Proc. Natl. Acad. Sci. USAVol. 79, pp. 7881-7885, December 1982Medical Sciences

Genetic and biochemical analysis of gonococcal IgAl protease:Cloning in Escherichia coli and construction of mutants ofgonococci that fail to produce the activity

(Neisseria gonorrhoeae/virulence-associated endopeptidase/recombinant DNA)

J. MICHAEL KOOMEY, RONALD E. GILL*, AND. STANLEY FALKOWDepartment of Medical Microbiology, Stanford University School of Medicine, Stanford, California 94305

Communicated by Stanley N. Cohen, August 30, 1982

ABSTRACT The biological significance of bacterial extracel-lular proteases that specifically cleave human IgAl is unknown.We have prepared a gene bank of gonococcal chromosomal DNAin Escherichia coli K-12 using a cosmid cloning system. Amongthese clones, we have identified and characterized an E. coli strainthat elaborates an extracellular endopeptidase that is indistin-guishable from gonococcal IgAl protease in its substrate specificityand action on human IgAl. Analysis of recombinant plasmids andexamination of plasmid-specific peptides in minicells have shownthat the IgAl protease activity in E. coli is associated with expres-sion of a Mr 140,000 peptide. We have isolated IgAl protease-de-ficient mutants of Neisseria gonorrhoeae by reintroduction ofphysically defined deletions of the cloned-gene into the gonococcalchromosome by transformation.

A number of strict human bacterial pathogens that colonizemucosal surfaces have been shown to elaborate an extracellularprotease that specifically cleaves human IgAl. These includeall known isolates of Neisseria gonorrhoeae, as well as Neisserianeningitidis (1), -Streptococcus pneumoniae (2-4), and Hae-mophilus influenzae (2-4), the three leading agents of bacterialmeningitis. In addition to these organisms, certain isolates ofStreptococcus sanguis and other oral streptococci implicated inperiodontal disease, plaque formation on teeth and occasionalsystemic disease, have been found to produce IgAl protease(5). Some isolates of Bacteroides melaninogenicus and Capno-cytophaga sp. also produce an IgAl-specific proteolytic activity(6). (For a recent review of bacterial IgA proteases, see ref. 7.)

IgA proteases have a unique substrate specificity for humanIgAl. Other human and animal serum proteins and Igs testedare not hydrolyzed (7). Cleavage of IgAl by these proteasesoccurs in the hinge region of the al heavy chain, located be-tween the first and second constant regions, yielding intactFdal and Fcal peptides (1, 8). Those effector functions of IgAlthat involve the integral coordination of Fab- and Fc-mediatedevents are presumably disrupted by such proteolytic cleavage.We describe here the isolation and characterization of Esch-

erichia coli strains bearing recombinant plasmids derived fromgonococcal chromosomal DNA that elaborate an extracellularendopeptidase activity indistinguishable from gonococcal IgAlprotease. We further show that derivatives of these recombi-nant sequences that have physically defined alterations in thestructural gene can be used as a site-specific mutagen to trans-form N. gonorrhoeae and that such transformants no longerproduce, IgAl protease.

MATERIALS AND METHODSStrains,, Plasmids, and Media. N. gonorrhoeae strain F62

(9) was used for preparation of chromosomal DNA and trans-

formation experiments. -E. coli strain HB101 was used as a re-cipient in transduction and transformation experiments. The E.coli-K-12 minicell-producing strain was DS410 (10). pREG152,a low-copy-number cosmid vector constructed by us, pBR322(11), pACYC184 (12), and pMR0360 (13), were used for molec-ular cloning. Gonococci were grown on GC agar base' (Difco)with 1% Isovitalex (Baltimore Biological Laboratories). pREG152confers resistance to trimethoprim via expression of dihydro-folate reductase II and E. coli containing this plasmid or its de-rivatives were selected and grown on L agar containing tri-methoprim lactate at 50 ,ug/ml and thymidine phosphorylaseat 0.03 unit/ml.

Materials. Trimethoprim lactate and thymidine phosphoryl-ase were a gift from Lynn B. Elwell (Burroughs Wellcome).Human IgAl myeloma protein (Par) was a gift from Mart Man-nick (Dept. of Rheumatology, University of Washington Med-ical Center). Human polyclonal IgG and pooled human IgMmyeloma protein were obtained from Cappel Laboratories(Cochranville, PA). Human IgA2 myeloma protein was fromTago (Burlingame, CA). Restriction endonucleases were ob-tained from New England BioLabs, Bethesda Research Labo-ratories, and Boehringer Mannheim. DNA ligase was a gift fromDavid Gelfand (Cetus). [35S]Methionine for labeling minicellswas obtained from Amersham (England).DNA Isolation and Manipulation. Isolation of high molecu-

lar weight chromosomal DNA, Sau3A DNA cleavage, and sizefractionation of DNA fragments were carried out as described(14). For cosmid cloning, 2 ,ug of Sau3A-cleaved gonococcalDNA, =35 kilobases (kb) long, was ligated to 0.50 ,ug ofBamHI-cleaved pREG152. This DNA was then packaged in vitro intoA particles by using the method of Sternberg (15). Plasmidscreening of recombinant clones was done using a minilysateprocedure (16) and preparative plasmid isolations were doneusing a scaled-up version of the same protocol. Transformationof E. coli strains was done as detailed (17). Transformation ofgonococci was carried out as described (18) with the exceptionthat piliated cells were suspended in GCH broth (9) and agaroverlays contained-ampicillin (Sigma) at 1 ,g/ml.

IgAl Protease Assay. The assay system used was a modifi-cation of that of Blake and Swanson (19). '"I-Radiolabeled hu-man IgAl was incubated with cell suspensions or culture su-pernatants of E. coli clones or gonococci. Reactions werestopped by boiling the mixtures with final sample buffer (20)and electrophoresis was on a 12.5% NaDodSO4/polyacryl-amide gel (21). Gels were then wrapped in plastic wrap and theintegrity of the IgAl-radiolabeled protein was analyzed by au-toradiography.

Abbreviation: kb, kilobase(s).* Present address: Dept. of Microbiology and Immunology, Universityof Colorado Health Sciences Center, Denver, CO 80262.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Analysis of Substrate Specificity of Proteolytic Activity.Proteolytic activity in L-broth culture supernatants of HB101(pVD100) was concentrated 50-fold by ammonium sulfate pre-cipitation as described (19) except that the precipitated materialwas resuspended and dialyzed against 0.05 M Tris HCI, pH 8.0/0.02 M KC1. Similarly treated supernatant fluid from HB101(pREG152) was used as a control. Fifteen-microliter aliquots ofsuch preparations were incubated for 4 hr at 370C with 15 ,gof various human immunoglobulins. Samples were analyzed byprotein gel electrophoresis and stained with Coomassie blue.

Analysis of Plasmid-Encoded Peptides. Minicells were pu-rified (22) from cultures of DS410. Labeling conditions andsample preparation have been described (20). Approximately100,000 cpm of acetone-precipitable material was applied toeach well of a 12.5% NaDodSO4/polyacrylamide gel, which wasthen electrophoresed until thew dye front reached the bottomof the gel. The gel was then stained with Coomassie blue andexamined by fluorography (23).

Nucleic Acid Hybridization. Restriction endonuclease frag-ments purified by electroelution were nick-translated (24) to aspecific activity of 2 x 107 cpm/,4g of DNA using [32P]de-oxyribonucleotides (New England Nuclear). One-microgramsamples of restriction endonuclease-cleaved chromosomalDNA were electrophoretically separated on a horizontal 0.70%agarose gel run in Tris acetate buffer (40 mM Tris/20 mMNaOAc/2 mM EDTA, pH 7.8). Transfer of DNA fragments andprocedures for the use of aminophenyl thioether paper werecarried out as directed (Schleicher & Schuell). Hybridizationswere carried out with 5 x 106 cpm of nick-translated probe in50% formamide/0.75 M NaCl/0.075 M Na citrate, pH 7, at420C. After washing, the paper was exposed to x-ray film in thepresence of Dupont Lightning Plus intensifying screens at700C.

RESULTSIdentification of a Clone Producing IgAl Protease Activity.

Size-fractionated chromosomal DNA from N. gonorrhoeae F62was ligated to pREG152 that had been linearized at the BamHIsite and the resulting products were packaged into A particles.Cosmid particles were absorbed to E. coli K-12 HB101 and in-cubated at 37°C for a short time to allow expression of antibioticresistance, and this mixture was then plated on trimethoprim-containing plates.

Trimethoprim-resistant transductants were obtained at a fre-quency of 104/,ug of ligated gonococcal DNA. Endonucleasecleavage and gel electrophoresis of the plasmids present inthese transductants showed that 90% of the transductants con-tained a 30- to 40-kb random insert of gonoccal DNA. The sizeof the gonococcal chromosome has been estimated to be 1,600kb (25). Based on this value and an average insert size of 30-40kb, identification of a specific clone would require the exami-nation of a relatively small number of transductants.

As a primary approach to identifying HB101 clones bearingthe gene sequences for gonococcal IgAl protease, we screenedthe cosmid transductants with a functional assay using 125I-la-beled human IgAl myeloma protein as a substrate. Cultures ofHB101 containing the vector pREG152 showed no discerniblecleavage or degradation of the IgAl myeloma protein when as-sayed in this- manner. Gel electrophoresis under reducing con-ditions showed that cleavage of IgAl myeloma (Par) by gono-coccal IgAl protease resulted in disappearance of the intact alheavy chain (Mr, 59,000) and the appearance of a new peptideband (Mr, 29,000). This broad new band represents two pep-tides, the Fdal and Fcal fragments. Gonococcal IgAl proteasecleaves the 472-amino acid-long al heavy chain between aminoacids 235 and 236.One clone was identified whose supernatant contained pro-

teolytic activity for the IgAl myeloma protein. This activityyielded an autoradiographic pattern of the IgAl protein sub-strate indistinguishable from that generated by treatment withgonococcal IgAl protease (Fig. 1). This clone was designatedVD100 and the 45-kb plasmid that it contained was calledpVD100. Restriction enzyme analysis ofthis plasmid confirmedthe presence of the 10-kb vector pREG152 joined to a 35-kbgonococcal DNA insert.

Analysis of VD100 grown in L broth with trimethoprimshowed that virtually all the IgAl protease activity was presentin culture supernatants with very little activity associated withcell pellets. Similarly, activity could be found in crude suspen-sions of colonies of VD100 grown on solid medium but wasbarely detectable in washed cells from such plates. The extra-cellular nature of the IgAl protease activity produced by VD100is consistent with the external localization of IgAl proteaseelaborated by gonococci (1) and other IgAl protease-producingbacteria (2-4). Preparations of the E. coli activity and gonococ-cal IgAl protease showed similar sensitivity to detergents,EDTA, and heat (data not shown). Moreover, the endopepti-dase activity in culture supernatants of HB101 (pVD100) waslimited solely to the al heavy chain of human IgAl (Fig. 2).Using end-point dilution, we compared the amount of IgAlproteolytic activity present in culture supernatants with that incultures of HB101 (pVD100). This showed that E. coli bearingpVD100 produce 0.5-2% of the extracellular activity in gono-coccal supernatants. The reasons for the difference in expres-sion are at present unclear.

Subcloning and Deletion Mapping of IgAl Protease-Encod-ing Sequences. Because the IgAl protease activity in the E. colicosmid clone was indistinguishable biochemically from the anal-ogous gonococcal activity, we sought to localize the DNA se-quences responsible for the proteolytic activity elaborated byVD100. pVD:00 was cleaved with restriction endonucleasesusing both single and double digestions. Fragments ofpVD100generated by this treatment were ligated to pBR322 and trans-formed into HB101. One protease-encoding plasmid derivative,pVD105 (formed by cloning fragments ofpVD100 digested withSal I/Xho I into pBR322) was chosen for further analysis. Aphysical map of this plasmid, constructed by restriction enzymedigestion and agarose gel electrophoresis, showed it to contain

1 2 3 1 2 3

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FIG. 1. IgAl protease activity in crude suspensions of N. gonor-rhoeae and HB101 (pVD100). The autoradiogram shows electropho-retic migration of 125I-labeled IgAl myeloma protein after incubationwith cell suspensions of N. gonorrhoeae strain F62 (lanes 1), HB101bearing the cloning vector pREG152 (lanes 2), and HB101 bearingpVD100 (lanes 3). (Left) Samples were prepared in the presence of 2-mercaptoethanol. (Right) Samples were prepared under nonreducingconditions. H and L, heavy and light chains of the IgAl myeloma pro-tein (Par); P, intact form of the myeloma protein; arrows, comigratingproteolytic fragments of Par.

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FIG. 2. Specificity of the proteolytic activity found in culture su-pernatants of HB101 (pVD100). Various human Igs were incubatedwith HB101 (pVD100) or HB101 (pREG152) and their integrity wasthen analyzed. Lanes: 1, IgAl myeloma protein/HB101 (pREG152); 2,IgAl myeloma protein/HB101 (pVD100); 3, IgA2 myeloma protein/HB101 (pREG152); 4, IgA2 myeloma protein/HB101 (pVD100); 5,polyclonal IgG/HB101 (pREG152); 6, polyclonal IgG/HB101 (pVD100);7, pooled IgM myeloma protein/HB101 (pREG152); 8, pooled IgM my-eloma protein/HB101 (pVD100). Proteolytic activity produced byHB101 (pVDlOO) is restricted to the IgAl heavy chain (lane 2). Num-bers on the right represent Mr standards (x 10-3): phosphorylase B,92,000; bovine serum albumin, 67,000; ovalbumin, 42,000; carbonicanhydrase, 30,000.

a 9.5-kb insert of gonococcal DNA.To elucidate the region in pVD105 associated with the pro-

tease activity, deletion derivatives ofpVD105 were constructed(Fig. 3). All derivatives described here were made by restrictionendonuclease cleavage of pVD105 followed by intramolecularligation of the fragment containing the plasmid replication andmaintenance regions and subsequent transformation into HB101.pVD106 was derived by Bgl II cleavage, pVD107 resulted fromHpa I cleavage, and pVD108 was constructed by Ava I diges-tion. HB101 transformants of these deleted subelones failed toelaborate IgAl proteolytic activity. The molecular nature of theenzymatic activity was explored by analysis ofplasmid-specifiedproteins of these clones.

Examination of Plasmid-Encoded Peptides. Minicell-pro-ducing strains of E. coli have been used extensively to prefer-entially label plasmid-encoded proteins. pVD105, pBR322, andthe deletion derivatives pVD106, pVD107, and pVD108 weretransformed into the K-12 minicell strain DS410. Minicellswere purified from the transformants and control plasmid-freeDS410 and labeled with [3S]methionine. Because plasmid-freeminicells incorporated so little 35S, we loaded 50% of the re-covered material, which represented 2,000-3,000 cpm on thegel. The results are shown in Fig. 4.

pVD105-containing minicells revealed three peptides notseen in the pBR322 sample. These were a large protein of Mr140,000 and two smaller proteins, one of Mr 36,000 and theother of Mr 28,000. Comparison with the plasmid-free controlshowed that the Mr 36,000 peptide was not unique to pVD105.This peptide probably represents one of the predominant outermembrane proteins in E. coli (26). Analysis ofpVD106 showedtwo peptides, a Mr 74,000 one and the Mr 28,000 one seen inpVD105 minicells; pVD107 specified this same Mr 28,000 pep-tide and a protein of Mr 34,000. The pVD108 sample showedno peptides unique to the inserted gonococcal DNA.

These results thus correlated the presence of the Mr 140,000peptide with the IgAl protease activity in E. coli. Assuming thatthe Mr 74,000 peptide of pVD106 and the Mr 34,000 peptideofpVD107 represent truncated variants of the Mr 140,000 pro-tease-associated peptide, the coding region for this peptidestarts somewhere between the central HindIII site and the AvaI site in the gonococcal DNA ofpVD105 and is transcribed fromthe right to the left relative to the map shown in Fig. 3. Thesedata have been corroborated by similar analysis ofTn5 insertionmutants ofpVD105 (unpublished data). Although the presenceof the intact Mr 28,000 peptide, which maps to the right of theMr 140,000 peptide, does not alone confer IgAl protease activ-ity, a role in the expression or processing of the proteolytic en-zyme cannot be ruled out.

Construction of IgAl Protease Mutants of N. gonorrhoeaeF62. We wished to isolate mutants of gonococci that failed toproduce IgAl protease because such strains were not currentlyavailable. We also wished to substantiate that the cloned se-quences in E. coli truly represented the gonococcal IgAl pro-tease gene and felt that both of these goals could be accom-plished by transforming deleted forms of the cloned gene fromE. coli into gonococci.

Piliated gonococci transform at a frequency of 1% using ho-mologous chromosomal DNA (18) and have a DNA-specific up-take system similar to, but distinct from, that described in Hae-mophilus influenzae (27). One could assume that, even with therestriction barrier, gonococcal DNA cloned in E. coli could bereintroduced into a gonococcal recipient at a reasonable fre-quency. However, the detection ofsuch transformants requiredthe association of a selectable marker with the cloned DNA se-quence. Selectable markers encoded by the gonococcal chro-mosome were ruled out because transformants could be gen-erated through recombination into the homologous sequencesfrom which they were derived as well as into the homologoussequences with which they were associated. Consequently, wechose to use the flactamase gene found on the R plasmids ofpenicillinase-producing gonococci (28).To construct the recombinant molecules necessary for this

transformation event, we cloned the 9-kb Cla I fragment frompVD105 into the cloning vector pACY184. This clone, pVD109,was chloramphenicol resistant and still encoded IgAl proteaseactivity in E. coli. The complete ,B lactamase sequence can be

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FIG. 3. Physical maps of pVD105 and the deletion derivatives pVD106, pVD107, and pVD108.

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FIG. 4. Detection of plasmid-encoded polypeptides: Autoradi-ogram of the 35S-labeled polypeptides derived from DS410 containingpBR322 (lanes A and D), pVD105 (lane B), plasmid-free control (laneC), pVD106 (lane E), pVDI07 (lane F), and pVD108 (lane G). Arrowsindicate peptides unique to cloned insert DNA. Numbers on the leftare Mr markers (x 10-3) for lanes A-C and those on the right are Mrmarkers (x 10-3) for lanes D-G: myosin, 200,000; ,Bgalactosidase,116,000; bovine serum albumin, 67,000; ovalbumin, 42,000; carbonicanhydrase, 30,000; ,B-lactoglobulin, 18,000.

isolated on a 2.2-kb BamHI fragment from the gonococcal Rplasmid pMR0360 propagated in E. coli. BamHI-cleavedpMR0360 plasmid DNA was ligated to Bgl II-cleaved pVD109DNA and transformed into HB101 selecting. for both chlor-amphenicol and ampicillin resistance. Plasmid DNA from suchtransformants was found to contain the 2.2-kb BamHI fragmentfrom pMR0360 inserted into the region of pVD109 previouslyoccupied by the 1.8-kb Bgl II fragment. Recombinant plasmidswith the ,B-lactamase fragment in both orientations relative topVD109 were isolated and designated pVD111 and pVD112.These plasmids have a deletion identical to that in pVD106 con-

current with the insertion of a gene encoding a selectablemarker. As expected, HB101 transformants of pVDl1l andpVD112 did not produce detectable protease activity. Physicalmaps of pVD1O9, pVD111, and pVD112 are presented in Fig.5.

For gonococcal transformation, plasmid DNAs (1 ug) frompVD11l and pVD112 were digested with a restriction enzymethat separated the insert DNA from the cloning vehicle; This

ensured that all transformants would result from a double cross-

over event from homologous recombination as opposed to a sin-gle crossover event (which might result ifcircular plasmid DNAwas used). Piliated gonococci were harvested from agar platesand suspended in supplemented GCH broth, and digestedpVD111 or pVD112 DNA was added. Control transformationsreceived no DNA. After incubation at 370C, the mixtures wereplated on GC-agar medium and incubated for 4 to 5 hr and thenan agar overlay of GC agar medium containing ampicillin at 1,pg/ml was added. The mixtures were further incubated, andampicillin-resistant colonies were found in cells that had re-

ceived the cleaved pVD111 or pVD112 but not among cells thathad not received DNA. Suspected transformants were streakedfrom these plates onto plates lacking ampicillin and then testedfor IgAl protease by using the "2I-labeled IgAl myeloma pro-

tein assay (NaDodSO4/polyacrylamide gel electrophoresis).Unlike the parental strain F62, gonococcal transformants de-rived from pVD111 and pVD112 DNA produced no detectableIgAl protease activity. Representative transformants derivedfrom pVD 11and pVD112 DNA were selected and designatedgonococcal strains VD111 and VD112, respectively.

Southern Blot Analysis of F62, VD111, and VD112 DNA.The fact that VD111 and VD112 were ampicillin resistant andno longer produced IgAl protease activity implied that thecloned sequences in pVD111 and pVD112 had been reintro-duced into the gonococcal chromosome by homologous recom-bination. To confirm this, we analyzed chromosomal DNA fromF62, VD111, and VD112 using the method of Southern (29).DNAs from these strains were digested individually with PstI, HindIII, and Pvu I, electrophoretically separated, and trans-ferred to activated aminophenyl thioether paper. The paper wasthen hybridized with a radiolabeled DNA probe made by nick-translation ofthe 4.2-kb HindIII fragment that spans the region,altered in pVD111 and pVD112 (Fig. 6 Left). In digests of F62DNA, the probe detected two Pst I fragments, one HindIII frag-ment (the chromosomal equivalent of the 4.2-kb probe frag-ment),and one Pvu I fragment. In digests ofVD111 and VD112DNA, the probe detected three Pst I fragments (two new frag-ments as compared with F62), a new single HindIII fragmentof4.6 kb, and two novel Pvu I fragments. These results are con-

sistent with the presence in the gonococcal chromosome of therecombinant sequences ofpVD111 and pVD112 because, in theconstruction of these plasmids, new Pst I and Pvu I sites wereintroduced and the 4.2-kb HindIII fragment was increased insize to 4.6 kb (resulting from the deletion ofa 1.8-kb Bgl II frag-ment and the insertion of a 2.2-kb BamHI fragment). The sizesof the new Pst I and Pvu I fragments in the transformants are

also consistent with the orientation of the 3lactamase gene inpVD111 and pVD112.

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FIG. 6. Analysis of IgAl protease-associated DNA sequences inF62, VD111, and VD112. Lanes: 1-3, Pst I-cleaved F62, VD111, andVD112, respectively; 4-6, HindIII-cleaved F62, VD111, and VD112,respectively; 7-9,Pvu I-cleaved F62, VD111, and VD112, respectively.(Left) Hybridization using the 4.2-kb Hindm fragment of PVD105 asa probe. (Right) Hybridization using the 2.2-kb BamHI fragment ofpMR0360. Sizes of restriction fragments (in kb) were determined bycomparison with A DNA cleaved with EcoRI, Hindu, and Sma I.

To ensure that these alterations were associated with thepresence of the ,B-lactamase-specific sequences, we eluted the4.2-kb HindIII probe from the paper and rehybridized with a

probe derived from the 2.2-kb BamHI fragment of pMR0360,the source ofthe ,-lactamase gene (Fig. 6 Right). F62 containedno sequences homologous to this probe. In VD11l and VD112,the fragments that were detected were the same as those iden-tified by using the 4.2-kb HindIII probe.

DISCUSSION

The data presented here indicate that we have successfully iso-lated hybrid plasmids that contain the IgAl protease gene ofN.gonorrhoeae. This conclusion is based on the following obser-vations: (i) the elaboration ofan extracellular proteolytic activityby E. coli bearing gonococcal-derived DNA sequences that isindistinguishable from the gonococcal IgAl protease and (ii) therecovery of mutants ofN. gonorrhoeae that fail to produce thisprotease following reintroduction into the chromosome of de-leted forms of these same cloned sequences.

Characterization ofplasmid-encoded proteins in E. coli mini-cells shows the presence of a cell-associated peptide of Mr140,000 that can be correlated with IgAl protease activity. Wehave, as yet, been unable to identify a specific labeled proteinin culture supernatants associated with protease activity and thequestion ofwhether the Mr 140,000 peptide we have identifiedrepresents the mature form of the protease or an unprocessedprecursor remains unanswered. M. Blake and E. Gotschlich(personal communication) have data that show that the IgAlprotease of N. gonorrhoeae as purified from culture superna-tants is a Mr 115,000-120,000 protein.

Cloning of the IgAl protease-specific sequences has allowedus to construct mutants of N. gonorrhoeae that have definedalterations in this gene. Thus, VDlll and VD112 representhomogeneic derivatives that differ from the parental strain F62solely in the production of IgAl protease (and the expressionof &lactamase). It should be noted that, in these strain con-

structions, no foreign DNA sequences were introduced becausethe cloned gene and the 13-lactamase fragment originated fromand are native to gonococci. F62, VD11, and VD112 show no

differences in total protein profiles as seen in NaDodSO4/

polyacrylamide gel electrophoresis, in viability or growth, orin phenotypic variation (piliation T± nonpiliation and opaque;± transparent colonial transitions). Despite the limitations im-posed by the specificity of the protease for human IgAl, webelieve that analysis of the parental and mutant strains in ap-propriate model systems will show that any discernible differ-ences reflect IgAl protease-mediated events or processes.

Several lines of evidence suggest that the ability ofgonococciand other bacterial agents of human disease to produce an ex-tracellular protease that specifically cleaves human IgAl maybe involved in the pathogenesis of infection. Cloning of thegonococcal IgAl protease gene in E. coli will facilitate the bio-chemical and genetic characterization ofthis endopeptidase andsimilar proteases elaborated by other bacteria. We hope thatthese experiments and analysis of the mutants we have de-scribed in appropriate model systems will provide insight intothe possible biological significance of IgAl protease as it per-tains to bacterial-host interrelationships.

We thank Lynn Elwell for providing the trimethoprim lactate andthymidine phosphorylase and Dr. Mart Mannick for the gift of IgAlmyeloma protein (Par). This work was supported by Program ProjectGrant AI-12192 from the National Institute for Allergy and InfectiousDisease.

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