INFECTION AND IMMUNITY, Apr. 1993, p. 1400-14050019-9567/93/041400-06$02.00/0Copyright ©D 1993, American Society for Microbiology

Site-Directed Mutagenesis of Glu-141 and His-223 in Pseudomonasaeruginosa Elastase: Catalytic Activity, Processing, and

Protective Activity of the Elastase againstPseudomonas Infection

SUSUMU KAWAMOTO,1* YUJI SHIBANO,2 JUN FUKUSHIMA,l NORIHISA ISHII,3KAZUYUKI MORIHARA,4 AND KENJI OKUDA'

Departments of Bactetiology1 and Dermatology,3 Yokohama City University School ofMedicine, 3-9 Fukuura,Kanazawa-ku, Yokohama 236; Institute for Fundamental Research, Suntory Limited, Shimamoto-cho,Mishima-gun, Osaka 6182; and Institute for Applied Life Science and Department of Enzymology,

University of East Asia, Ichinomiya, Shimonoseki, Yamaguchi 751,4 Japan

Received 29 September 1992/Accepted 7 January 1993

Both Pseudomonas aeruginosa elastase and Bacillus thermoproteolyticus thermolysin are zinc metallopro-teases. On the basis of the high homology of the P. aeruginosa elastase with the BaciUlus thermolysin, we

hypothesized that Glu-141 and His-223 are the key residues for catalytic activity of the Pseudomonas elastase.To test this possibility, we replaced Glu-141 with Asp, Gln, and Gly and His-223 with Gly, Glu, and Leu bysite-directed mutagenesis. These substitutions dramatically diminished the proteolytic activities of the mutantelastases when they were expressed in Escherichia coli cells. Although these mutant elastase precursors

(proelastases) were produced, no appreciable processing was observed with these mutants. The possibility thatautocatalysis is involved in both the processing and activation of elastase is discussed. Furthermore, byimmunizing mice with vaccines made from these mutant elastase, we were able to obtain good protectionagainst an intraperitoneal P. aeruginosa challenge.

Pseudomonas aeruginosa is an opportunistic pathogenwhich can cause fatal infection in vulnerable hosts. Severalcomponents related to its virulence have been identified andcharacterized (29). Elastase acts to inactivate a variety ofbiologically important proteins and processes (24). In addi-tion, there is strong evidence that elastase plays an impor-tant role in the pathogenesis of P. aeruginosa infections (14,27). We and another group have independently cloned (32,41) and analyzed (2, 10, 23) the elastase structural gene(lasB) from the elastase-producing strains P. aeruginosa IFO3455 and PAO1, respectively. We also cloned and analyzedthe elastase gene from several non-elastase-producingPseudomonas strains (36) and other metalloprotease genes(Pseudomonas alkaline proteinase [1, 28] and Vibrio collag-enase [9, 35]). Recently, the elastase-related lasA gene (30,33, 40), the existence of preproelastase and proelastase (2,10, 18), a transcriptional activator (lasR gene) (12), andtranslational control (4) of elastase expression have beendescribed (see reference 11 for a review). Moreover, P.aeruginosa elastase has been crystallized and the three-dimensional structure of the protein has been determined(37).Among the metalloproteases, the thermolysin from Bacil-

lus thermoproteolyticus has been the most extensively stud-ied. A possible catalytic mechanism for this enzyme hasbeen suggested by Matthews and coworkers on the basis ofcrystallographic analyses (13, 20, 21). According to theirfindings, in native thermolysin, the zinc ion is coordinated tothree ligands (His-142, His-146, and Glu-166) provided bythe protein molecule. With their model of thermolysin andthe tertiary structure of Pseudomonas elastase (37) in mind,we hypothesized an essential role for some key residues in

* Corresponding author.

elastase activity. In the present study, using the data for thethermolysin and elastase, we replaced two amino acid resi-dues (Glu-141 and His-223) in the elastase to confirm theactive center of catalytic activity. We also confirmed theprotective activity of the mutated elastase against Pseudo-monas infection.

MATERIALS AND METHODS

Strains and plasmids. P. aeruginosa IFO 3455 was ob-tained from the Institute for Fermentation, Osaka, Japan(24). M13mpl8 phage were used for DNA sequencing.Escherichia coli JM109, plasmid vector pUC18, a reactionkit for site-directed mutagenesis, and restriction enzymeswere obtained from Takara Shuzo Co., Ltd., Kyoto, Japan.Trypsin from bovine pancreas and trypsin inhibitor fromsoybean came from Sigma (St. Louis, Mo.). BALB/c andC57BL/6 mice (6 to 8 weeks old) were purchased fromShizuoka Experimental Animal Co., Ltd., Shizuoka, Japan.

Site-directed mutagenesis. Site-directed mutagenesis wascarried out by the protocol described by Kunkel (19).Plasmid pELP1 was constructed by inserting an EcoRI-PstIfragment (2.6 kb) derived from pELK6 (41) and used forelastase expression. The fragment contains coding regionsfor the elastase signal peptide, proregion peptide (propep-tide), and mature protein. A SalI-SalI fragment (0.6 kb) frompELP1 including the codons to be mutagenized was insertedinto M13mpl8 RF DNA. Single-stranded DNA was preparedand subjected to site-directed mutagenesis (19) with thefollowing oligonucleotides: 5'-CCCACGACGTCAGCC-3'(Glu-141--*Asp), 5'-GGCCCACCAGGTCAG-3' (Glu-141-*Gln), 5'-GCCCACGCIGiGTCAGC-3' (Glu-141- Gly), 5'-CGACGTGGGCCACTCC-3' (His-223-*Gly), 5'-CGACGTG£AGCACTCCAG-3' (His-223--*Glu), and 5'-CGACGTGCTCCACTCC-3' (His-223-*Leu). From the phage clones ob-

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tained, we prepared single-stranded DNA. RF DNA wasprepared from the mutagenized phage clones, and the mu-tated Sall-SalI fragment was ligated to the expression vectorpELP1 which lacked the corresponding SalI-Sall fragment.The mutations were finally confirmed by DNA sequencing,and E. coli cells were transformed with these vectors formutant elastase expression.DNA sequencing. Oligonucleotide primers were synthe-

sized with an automatic DNA synthesizer (381A DNAsynthesizer; Applied Biosystems, Foster City, Calif.). DNAwas sequenced with universal or synthetic oligonucleotideprimers by the dideoxy chain-termination method (31) with2P-labeled nucleotides.Detection of proteins. Transformants carrying wild-type or

mutant elastase genes were cultured in Luria-Bertani broth.Isopropyl-13-D-thiogalactopyranoside (IPTG) (1 mM) wasadded at mid-log phase, and cultivation was continued for 16h. Cells were collected, washed, and resuspended in 50 mMTris-HCl (pH 8.0) containing 1 mM CaCl2. Sonicated bacte-rial suspensions or samples fractionated by centrifugationwere analyzed by sodium dodecyl sulfate (SDS)-12.5%polyacrylamide gel electrophoresis. Proteins were visualizedby staining with Coomassie brilliant blue. To confirm thepresence of elastase protein, Western immunoblotting wasperformed by the procedure of Burnette (5). Antielastaseantibody has been described previously (36).

Proteolytic assays. Total proteolytic activity was deter-mined by a modification of the method described previously(36), with a 30-min digestion with azocasein (Sigma) at 37°Cin 30 mM Tris-HCl (pH 8.0) containing 1 mM CaCl2. Oneunit of proteolytic activity was defined as the amount thatincreased culture density of 1 A440 unit per minute. Elas-tolytic activity was determined by the method of Bjorn et al.(3). Digestion of elastin Congo red (Sigma) suspended in 30mM Tris-HCl (pH 8.0)-i mM CaCl2 was carried out for 20 to24 h. The degree of digestion was determined by measuringthe diameter of the lytic ring in the agar plate.

Preparation of inactive Pseudomonas elastase. CrystallizedPseudomonas elastase was obtained from Nagase Biochem-ical Co., Fukuchiyama, Kyoto, Japan. The irreversibleinhibitor of Pseudomonas elastase, ClCH2CO-HO-Leu-Ala-Gly-NH2, was obtained from the Protein Research Founda-tion, Minoh, Osaka, Japan. Inactive Pseudomonas elastasewas prepared as described previously (10, 25).

Preparation of elastase from E. coli. Mutant elastase waspartially purified with a Sepharose 4B column. The superna-tant from the sonicated bacterial suspension was applied toan antielastase-coated Sepharose 4B column. After elutionwith 100 mM glycine buffer, pH 2.8, we dialyzed and furtherpurified the preparation with a Sephadex G-100 column.

Protective effect of mutant elastase. Each mouse was sub-cutaneously immunized with 10 ,ug of inactivated elastase(elastase toxoid) or partially purified mutant elastase, and 2weeks later, the same dose of antigen was again injected intothe same mouse. After 1 week, immunized and unimmunizedcontrol mice were intraperitoneally injected with a virulentstrain of P. aeruginosa (IFO 3455), and the 50% lethal dose(LD50) was determined. Statistical analysis of the experi-mental data was done by the two-tailed Student t test.

RESULTS

Comparison of elastase and thermolysin amino acid se-quences. Initially, we compared the primary structure ofelastase with that of thermolysin, which has already beendetermined at the tertiary structural level. Except for the N-

and C-terminal regions, there is a high level of homology(33% identical and 53% similar residues) (data not shown).Moreover, by using the program FRODO (15), we simulateda three-dimensional model, which shows how the catalyticsites of Pseudomonas elastase and thermolysin superimposeextremely well (data not shown). The key roles played byGlu-143 and His-231 in thermolysin catalysis were proposedby Matthews and his coworkers on the basis of crystallo-graphic analyses (13, 20, 21), and the results of site-directedmutagenesis confirmed that the corresponding residues arealso the key residues for the catalytic activity of Bacillussubtilis neutral protease (38).

In the course of our present study, the tertiary structure ofPseudomonas elastase was reported (37), and comparativeanalyses of both the sequence (2, 10, 23, 37) and the tertiarystructure (37) of thermolysin and elastase resulted in identi-fication of the probable key residues in the active site ofelastase, including Glu-141 and His-223, which in thermol-ysin are at positions 143 and 231, respectively. Thus, ourcomputer graphic data were confirmed by the crystallo-graphic analysis of elastase, and it has been proposed thatGlu-141 and His-223 are the key residues in the active site. Inthe present study, in order to provide more direct experi-mental confirmation of the strong similarity of the catalyticsites of elastase and thermolysin, we decided to replaceresidues Glu-141 and His-223 in Pseudomonas elastase withother amino acids by site-directed mutagenesis.

Isolation of mutants. The Sall-SalI fragment of plasmidpELP1, containing the coding sequences for the residues tobe mutated, was inserted into the multicloning site ofM13mpl8, and single-stranded DNA was prepared. Thissingle-stranded DNA was annealed to each of the six primersfor site-directed mutagenesis, and the double-stranded mol-ecules obtained after polymerization and ligation reactions(19) were used to construct expression vectors for mutantelastase. As determined by sequence analysis, three Glu-141mutants (Glu--Asp, Glu--Gln, and Glu--Gly) and threeHis-223 mutants (His--Gly, His--Glu, and His-*Leu) wereisolated (see Materials and Methods).

Analysis of the elastase gene products. The mutant elastasegenes were expressed by cultivating the E. coli transfor-mants in the presence of IPTG. After a 24-h incubation, thesonicated cell suspensions were analyzed by SDS-polyacryl-amide gel electrophoresis and Western blotting. We ob-served major species of approximately 33 and 51 kDa in E.coli transformants carrying the wild-type elastase gene [E.coli(pELP1); see Materials and Methods] and E. coli trans-formants carrying the mutant elastase genes respectively(data not shown). The 33-kDa protein is the same size asmature elastase, and the 51-kDa protein is the size ofproelastase, the precursor form of elastase (2, 10, 18). Noneof the transformants released elastase into the culture me-dium, as tested by Western blotting and proteolytic activityassays (data not shown).To examine the localization of expression products in E.

coli cells carrying the wild-type or mutant elastase genes, wefractionated sonicated cell suspensions by centrifugation at10,000 x g for 10 min. As shown in Fig. 1A, wild-typeelastase was expressed mainly in the soluble fraction, prob-ably in the periplasmic fraction, as has been assumed (11), asa 33-kDa protein. With the mutant elastases, we observed amajor band of 51 kDa, mainly in the insoluble fraction (Fig.2A). The apparent size of the mutant proteins appears toreflect removal of the signal peptide. This result suggeststhat the mutant elastase expressed in E. coli cells remainsproelastase and is located mainly in the insoluble fraction,

VOL. 61, 1993

1402 KAWAMOTO ET AL.

A no incubation1 2 3 4 5 6 7

kd200

92.5*-- 69

46

1 2 3 4 5 6 7

30

21.5B 25-C, 16hr

1 2 3 4 5 6 7

jz , , .-

kd200

-92.5- 69

46 --

1 2 3 4 5 6 7

~~~~t 30130

FIG. 1. Elastase gene products in the soluble fraction from E.coli cells before (A) and after (B) a 16-h incubation at 25°C. Lanes:1, wild-type elastase; 2, mutant Glu-141- Asp; 3, mutant Glu-141- Gln; 4, mutant Glu-141--Gly; 5, mutant His-223-->Gly; 6,mutant His-223--Glu; 7, mutant His-223->Leu. The soluble bacte-rial cell fraction (30 ,ug of protein) was analyzed by SDS-polyacryl-amide gel electrophoresis. The panels show Coomassie blue staining(left) and Western blotting (right). Numbers in both sets of panelsrepresent identical samples. Arrowheads show the positions of51-kDa (upper) and 33-kDa (lower) molecules.

probably in the periplasm, as insoluble aggregates or mem-brane-bound material.

Catalytic activity of elastase expressed in E. coli. We nextassayed the catalytic activity of elastase expressed in E. colicells. When the proteolytic and elastolytic activities in thesupernatant fraction of E. coli cells carrying the wild-type ormutant elastase gene were measured, no supernatant frac-tion had any activity. However, after a 16-h incubation at25°C, the supernatant fraction from E. coli carrying thewild-type elastase gene showed a high level of proteolytic(14.7 U/mg of protein) and elastolytic (29-mm-diameter lyticring) activities, indicating that the wild-type elastase wasactivated by this treatment. After the incubation, Westernblotting of each sample was also performed, but no apparentband shift was observed for E. coli cells carrying either thewild-type or mutant elastase gene (Fig. 1B). The insolublefraction of each E. coli strain was also analyzed. None of theinsoluble fractions from E. coli cells carrying a mutantelastase gene showed any proteolytic activity with or with-out a 16-h incubation at 25°C (data not shown), and noapparent band shifts were observed in Western blotting (Fig.2B).To test the possibility of a self-activating event in elastase,

each sonicated fraction was incubated with an autoactivatedsupernatant fraction from the wild-type-elastase gene-carry-ing bacterial clone. To 100 ,ul of sonicated fraction (about 2mg of protein) containing both soluble and insoluble frac-tions from E. coli cells carrying the wild-type or mutantelastase gene was added 20 RI of preactivated (25°C, 2 days)supernatant fraction (1.34 mg of protein per ml) from E. colicells carrying the wild-type elastase gene. After 1 h ofincubation at 37°C, these samples were assayed for proteo-

A no incubationt 2 3 4 5 6 7

kd200

: 92.5- 69

. 46

- 30

21.5

B 25t, 16hr

1 2 3 4 5 6 7 kd-200

-92.569

1 2 3 4 5 6 7_/A -' * : 4,o*_--

1 2 3 4 5 6 7

46

-30-

FIG. 2. Elastase gene products in the insoluble fraction from E.coli cells before (A) and after (B) a 16-h incubation at 25°C. Theinsoluble bacterial cell fraction (20 ,ug of protein) was analyzed bySDS-polyacrylamide gel electrophoresis. The panels show Coo-massie blue staining (left) and Western blotting (right). Lane assign-ments and marker positions are as in Fig. 1.

lytic activity. After activated elastase treatment, only thesonicated fraction from E. coli cells carrying the wild-typeelastase gene showed proteolytic activity (14.0 U/mg ofprotein). Western blotting was also performed, but no clearband shift was observed (data not shown). On the otherhand, when the sonicated fraction from E. coli cells carryinga mutant elastase gene was treated with activated solublefraction, neither proteolytic activity nor processing (bandshift on Western blotting) (data not shown) was observed.

Furthermore, after incubation with a limited amount (0.1mg/ml) of trypsin, which has been shown to activate theelastase precursor (16), only the sonicated fraction from E.coli cells carrying the wild-type elastase gene showed pro-teolytic activity (14.7 U/mg of protein). However, none ofthe E. coli cells carrying a mutant elastase gene showed anyactivity even after trypsin treatment. These findings suggestthe involvement of limited proteolysis in elastase activation.A more detailed examination of activated elastase and

trypsin treatment was made (Table 1). Again, addition ofactivated elastase or trypsin to the inactive supernatantfraction of E. coli cells carrying the wild-type elastase generesulted in dramatic activation. EDTA, an inhibitor of elas-tase (26), inhibited activation by activated elastase treatmentbut not by trypsin treatment. A small amount of the acti-vated elastase may trigger the activation, and a limitedamount of trypsin may substitute for elastase in the activa-tion process.

Protective activity of mutant elastase. One of the objectivesof the present study was to test the possibility of producingvaccines by molecular genetic techniques. Since mutantelastases have no catalytic activity and have almost the sameantigenicity as the original wild-type elastase, as evidencedby the Western blot analyses, the protective activity againstPseudomonas infection of the mutant elastase with theHis-223---Glu mutation was tested. As shown in Table 2, an

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SITE-DIRECTED MUTAGENESIS OF PSEUDOMONAS ELASTASE 1403

TABLE 1. Activation of elastase in the soluble fraction of E. colicells carrying the wild-type elastase genea

Addition RelativeSoluble prelyticfraction Activated Trypsin EDTA activityb (%)

- + - - 0.7- - + - 0.1+ - - - 0+ + - - 89.4+ + - + 13.9+ - + - 98.1+ _ + + 75.5

a To 50 ,ul of the soluble fraction (about 0.46 mg of protein) was added 10 ,ulof activated (25'C, 2 days) supernatant fraction (0.13 mg of protein per ml)from E. coli cells carrying the wild-type elastase gene, trypsin (0.1 mg/ml), orEDTA (20 mM). After 1 h of incubation at 37°C, 10 p.l of trypsin inhibitor (1mg/ml) or CaC12 (20 mM) was added to the trypsin and EDTA incubations,respectively. Proteolytic activity was determined as described in Materialsand Methods.

b The activity of the soluble fraction after 16 h of incubation at 25'C wasdefined as 100%.

approximately 10-fold increase in the LD50 relative to theLD50 for unvaccinated mice was observed in both BALB/cand C57BL/6 mice when 20 ,ug of partially purified mutantelastase was injected subcutaneously per mouse. The levelof protective activity was almost the same as that obtainedwith elastase toxoid (elastase inactivated by inhibitor treat-ment [10, 25]), as described previously (24).

DISCUSSION

Pseudomonas elastase shows a high degree of amino acidhomology (33%) to thermolysin. The primary amino acidsequences confirm the similarity of the active centers ofthermolysin and elastase and support the model for themechanism of action of thermolysin described by Matthewsand coworkers (13, 20, 21). The glutamic acid at position 141and histidine at position 223 appear to be key residues for thecatalytic activity of the Pseudomonas elastase, as is the casewith the corresponding residues in Bacillus thermolysin (13,20, 21) and Bacillus neutral protease (38), since their replace-ment with other amino acids causes a dramatic decrease inelastase activity. Therefore, the roles of Glu-141 and His-223in elastase activity, as determined experimentally, are in

TABLE 2. Protective effect of mutant elastase antigena

Mouse Immunizing LD50strain antigen (10" live cells)

BALB/c Mutant elastase 8.5Control 0.31Elastase toxoid 5.1None 0.31

C57BL/6 Mutant elastase 18.5Control 0.27Elastase toxoid 8.5None 0.27

a Partially purified His-223--*Glu mutant elastase, the corresponding frac-tion from E. coli cells containing the cloning vector (pUC18) alone (control),or elastase toxoid (elastase inactivated by inhibitor treatment) was used as theimmunizing antigen. Twice-immunized mice (4 or 5 per group) were chal-lenged intraperitoneally with a virulent strain of P. aeruginosa (IFO 3455).After 8 days, the number of surviving mice was determined and the LD50 wascalculated.

agreement with the proposed model of thermolysin activity,in which the glutamic acid is directly responsible for nucleo-philic attack on the water molecule in the scissile bond of thesubstrate and histidine helps to stabilize the transition stateof the reaction. The similarity between thermolysin andPseudomonas elastase is also evident in the very recentanalysis of their three-dimensional structures (37).Many secreted proteases are synthesized as inactive larger

precursors, which are subsequently cleaved as they passthrough the bacterial membrane to give fully active matureproteases (39). Moreover, it has been reported that thematuration and release of some proteases from the mem-brane involve an autocatalytic process (6, 22). Recent stud-ies have given us some information about the processing,activation, and secretion of elastase. The existence of pre-proelastase and proelastase has been described (2, 10, 18),and it was suggested that the inactive form of elastase isactivated by limited proteolysis or by dissociation from anoncovalently bound "inhibitor," which may be thepolypeptide cleaved from proelastase (8, 17). In the presentstudy, a 33-kDa band was observed mainly in the solublefraction of E. coli cells carrying the wild-type elastase gene,but a 51-kDa band was found mainly in the insoluble fractionof E. coli cells carrying a mutant elastase gene (Fig. 1, and2); both were probably located in the periplasmic space.Previous studies (2, 10, 18) suggest that the 51-kDa moleculeis the inactive precursor of elastase (proelastase) and the33-kDa molecule corresponds to mature active elastase.The processing of proelastase (51 kDa) to mature elastase

(33 kDa) may be self-catalyzed by its own proteolyticactivity. It is likely that mutant elastase protein, which hadno catalytic activity and could not be self-processed, re-mained as a 51-kDa proelastase. Furthermore, E. coli cellscarrying the wild-type elastase gene showed a 33-kDa ma-ture-sized band, but the untreated sample had no proteolyticactivity, suggesting that some process, probably causing nochange in molecular size, is required for activation of the33-kDa protein. Our data suggest that elastase may beautocatalyzed to yield active mature elastase. In addition toa 16-h incubation at 25°C, limited proteolysis induced withtrace amounts of trypsin and activated elastase was effectivein elastase activation. Limited autoproteolysis may be in-volved in activation of the inactive elastase molecule. How-ever, the possibility also exists that another protease in E.coli is involved in elastase activation. Proteolysis mightoccur in the 18-kDa propeptide molecule, which is processedfrom the 51-kDa proelastase but is bound tightly to the33-kDa elastase molecule, since no appreciable change inmolecular size was observed after the treatments. Theprevious data (16) also suggest that the proregion peptide isassociated with the inactive elastase molecule and is sensi-tive to trypsin, to the activated elastase, or possibly to both.More experiments are necessary to clarify this issue.

In summary, from these results and those of previousstudies (2, 8, 10, 16-18), we propose a model for theprocessing of Pseudomonas elastase in E. coli cells (Fig. 3)as a working hypothesis. Elastase is synthesized as a 53.6-kDa preproelastase, and proelastase (51.2 kDa) is generatedafter removal of the signal peptide (2.4 kDa) from the initialgene product. In P. aeruginosa, the PilD peptidase, mutationof which prevents the export of a wide range of extracellularpolypeptides, including elastase, may be involved in theprocess (34). Proelastase protein proceeds to an apparentlymature product (33 kDa) by autoproteolytic processing.However, possibly because the 33-kDa protein is boundtightly to the proregion peptide (propeptide) (18.2 kDa), the

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1404 KAWAMOTO ET AL.

(Elastase gene)

4Preproelastase

(53.6 kDa)

[Mutant]

(Inactive)

FIG. 3. Schematic diagram illustrating the sequence of possibleevents involved in elastase processing in E. coli.

33-kDa protein associated with the proregion peptide showsno activity. Further limited proteolysis is required for acti-vation of the mature-sized elastase to yield the fully activeform, which is completely dissociated from the 18-kDamolecule.

Elastase may be activated by its own proteolytic activity.Thus, autoproteolysis of elastase may be involved in boththe processing and activation of elastase, although autoca-talysis is not the only possible explanation, since the mutantelastases could not be processed or activated by treatmentwith activated elastase. There is also a possibility that acertain protease of the E. coli host cell can play an alterna-tive role in elastase processing, activation, or both. Ifautocatalysis is indeed the case, once a very small amount ofelastase becomes active, proelastase is converted to activeelastase by this small amount of elastase. That activationresults in the production of active elastase, which can in turnactivate more proelastase. This results in a cascade effectwhich accelerates autoprocessing and autoactivation of elas-tase. It seems that LasA protein in P. aeruginosa actsdirectly on the elastin substrate and does not interact di-rectly with elastase or affect its secretion (11, 40). Our modelis basically compatible with the hypothesis put forwardpreviously (11, 17).

Unlike most gram-negative bacteria, P. aeruginosa se-cretes several proteins into the medium. Actually, P. aerug-inosa secretes mature active elastase, which has been as-sumed to remain in the periplasm in E. coli cells expressingthe wild-type elastase gene (11). The mechanism of proteinsecretion in gram-negative bacteria, which involves thecrossing of both the inner cytoplasmic and outer mem-

branes, is still poorly understood. Periplasmic elastase isinactive in P. aeruginosa, yet E. coli is capable of processingelastase to its active form. At present, we cannot adequatelyexplain the reasons for the difference between P. aeruginosaand E. coli in elastase processing and secretion. Elastaseexpression studies with non-elastase-producing Pseudo-monas strains (36) instead of E. coli cells (this study) areunder way.We also tested the protective activity of one of mutant

elastases, since an active-site mutation of exotoxin A of P.aeruginosa which abolished enzymatic activity but did notsignificantly alter immunogenicity yielded an efficaciousvaccine (7). As shown in Table 2, the mutant elastase in

which His-223 had been replaced by Glu showed goodprotective activity against Pseudomonas infection. In thepresent experiment, we only tested the protective ability ofthese vaccines against intraperitoneal challenge with a viru-lent strain. Experiments with the burned-mouse infectionsystem (14, 24) are now in progress to study this protectiveactivity further. With the system we used, we observedalmost the same level of protection as was found with theelastase toxoid. Moreover, it was unnecessary to inactivatethese elastases for protection, which was of great advantagesince the preparation of elastase toxoid (elastase inactivatedby Formalin or inhibitor treatment [25]) is very complicated.These results suggest that site-directed mutated elastase isuseful for preparing toxoid vaccines.

ACKNOWLEDGMENTS

We thank Tamiko Kaneko for her technical assistance and Fu-miko Atsumi and Kayoko Chiba for their secretarial assistance.

This work was supported in part by a Grant-in-Aid for ScientificResearch (no. 03670219) from the Ministry of Education, Scienceand Culture of Japan.

ADDENDUM IN PROOF

We have recently reexamined the nucleotide sequence ofP. aeruginosa IFO 3455 elastase and have discovered anerror in the sequence published previously (10). The nucle-otide at position 382 should be A (not G), and the corre-sponding amino acid should be Met-128 (not Val). Thecorrect sequence has been deposited in GenBank (accessionnumber M24531).

REFERENCES1. Atsumi, Y., S. Yamamoto, K. Morihara, J. Fukushima, H.

Takeuchi, N. Mizuki, S. Kawamoto, and K. Okuda. 1989.Cloning and expression of the alkaline proteinase gene fromPseudomonas aeruginosa IFO 3455. J. Bacteriol. 171:5173-5175.

2. Bever, R. A., and B. H. Iglewski. 1988. Molecular characteriza-tion and nucleotide sequence of the Pseudomonas aenrginosaelastase structural gene. J. Bacteriol. 170:4309-4314.

3. Bjorn, M. J., P. A. Sokol, and B. H. Iglewski. 1979. Influence ofiron on yields of extracellular products in Pseudomonas aerug-inosa cultures. J. Bacteriol. 138:193-200.

4. Brumlik, M. J., and D. G. Storey. 1992. Zinc and iron regulatetranslation of the gene encoding Pseudomonas aeruginosa elas-tase. Mol. Microbiol. 6:337-344.

5. Burnette, W. N. 1981. "Western blotting": electrophoretictransfer of proteins from sodium dodecyl sulfate-polyacryl-amide gels to unmodified nitrocellulose and radiographic detec-tion with antibody and radioiodinated protein A. Anal. Bio-chem. 112:195-203.

6. Chang, P.-C., and Y.-H. W. Lee. 1992. Extracellular autopro-cessing of a metalloprotease from Streptomyces cacaoi. J. Biol.Chem. 267:3952-3958.

7. Douglas, C. M., and R. J. Collier. 1987. Exotoxin A of Pseudo-monas aeruginosa: substitution of glutamic acid 553 with aspar-tic acid drastically reduces toxicity and enzymatic activity. J.Bacteriol. 169:4967-4971.

8. Fecycz, I. T., and J. N. Campbell. 1985. Mechanisms of activa-tion and secretion of a cell-associated precursor of an exocellu-lar protease of Pseudomonas aenrginosa 34362A. Eur. J. Bio-chem. 146:35-42.

9. Fukushima, J., H. Takeuchi, E. Tanaka, K. Hamajima, Y. Sato,S. Kawamoto, K. Morihara, B. Keil, and K. Okuda. 1990.Molecular cloning and partial DNA sequencing of the collage-nase gene of Vibrio alginolyticus. Microbiol. Immunol. 34:977-984.

10. Fukushima, J., S. Yamamoto, K. Morihara, Y. Atsumi, H.

INFECT. IMMUN.

SITE-DIRECTED MUTAGENESIS OF PSEUDOMONAS ELASTASE 1405

Takeuchi, S. Kawamoto, and K. Okuda. 1989. Structural geneand complete amino acid sequence of Pseudomonas aeruginosaIFO 3455 elastase. J. Bacteriol. 171:1698-1704.

11. Galloway, D. R. 1991. Pseudomonas aeruginosa elastase andelastolysis revisited: recent developments. Mol. Microbiol.5:2315-2321.

12. Gambello, M. J., and B. H. Iglewski. 1991. Cloning and charac-terization of the Pseudomonas aeruginosa lasR gene, a tran-scriptional activator of elastase expression. J. Bacteriol. 173:3000-3009.

13. Hangauer, D. G., A. F. Monzingo, and B. W. Matthews. 1984.An interactive computer graphics study of thermolysin-cata-lyzed peptide cleavage and inhibition by N-carboxymethyldipeptides. Biochemistry 23:5730-5741.

14. Holder, I. A. 1985. The pathogenesis of infections owing toPseudomonas aeruginosa using the burned mouse model: ex-perimental studies from the Shriners Burns Institute, Cincin-nati. Can. J. Microbiol. 31:393-402.

15. Jones, T. A. 1985. Interactive computer graphics: FRODO.Methods Enzymol. 115:157-171.

16. Kessler, E., and M. Safrin. 1988. Partial purification and char-acterization of an inactive precursor of Pseudomonas aerugi-nosa elastase. J. Bacteriol. 170:1215-1219.

17. Kessler, E., and M. Safrin. 1988. Synthesis, processing, andtransport of Pseudomonas aeruginosa elastase. J. Bacteriol.170:5241-5247.

18. Kessler, E., M. Safrin, M. Peretz, and Y. Burstein. 1992.Identification of cleavage sites involved in proteolytic process-ing of Pseudomonas aenrginosa preproelastase. FEBS Lett.299:291-293.

19. Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesiswithout phenotypic selection. Proc. Natl. Acad. Sci. USA82:488-492.

20. Matthews, B. W., P. M. Colman, J. N. Jansonius, K. Titani,K. A. Walsh, and H. Neurath. 1972. Structure of thermolysin.Nature (London) New Biol. 238:41-43.

21. Matthews, B. W., J. N. Jansonius, P. M. Colman, B. P. Schoen-born, and D. Dupourque. 1972. Three-dimensional structure ofthermolysin. Nature (London) New Biol. 238:37-41.

22. Miyazaki, H., N. Yanagida, S. Horinouchi, and T. Beppu. 1989.Characterization of the precursor of Serratia marcescens serineprotease and COOH-terminal processing of the precursor duringits excretion through the outer membrane of Eschenchia coli. J.Bacteriol. 171:6566-6572.

23. Morihara, K., J. Fukushima, and K. Okuda. 1989. Primarystructure of Pseudomonas aeruginosa elastase: a comparisonwith thermolysin. Antibiot. Chemother. (Basel) 42:36-41.

24. Morihara, K., and J. Y. Homma. 1985. Pseudomonas proteases,p. 41-79. In I. A. Holder (ed.), Bacterial enzymes and virulence.CRC Press, Inc., Boca Raton, Fla.

25. Morihara, K., and J. Y. Homma. 1986. New method of prepar-

ing elastase toxoid from Pseudomonas aeruginosa. J. Clin.Microbiol. 23:53-55.

26. Morihara, K., H. Tsuzuki, T. Oka, H. Inoue, and M. Ebata.1965. Pseudomonas aeruginosa elastase. J. Biol. Chem. 240:3295-3304.

27. Nicas, T. I., and B. H. Iglewski. 1985. The contribution of

exoproducts to virulence of Pseudomonas aeruginosa. Can. J.Microbiol. 31:387-392.

28. Okuda, K., K. Morihara, Y. Atsumi, H. Takeuchi, S. Kawa-moto, H. Kawasaki, K. Suzuki, and J. Fukushima. 1990. Com-plete nucleotide sequence of the structural gene for alkalineproteinase from Pseudomonas aeruginosa IFO 3455. Infect.Immun. 58:4083-4088.

29. Pavlovskis, 0. R., and B. Wretlind. 1982. Pseudomonas aerug-inosa toxin, p. 97-128. In C. S. F. Easmon and J. Jeijaszewicx(ed.), Medical microbiology, vol. 1. Academic Press, London.

30. Peters, J. E., and D. R. Galloway. 1990. Purification andcharacterization of an active fragment of the LasA protein fromPseudomonas aeruginosa: enhancement of elastase activity. J.Bacteriol. 172:2236-2240.

31. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci.USA 74:5463-5467.

32. Schad, P. A., R. A. Bever, T. I. Nicas, F. Leduc, L. F. Hanne,and B. H. Iglewski. 1987. Cloning and characterization ofelastase genes from Pseudomonas aeruginosa. J. Bacteriol.169:2691-2696.

33. Schad, P. A., and B. H. Iglewski. 1988. Nucleotide sequence andexpression in Escherichia coli of the Pseudomonas aeruginosalasA gene. J. Bacteriol. 170:2784-2789.

34. Strom, M. S., D. Nunn, and S. Lory. 1991. Multiple roles of thepilus biogenesis protein PiID: involvement of PilD in excretionof enzymes from Pseudomonas aeruginosa. J. Bacteriol. 173:1175-1180.

35. Takeuchi, H., Y. Shibano, K. Morihara, J. Fukushima, S. Inami,B. Keil, A.-M. Gilles, S. Kawamoto, and K. Okuda. 1992.Structural gene and complete amino acid sequence of Vibrioalginolyticus collagenase. Biochem. J. 281:703-708.

36. Tanaka, E., S. Kawamoto, J. Fukushima, K. Hamajima, H.Onishi, Y. Miyagi, S. Inami, K. Morihara, and K. Okuda. 1991.Detection of elastase production in Escherichia coli with theelastase structural gene from several non-elastase-producingstrains of Pseudomonas aeruginosa. J. Bacteriol. 173:6153-6158.

37. Thayer, M. M., K. M. Flaherty, and D. B. McKay. 1991.Three-dimensional structure of the elastase of Pseudomonasaeruginosa at 1.5-A resolution. J. Biol. Chem. 266:2864-2871.

38. Toma, S., S. Campagnoli, E. D. Gregoriis, R. Gianna, I. Mar-garit, M. Zamai, and G. Grandi. 1989. Effect of Glu-143 andHis-231 substitutions on the catalytic activity and secretion ofBacillus subtilis neutral protease. Protein Eng. 2:359-364.

39. Wandersman, C. 1989. Secretion, processing and activation ofbacterial extracellular proteases. Mol. Microbiol. 3:1825-1831.

40. Wolz, C., E. Hellstern, M. Haug, D. R. Galloway, M. L. Vasil,and G. Doring. 1991. Pseudomonas aeruginosa LasB mutantconstructed by insertional mutagenesis reveals elastolytic activ-ity due to alkaline proteinase and the LasA fragment. Mol.Microbiol. 5:2125-2131.

41. Yamamoto, S., J. Fukushima, Y. Atsumi, H. Takeuchi, S.Kawamoto, K. Okuda, and K. Morihara. 1988. Cloning andcharacterization of elastase structural gene from Pseudomonasaeruginosa IFO 3455. Biochem. Biophys. Res. Commun. 152:1117-1122.

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