processing site in the Pla molecule. This site is different specific plasmids, of which pPCP1 encodes Pla. The from the residue K217 within L4, which was proposed by predicted sequence ofPla is more identical to the PgtE Kramer et al. 2000b) as the autoprocessing site in OmpT. sequence of S. typhimurium than to the sequences of The substitutions E217S and F215Y in L4 abolished or other omptins, which has led to the suggestion that Y. decreased the autoprocessing into b-Pla, which indicates pestis acquired Pla by horizontal gene transfer from S. that the negatively charged glutamate-217 and the lack ofa typhimurium Sodeinde and Goguen, 1989). Our results hydroxyl group in phenylalanine-215 are important in the support this hypothesis. The sequences ofthe C-terminal self-recognition of L5 by Pla. We found that the hybrid regions in Pla that we found to be important for protein, Hy7, which is OmpT with L4 and L5 from Pla, plasminogen activation, i.e. L3, L4 and L5, are more formed the b-form M. Kukkonen and H. LaÊng, unpublished similar to those in PgtE than to other omptins. The surface data), whereas Hy8 was not autoprocessed. These findings expression ofPgtE in S. typhimurium is affected by the indicate that the autoprocessing specificity of Pla is dictated two-component regulatory system, PhoPQ, which has a by its L4 and L5 surface loops. It was somewhat surprising role in adaptation ofthe bacteria to intracellular environ- that prevention of autoprocessing had no significant effect ments and for survival within macrophages Guina et al., on plasminogen activation or cleavage of a2AP by Pla, and 2000). The presence ofthe PhoPQ signal transduction the biological function of omptin autoprocessing remains to system in Y. pestis was reported recently Oyston et al., be identified. The lowered plasminogen activation observed 2000), but it is not yet known whether the expression of with the Pla±OmpT hybrid Hy1 may result from steric Pla is controlled by this system. hindrance by the unprocessed L5 from OmpT that differs in Several bacterial pathogens have been reported to sequence from L5 of Pla. inactivate a2AP or other protease inhibitors reviewed by Using the information obtained with Pla substitution Travis et al., 1995). Inactivation results from cleavage at mutants and Pla±OmpT hybrid molecules, we decided to or near the reactive site ofthe antiprotease Rapala-Kozik

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 1108 M. Kukkonen et al. et al., 1999). We are currently analysing the mechanisms by polymerase chain reaction PCR) and cloned into pSE380. ofantiprotease inactivation by Pla, and our ongoing work The primers were designed on the basis ofthe nucleotide has shown that Pla also inactivates another plasmin sequences of pla Sodeinde and Goguen, 1989) and ompT Grodberg et al., 1988) and contained an NcoI restriction site inhibitor, a -macroglobulin K. LaÈhteenmaÈki, unpub- 2 at the 50 ends. In order to maintain the correct reading lished). Generation ofplasmin via expression ofeither frames, the forward primer contained the trinucleotide GCA plasminogen activators or plasminogen receptors has immediately after the initiation codon ATG. E. coli strains XL1 been shown to potentiate the degradation ofextracellular and BL21 were used as expression hosts for the full-length matrices and bacterial spread through tissue barriers pla and ompT. In order to produce the His6-tag fusion reviewed by LaÈhteenmaÈki et al., 2000). The virulence proteins, DNA regions of pla and ompT encoding the mature function of Pla has been attributed to plasminogen omptins were PCR amplified and cloned into the pQE30 expression vector. The primers contained a 50 BbuI restric- activation Goguen et al., 2000), and inactivation of tion site in the forward primer and a HindIII restriction site circulating plasmin inhibitors by Pla may create uncon- after the stop codon in the reverse primer. The His6±Pla and trolled proteolysis and contribute to the exceptional His6±OmpT proteins were isolated by affinity chromatogra- virulence potential of Y. pestis. phy from the cytoplasm of E. coli strain BL1 pREP4) using denaturing conditions according to the protocol ofthe QIAexpressionist kit Qiagen). Experimental procedures Strains, plasmids and cultivation of bacteria The bacteria and plasmids used in this study are listed in Hybrid Pla±OmpT proteins and mutagenesis of Pla and Table 2. The Y. pestis strains were cultivated on brain±heart OmpT infusion BHI) agar plates at 288C and then in BHI broth with The nucleotide sequence of pla from plasmid pC4006 was shaking overnight at 378C. Bacteria were washed once with determined by dye terminator cycle sequencing BigDye phosphate-buffered saline PBS), pH 7.1, and suspended in Terminator cycle sequencing kit, ABI Prism 310 genetic PBS for the assays. The recombinant E. coli strains were analyser; PE Applied Biosystems) and found to be identical to cultivated in Luria broth supplemented with glucose 0.2%), the sequences of pla from Y. pestis EV76-6 and pPCP1 ampicillin 100 mgml21) and tetracycline 12.5 mgml21)or deposited at the GenBank under accession numbers X15136 kanamycin 25 mgml21) with shaking overnight at 378C. For and AF053945. The hybrid Pla±OmpT proteins and point induction ofrecombinant pla or ompT, cells from 10 ml of the mutations, deletions and insertions in Pla or OmpT were medium were collected, suspended in 150 ml ofPBS, and created by a recombinant PCR amplification procedure 100 ml ofthe suspension was plated on Luria agar containing Higuchi et al., 1988), in which the desired switch site in the 5 mM IPTG Promega) and antibiotics. After overnight hybrids or the mutation site were incorporated in the internal cultivation, the cells were collected, washed once with PBS primer pair. The outside primer pair was the same as used in and suspended in PBS to a cell density of2 Â 109 cells ml21. amplifying entire pla or ompT, and the internal primer sequences were designed on the basis of pla and ompT Expression of pla and ompT sequences and the desired mutation. The switch sites in the Pla±OmpT hybrids were created at nucleotides 473, 745, Pla and OmpT were expressed from the plasmid pSE380 902 and 1049 of pla and corresponding nucleotides in ompT. with an IPTG-inducible trc promoter or from the plasmid The resulting fusion sites were at residues 46, 136, 190 and pQE30 as N-terminal His-tag fusions. The complete reading 241 ofPla. These sites were chosen as they share high frames of pla from plasmid pC4006 and ompT from the sequence identity and, excepting the Pla loop 1 substitutes, genomic DNA ofthe strain E. coli AAEC072 were amplified encode predicted transmembrane b-strands. The correct

Table 2. Bacterial strains and plasmids.

Strain or plasmid Characteristics Reference or source

Y. pestis KIM D27 pPCP11 pgm pYV1 derivative of Y. pestis KIM-10 Finegold 1968); Une and Brubaker 1984) Y. pestis KIM D34 pPCP12 pgm pYV1 derivative of Y. pestis KIM-10 Finegold 1968); Une and Brubaker 1984); Sample et al. 1987) E. coli AAEC072 Dfim derivative of E. coli MG1655 Blomfield et al. 1991) E. coli XL1 Blue MRF0 D7mcrA)183 D7mcrCB-hsdSMR-mrr) Stratagene 173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F0 proAB lacIqZDM15 Tn10 7tet)] E. coli BL21 lDE3 ompT lon Novagen pSE380 lacO operator and lacIq, trc promoter Invitrogen pQE30 lac operator, T5 promoter Qiagen pREP4 lacI Qiagen pC4006 pla in a 1.2 kb fragment from Yersinia pestis Kienle et al. 1992) pMRK1 pla in pSE380 This study pMRK2 ompT in pSE380 This study

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 Plasminogen activator Pla of Yersinia pestis 1109 nucleotide sequence was ascertained at each substitution or chromogenic substrate was measured at 405 nm in a mutation site before testing the mutated proteins. microtitre plate reader Labsystems Multiskan). The rate of plasmin formation was analysed as described by Mangel et al. 1994) for a coupled assay of omptin activity. We used Immunological methods absorbance values at the time interval 15±60 min to compare activities ofthe modifiedPla proteins. The results are given To obtain the anti-His6±Pla and anti-His6±OmpT antisera, as a percentage ofthe activity shown by E. coli XL1 pMRK1) 400 mg ofthe fusionproteins was subcutaneously injected and are means from four independent assays with duplicates. with Freund's complete adjuvant into New Zealand white The range gives the lowest and highest value in the assays, rabbits; three boosters with 200 mg ofantigen and adjuvant in which the activity shown by E. coli XL1 pMRK1) was were given at weeks 4, 6 and 10, and the antisera were assigned as 100% in each individual test. The effect of a2AP collected 2 weeks after the last booster. on plasmin activity was measured at 23 mgml21, which is a The following peptides were used as immunogens to obtain 1.5-foldexcess ofthe concentration ofplasminogen in the antisera recognizing the loops in Pla: L1, amino acid residues assay on a molar basis. Plasmin Sigma) was used at a 24±38; L2, residues 74±104; L3, residues 149±163; L4, concentration of20 mgml21. Plasminogen activation was residues 201±220; and L5, 249±278. The peptides were also assessed with cell envelope fractions prepared from synthesized using an automatic peptide synthesizer with sonicated E. coli as described by Palva 1978). We used an customized protocols Genosphere Technologies) and con- amount ofcell envelopes that corresponded to 8 Â 107 cells, jugated to keyhole limpet haemocyanin KLH). Molecular and the assay was essentially performed as for whole weight and purity ofthe products 95%) was confirmedby bacteria. reverse-phase high-pressure liquid chromatography HPLC) as well as mass spectrometry, and they were provided to us in lyophilized form. Each peptide±KLH conjugate was used to immunize two rabbits subcutaneously at 3 week intervals Cleavage of plasminogen and a2AP with a dose of200 mg ofthe conjugate and Freund's complete adjuvant. Three booster injections, each containing For cleavage assays, 2 Â 108 bacteria in 100 ml ofPBS were 100 mg ofthe antigen, were given with Freund's incomplete incubated with 5 mg ofhuman Glu-plasminogen or a2AP adjuvant. The antisera were collected 3 weeks after the final Calbiochem) at 378C. In inhibition assays, a2AP was added booster. The antisera were produced by the Laboratory at 60 mgml21. Samples of40 ml were taken for analysis at Animal Center, University ofHelsinki, and by MedProbe, 4 h and 22 h. After adding 20 mlof2Â SDS±PAGE sample Norway. The animals used in this study were treated in buffer and boiling for 10 min, 15 ml ofthe mixture was accordance with the guidelines ofthe institutional animal care resolved on 12% SDS±PAGE. The peptide bands were and use committee. transferred onto nitrocellulose membrane pore size 0.2 mm) In order to analyse Pla and OmpT in cell envelope and probed with rabbit anti-human plasminogen IgG 9 21 preparations, cell suspensions 4 Â 10 cells in 2 ml of 2.7 mgml ; Dakopatts) or rabbit anti-human a2-antiplasmin PBS) were sonicated four times for 30 s over crushed ice, the IgG diluted 1:750; Calbiochem). The peptides were detected unbroken cells were removed, and the cell envelopes were by alkaline phosphatase-conjugated anti- rabbit IgG and the pelleted and suspended in 40 ml ofPBS Palva, 1978). After phosphatase substrate. The intensity ofthe peptide stain on electrophoretic separation by SDS±PAGE gel 12%), the nitrocellulose membranes was quantified using the TINA proteins were transferred onto nitrocellulose membrane version 2.09c) image analysis program Raytest Isotopen- Hybond), and the Pla and OmpT peptides were detected messgeraÈte). with primary anti-His6±Pla or anti-His6±OmpT antisera diluted 1:500) and secondary swine anti-rabbit IgG±alkaline phosphatase conjugate Dako; diluted 1:2500) followed by the phosphatase substrate Towbin et al., 1979). Sequence comparisons and construction of the Pla model For immunofluorescence staining of bacterial cells, drops ofsuspensions were air dried on glass slides, fixedwith 3.5% Alignment ofPla with OmpT Sugimura and Nishihara, 1988) paraformaldehyde in PBS, washed and stained with a 1:32 and with OmpP Kaufmann et al., 1994), PgtE Guina et al., 2000) and SopA Egile et al., 1997) was made with the dilution ofthe anti-His 6±Pla, the anti-His6±OmpT or an antipeptide antiserum and fluorescein isothiocyanate FITC)- CLUSTALW multiple alignment program http://dot.imgen. conjugated swine anti-rabbit IgG Dako; diluted 1:50) using bcm.tmc.edu:9331/multialign.html). The location ofthe b- routine procedures Pere et al., 1987). strands ofPla was predicted based on homology to these other proteins ofthe omptin family.The method has been used before to deduce the topology of OmpF and LamB Plasminogen activation proteins Ferenci, 1994; LaÊng and Ferenci, 1995). The exact ends ofthe b-strands and the beginnings ofthe surfaceloops Kinetic measurement ofplasminogen activation Parkkinen were derived by comparing Pla amino acid sequence with the et al., 1991) was performed by incubating 8 Â 107 bacteria, recent topology model ofOmpT Kramer et al., 2000a). 4 mg ofhuman Glu-plasminogen American Diagnostica) and Similarity ofthe regions in Pla was compared with the the chromogenic plasmin substrate S-2251 0.45 mM Val- sequences ofthe active sites oftPA and uPA using the Leu-Lys-p-nitroaniline dihydrochloride; Chromogenix) in a Genestream ALIGN program http://www.expasy.ch/tools/ total volume of200 mlat378C. Breakdown ofthe #align).

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 40, 1097±1111 1110 M. Kukkonen et al.

Amino acid sequencing Goguen, J.D., Bugge, T., and Degen, J.L. 2000) Role ofthe pleiotropic effects of plasminogen deficiency in infection For N-terminal sequence analysis, the b-Pla peptide was experiments with plasminogen-deficient mice. Methods 21: separated by 12% SDS±PAGE followed by electroblotting 179±183. Matsudaira, 1987) onto ProBlott polyvinylidene difluoride Grodberg, J., and Dunn, J.J. 1988) ompT encodes the PVDF) membrane Applied Biosystems) and visualized by Escherichia coli outer membrane protease that cleaves T7 Coomassie brilliant blue staining. The stained protein band RNA polymerase during purification. J Bacteriol 170: was cut out and loaded to a Procise 494A sequencer Applied 1245±1253. Biosystems, Perkin-Elmer). Grodberg, J., Lundrigan, M.D., Toledo, D.L., Mangel, W.F., and Dunn, J.J. 1988) Complete nucleotide sequence and Acknowledgements deduced amino acid sequence ofthe ompT gene of Escherichia coli K12. Nucleic Acids Res 16: 1209. This study was supported by the Academy ofSciences Guina, T., Yi, E.C., Wang, H., Hackett, M., and Miller, S.I. project numbers 42103, 164916 and 166215) and the 2000) A PhoP-regulated outer membrane protease of University ofHelsinki. We thank Raili Lameranta, Pia serovar Typhimurium promotes resis- Salomaa, Riku Fagerlund and Silja Jaatinen for technical tance to alpha-helical antimicrobial peptides. J Bacteriol assistance. 182: 4077±4086. Higuchi, R., Krummel, B., and Saiki, R.K. 1988) A general method of in vitro preparation and specific mutagenesis of References DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16: 7351±7367. Achtman, M., Zurth, K., Morelli, G., Torrea, G., Guiyoule, A., and Carniel, E. 1999) Yersinia pestis, the cause of plague, Hinnebusch, B.J., Fischer, E.R., and Schwan, T.G. 1998) is a recently emerged clone of Yersinia pseudotubercu- Evaluation ofthe role ofthe Yersinia pestis plasminogen losis. 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