Cloning Expression of Treponema Antigen Genes Escherichia · 436 NORGARD ANDMILLER purification, and physicochemical characteriza-tion cannot be overemphasized. Inaneffort to circumventthe

INFECTION AND IMMUNITY, Nov. 1983, p. 435-445 Vol. 42, No. 20019-9567/83/110435-11$02.00/0Copyright © 1983, American Society for Microbiology

Cloning and Expression of Treponema pallidum (Nichols)Antigen Genes in Escherichia coliMICHAEL V. NORGARD1* AND JAMES N. MILLER2

Department of Microbiology, University of Texas Health Science Center, Dallas, Texas 75235' andDepartment of Microbiology and Immunology, University of California at Los Angeles School of Medicine,

Los Angeles, California 900242

Received 16 May 1983/Accepted 8 August 1983

Hybrid pBR322 plasmid clone banks comprised of more than 125,000 recombi-nant DNA clones and representing the entire Treponema pallidum Nicholsgenome were constructed in Escherichia coli K-12 RR1. The two clone banksindividually contain over 53,000 and 72,000 recombinant clones. The numberaverage and mass average sizes of the cloned DNA inserts were found to beapproximately 12 and 13 kilobase pairs, respectively, indicating the presence oflarge treponemal DNA inserts in a majority of recombinant clones. To detect E.coli clones synthesizing T. pallidum antigens as hybrid plasmid gene translationproducts in the clone bank, a simplified, direct, solid-phase radioimmuno-colonyblot (RICB) assay was developed employing immunoglobulin G antibody isolatedfrom anti-T. pallidum immune rabbit serum. Clones with positive reactivities inthe RICB assay were isolated at frequencies of 0.1 to 0.2%. One isolated RICB-positive clone, designated RICB2-1, produced a very strong signal in the RICBassay and was subsequently found through E. coli cell-free in vitro transcription-/translation analysis to encode the synthesis of two gene translation products withapparent molecular weights of 77,000 and 44,000. The 44,000-dalton protein waseffectively immunoprecipitated from [35S]methionine-labeled E. coli clone cells byusing either immune rabbit serum (preabsorbed with Treponema phagedenisbiotype Reiter antigens) or selected human syphilitic serum, whereas the 77,000-dalton protein was never immunoprecipitable by similar methods. Purifiedplasmid DNA from clone RICB2-1 contained a treponemal DNA insert of 3.70kilobase pairs, which was of suitable size to code for the 121-dalton (44 + 77)protein. The insert was also flanked on each end by PstI sites and possessed threeinternal PstI sites with fragment sizes of 2.15, 1.18, 0.20, and 0.17 kilobase pairs.Purified clone RICB2-1 plasmid DNA was capable of transforming recipient E.coli cells to virtually 100% RICB reactivity, thus substantiating the plasmid-encoded characteristic. Further experiments employing various antisera in ra-dioimmunoprecipitation systems utilizing cell-free in vitro synthesized genetranslation products from clone RICB2-1 also provided the first evidence that E.coli may be capable of using endogenous T. pallidum DNA promotors for geneticexpression. These studies, amplified by the isolation of a potentially significantimmunoprecipitable 44,000-dalton recombinant protein antigen, point to theimportance of the "cloned antigen gene" approach for the eventual eludication ofspecific antigens or immunogens operative in the pathogenesis, immunology, andserodiagnosis of T. pallidum infection.

A satisfactory explanation of the events which pathogenesis, immunology, and serodiagnosis ofoccur after host-Treponema pallidum interac- syphilis. Despite this limitation, there is increas-tion remains obscure (15). One of the major ing direct and indirect evidence that surfacefactors which contributes to this enigma contin- proteins, glycoproteins, and/or polysaccharidesues to be the inability to sustain the growth of may play a key role in the disease process, thethe organism in vitro (13, 14, 44); this limitation stimulation of acquired resistance and the devel-has severely hampered the isolation and purifi- opment of specific and sensitive serodiagnosticcation of sufficient quantities of "purified" (free procedures (1, 2, 4, 6, 7, 10, 15-18, 21, 23, 29,from rabbit host testicular tissue) constituent 30, 33, 34, 36-38, 54-57, 60; unpublished data);antigens of T. pallidum for studies relating to the the importance associated with their isolation,

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436 NORGARD AND MILLER

purification, and physicochemical characteriza-tion cannot be overemphasized.

In an effort to circumvent the historical limita-tion of in vivo cultivation, this laboratory wasamong the first to initiate and describe thecloning and expression of T. pallidum antigengenes in an in vitro-cultivable host (Escherichiacoli) (50, 51, 59; M. V. Norgard and J. N. Miller,Abstr. Annu. Meet, Am. Soc. Microbiol. 1982,D38, p.53). Using this strategy, we describe herea cloned T. pallidum DNA hybrid gene transla-tion product (synthesized in E. coli) that specifi-cally reacts with T. pallidum antibodies found inthe serum of infected rabbits or humans; thisrecombinant T. pallidum antigen therefore canpotentially be isolated by affinity purification orother isolation methods, thereby further clarify-ing the overall long-term feasibility of employingthe "cloned antigen gene" approach, for futurestudies on the pathogenesis, immunology, andserodiagnosis of T. pallidum infection.

MATERIALS AND METHODSBacterial strains and plasmids. The virulent Nichols

strain of T. pallidum (42) was used as the repre-sentative pathogen in this study and was maintainedand cultivated in the testicles of rabbits as previouslydescribed (42, 47). Treponema phagedenis biotypeReiter (Reiter strain), cultivated and prepared as de-scribed earlier (47), was used for sera absorptions. E.coli RR1 (8) was used as the recipient host strain fortransformation (41). The Cowan I strain of Staphylo-coccus aureus was used in radioimmunoprecipitation(RIP) experiments to collect antigen-antibody com-plexes. The plasmid vector pBR322 was maintained inE. coli RR1 and isolated and purified as described byNorgard (39).

Establishment of a T. pallidum clone bank in E. coliRR1. The DNA of T. pallidum was isolated, purified,and shown to be virtually free of contaminating rabbithost DNA by both Cot (43) and Southern blot analyses(49), as previously described (42). Two 20-p.g quanti-ties of T. pallidum DNA were partially digested indi-vidually with the restriction enzymes AluI and HaeIII(New England Biolabs, Beverly, Mass.) by the methodof Maniatis et al. (31), mixed, and subjected to neutral(pH 8.0) sucrose density gradient velocity centrifuga-tion for size selection, using 3H-labeled X HindIllmolecular weight markers (New England NuclearCorp., Boston, Mass.) as reference standards (43).Centrifugation was carried out with a twice frozen andthawed 12-ml sucrose gradient in a Beckman SW40 Tirotor (5°C) at 38,000 rpm for 13 h (43). Fractionscontaining T. pallidum DNA partial fragments with amass average of 13 kilobase pairs (kb) were collected,pooled, and precipitated in standard fashion (43). Theresulting material was then tailed with dCTP residuesusing terminal deoxynucleotidyl transferase (TdT) andfurther purified as previously described (43), for a totalyield of approximately 6 ,ug of dC-tailed T. pallidumDNA, as estimated by ethidium bromide microdotanalysis.For annealing to form hybrid plasmid DNAs, ap-

proximately equimolar quantities of 6 ,ug of the dC-

tailed T. pallidum DNA inserts and 2 ,ug of dG-tailedpBR322 (tailed at the PstI site) (43) were mixed in a 4-ml reaction annealing volume as described previously(43) and allowed to hybridize at 60°C for 1 h (45). Theresulting hybrids were rapidly chilled in an ice waterbath to be used as donor DNA for the transformationof E. coli RR1.

E. coli RR1 cells were transformed by a modifica-tion of the methods of Norgard et al. (41). and Villa-Komaroff et al. (58). Briefly, RR1 cells grown to aculture density absorbance (550 nm) of 0.3 were har-vested from 4 liters of L broth (41) and washed twiceby centrifugation in 800 ml of cold, sterile wash buffer(0.1 M NaCl, 5 mM MgCl2, 5 mM Tris-hydrochloride[pH 7.6]). Cells were suspended in 400 ml of coldCaC12 buffer (70 mM MnCl2, 40 mM sodium acetate,30 mM CaC12 [pH 5.6]) for 20 min on ice, collected bycentrifugation, and resuspended in 10 ml of freshCaCI2 buffer. The 10-ml RR1 host cell suspension wasthen mixed with 4 ml of annealed hybrid plasmidDNAs and incubated on ice for a total of 60 min withoccasional mixing. The transformation mixture washeat shocked at 37°C for 4 min and plated onto 14 largePyrex (22 by 32 cm) pans of L agar containing 12.5 F.gof tetracycline per ml (L+Tet agar) (41). After incuba-tion for 48 h at 37°C, over 80,000 Tp recombinant DNAclones were observed, collected (pooled), washed, andstored (43) as a T. pallidum DNA total clones bank.RICB assay to detect expressed antigens. Previously

described methods for the detection of bacterial clonesproducing various antigenic products (9, 12) wereattempted unsuccessfully. Consequently, a more sim-plified solid-phase radioimmunoassay for the detectionof T. pallidum antigens being synthesized as hybridplasmid gene translation products by E. coli clonecolonies was developed from modifications of proce-dures described by Henning et al. (24) and Raetz (46).This modified technique was termed the radioimmuno-colony blot (RICB) assay. Standard petri dishes (100mm) containing 9 ml of L+Tet selective agar mediumwere prepared to restrict clone colony size. Cells fromthe Tp total clones bank were diluted appropriately incold L broth and plated on the thin agar plates at adensity of approximately 500 to 1,000 CFU per plate.After incubation at 37°C for not more than 14 to 16 h,the clone colonies were lifted gently onto keyed(marked) 8.26-cm disks of sterile, dry, Whatman no.42 filter paper; filters were placed onto the colonies,smoothed down, and gently lifted. The master plateswere incubated for an additional several hours at 37°Cto allow minor regrowth of the colonies, followed bystorage at 4°C. (Not all of the colonies will visuallyrecover; indentations in the agar where colonies wereoriginally lifted can be identified and will harbor livebacteria.)Each filter was then placed into a separate petri

dish, and 2 ml of lysis buffer (20 mM Tris-hydrochlo-ride, 10 mM EDTA, 10 mg of lysozyme per ml [pH8.0]) was gently added to each dish. After incubationat room temperature (RT) for 30 min, an equal volume(2 ml) of 0.2% (vol/vol) Triton X-100 was added, theplates were rotated gently to promote mixing, andincubation at RT was allowed to continue for anadditional 15 to 30 min. At this time, the coloniesbecame more raised and glistened with a swollenappearance; the colonies also became very "stringy"and viscous when touched with a sterile probe, indi-

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CLONING OF T. PALLIDUM ANTIGEN GENES 437

cating cell lysis. The filters were removed, gentlyrinsed (dipped) in 0.1 x phosphate-buffered saline(PBS) (1 mM sodium phosphate buffer, 0.085% NaCl),and drained at RT for 30 min on dry paper towels, withthe colony side up. For a positive control, filters weremoved to plastic wrap, and 1 ,ul of a T. pallidumsonicate (5 x 108 cells per ml) (47) was spotted andallowed to air dry for 15 min at RT. To aid in fixation,the filters were immersed briefly in 10% ethanol (47)and then allowed to air dry for 1 h on fresh dry papertowels at RT. Filters were then placed into clean petridishes and presoaked for 3 h at 4°C in 20 ml of PBScontaining 2% (vol/vol) fetal calf serum and 1 mMsodium iodide. The presoak buffer was then eitherdecanted or the filters were placed into clean petridishes, followed by the addition of 20 ml of freshpresoak buffer containing 105 cpm of 125I-labeled (25)purified (19) pooled immune rabbit immunoglobulin G(IgG) (6 to 9 months postinfection with T. pallidum)per ml (total of 2 x 106 cpm per filter). The labeled IgGprobe was freshly preabsorbed for 3 h on ice beforeaddition to the presoak buffer with a fresh preparationof PBS-washed E. coli RR1 cells (harboring pBR322)to reduce natural E. coli antibodies present in thepurified IgG preparation (M. V. Norgard, unpublishedobservations). The use of higher levels of probe onlyresulted in increased background, with no discernibleincrease in the signal, as observed by Henning et al.(24). Filters were immersed (colony side up) in theprobe solution and incubated at 4°C overnight withvery mild rocking to evenly distribute the probe andimpede edge drying. Groups of 12 filters were washedgently but extensively in one vessel with four changesof 4 liters of PBS, each washing step being performedwith very mild rocking at RT for a minimum of 1 hbetween washes. Generally, the first two washes wereperformed for 1.5 h each, with the remaining twowashes each being carried out for an additional 1 h.The filters were then removed, blotted on paper tow-els, and dried by warm air. After mounting on card-board, autoradiography was performed at -70°C for 3to 7 days using Kodak XAR-5 or Fuji X-ray film.Potential positives were picked from the master plates,retested in the RICB assay, streaked for isolation andpurification of the-elone, and again tested to ensureRICB reactivity.

Sera and antisera preparations. Normal rabbit serum(NRS) was collected from Venereal Disease ResearchLaboratory nonreactive New Zealand white male rab-bits. Pooled immune rabbit serum (IRS) was obtainedfrom animals 6 to 9 months after intratesticular infec-tion with T. pallidum Nichols; immune rabbits wereshown to be "chancre immune" when challengedintradermally with 105 motile T. pallidum Nichols cellsper site (6).

All normal and immune rabbit sera tested possessedhigh quantities of naturally occurring E. coli antibodieswhich interfered with the interpretations of RIP ex-periments. Consequently, sera were freshly preab-sorbed with either intact or sonicated preparations ofE. coli RR1 containing pBR322. To prepare the E. colisorbents, confluent lawns of E. coli RR1 (pBR322)growing on L+Tet agar (41) were harvested andwashed three times by centrifugation in cold PBScontaining 0.02% sodium azide. The resulting cellpellet volume was suspended in 4 volumes of PBS plusazide and used as either intact organisms, or it was

sonicated, followed by freezing the preparations at-60°C. Sonication of the viscous bacterial suspensionrequired 18 min of sonication (on ice) at a 50o pulse(47). For absorptions using sonicated sorbent, equalvolumes of serum and sonicated sorbent were incubat-ed in an ice water bath for 2 h and centrifuged at 21,000x g for 30 min (4C) to remove precipitating antigen-antibody complexes, and the process was repeatedonce to yield a final preparation of 33% serum in PBSplus azide. This procedure appeared to remove morethan 90%o or more of the E. coli antibodies. Where thepossibility existed that active proteases liberated fromthe sonicated E. coli cells might dramatically affectcertain experiments, some sera were absorbed againstwhole E. coli cells. Cells from intact E. coli to be usedas sorbent were collected by centrifugation and mixedwith an equal starting volume of serum on ice for 2 h,followed by removal of the E. coli cells by centrifuga-tion and a repeat of the absorption process. Thistechnique was not as effective as employing sonicatedE. coli sorbents, but it was adequate for some experi-mental analyses. To facilitate the removal of antibod-ies directed against common treponemal group anti-gens, both NRS and IRS were also preabsorbed withthe particulate fraction of 5 x 109 sonicated T. phage-denis biotype Reiter cells (47) per ml of serum insimilar fashion as intact E. coli sorbent.

sS labeling of E. coli ceUs. Strains of E. coli RR1(pBR322) and selected recombinant DNA clones werelabeled with L-[35S]methionine through cultivation in20 ml of M9 minimal medium (40) supplemented withone-quarter strength methionine assay medium (DifcoLaboratories, Detroit, Mich.) and 0.1 mCi of[35Slmethionine (NEG-009T; ca. 1,000 Ci/mmol; NewEngland Nuclear Corp., Boston, Mass.) per ml oflabeling medium. After 13 h of incubation at 37°C, thelabeled cells were chilled on ice, collected by centrifu-gation, and washed four times by centrifugation in 25-ml portions of cold PBS, followed by final suspensionin 4 ml of cold PBS. The preparation was divided into0.4-ml portions and stored at -60°C. Preparationsmade in this fashion routinely contained 6 x 10' cpm/

1L, and 0.1-ml portions were used per RIP test.In vitro E. coli cell-free transcription/translation as-

says. In vitro protein synthesis was performed withvarious template plasmid DNAs, as described byKung et al. (26, 27). The system (35 ,ul) contained 15mM Tris-acetate (pH 8.2), 11 mM sodium dimethylglu-tarate, 35 mM ammonium acetate, 65 mM potassiumacetate, 10 mM magnesium acetate, 0.8 mM spermi-dine hydrochloride, 2.4 mM dithiothreitol, 0.93 mMeach of UTP, CTP, and GTP, 3.0 mM ATP, 24 mMphosphoenolpyruvate, 0.2 ,ug of pyruvate kinase, 0.05mM ppGpp, 0.7 mM 3'5'-cAMP, 1 mM calcium leuco-vorin, 12.5 ,ug of E. coli tRNA, 0.63 mg of polyethyl-ene glycol 6000, approximately 6 ,ug of plasmid DNAtemplate, 0.4 mM isopropyl-thio-,-D-galactopyrano-side, 0.112 mM each of every 20 amino acid, 65 ,uCi[355]methionine, 1.2 absorbance units (at 260 nm) ofammonium chloride-washed ribosomes, ribosomalwash (50 jig of protein), a 0.25 M DEAE salt eluate(110 ,ug of protein), a 1 M DEAE salt eluate (7.5 jig ofprotein), elongation factor-Tu (3 jig) and Ehrlich asci-tes S-100 (26 jig). The reaction was carried out at 37°Cfor 90 min. The in vitro synthesis products wereanalyzed by sodium dodecyl sulfate-polyacrylamidegel (10%) electrophoresis (SDS-PAGE) (28). Samples

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(3 ,u) were dissolved in 10 p.l of sample buffer (28) andboiled for 2 min before application to the gels. Gelswere processed in the same way as those used in RIPs.

RIPs. RIPs performed on presolubilized E. coliclones or on transcription/translation assay mixtureswere carried out essentially as described by Hansen etal. (22) and Gulig et al. (20) with minor modifications.Briefly, 0.1 ml of "S-labeled E. coli cells was mixedwith 10 ml of solubilization buffer (10 mM Tris-hydrochloride, 150 mM NaCl, 10 mM EDTA, 1%[vol/vol] Triton X-100, 0.2% [wt/vol] sodium deoxy-cholate, 0.1% [wt/vol] sodium dodecyl sulfate [pH7.8]). For RIPs of in vitro transcription/translationassay mixtures, 10 ,ul of assay mixture was added to 5ml of solubilization buffer. Solubilization mixtureswere incubated at 37°C for 1 h with periodic mixing,and insoluble material was removed by centrifugationat 17,000 rpm in a Sorvall SS-34 rotor for 1 h at 22°C.Samples harvested for liquid scintillation countingbefore and after centrifugation indicated that E. colicells were routinely 80% solubilized. To each sampletube was then added the equivalent of 0.5 ml of serum(e.g., 1.5 ml of 33% serum resulting from preabsorp-tion with E. coli sonicates, etc.) followed by rocking atRT for 2 h. Samples (0.2 ml) of Formalin-fixed andprewashed protein A-bearing S. aureus Cowan I wereadded to each tube, and tubes were incubated at 4°Cwith rocking for an additional 1 h. The resultingprotein A-bearing S. aureus-antibody-antigen com-plexes were collected by centrifugation and washedfive times in 1-ml portions of solubilization buffer,followed by dissociation of the complexes upon boilingin digestion buffer (20), reduction with 2-mercaptoeth-anol, and resolution on SDS-PAGE (28). Radiolabeledproteins found in SDS-PAGE gels were identified byusing EnHance fluorography (New England NuclearCorp.), as described by the manufacturer.

RESULTSCharacteristics of the clone bank. The cloning

procedures resulted in the recovery of over80,000 clone colonies arising on L+Tet agarselective medium. Replica plating of 200 ran-domly selected clones on L+Tet and L+Ampmedia (41) indicated an estimated background ofclones without inserts to be 9.5%. Thus, over72,000 actual recombinant clones comprise thisclone bank. An earlier constructed T. pallidumclone bank, comprised of over 53,000 clones, isalso on hand in this laboratory (M. V. Norgardand J. N. Miller, Abstr. Annu. Meet. Am. Soc.Microbiol. 1982, D38, p. 53). Figure 1 (lane 3)demonstrates by agarose gel (1%) electrophore-sis and staining with ethidium bromide that totalclones plasmid DNA grown and amplified bychloramphenicol and isolated from the describedtotal clones bank (39) contained a broad spec-trum of cloned insert sizes, as expected anddemonstrated by the continuous smear withinthe length of the gel track lane. In considerationof the fact that background pBR322 componentsand chimera plasmids with small inserts in thebank characteristically amplify better than those

1 2 3-I

I CHIMERASo- pBR322I DIMER

--- pBR322MONOMER

FIG. 1. Agarose gel (1%) electrophoresis of T. pal-lidum total clones plasmid DNA. Lane 1, bacterio-phage lambda-HindIII molecular weight markers; lane2, covalently closed circular pBR322 plasmid DNA;lane 3, T. pallidum total clones plasmid DNA. Thesmear of fluorescent plasmids between the majorpBR322 bands indicates the presence of high-molecu-lar-weight recombinant plasmids.

chimeras possessing large inserts, the clonebank apparently contains a high proportion ofclones with very large inserts. The increasingintensity of fluorescence observed at the higher-molecular-weight regions of the gel is consistentwith this finding as well as with the overallpreselection of inserts on sucrose gradients be-fore cloning into the pBR322 vector. Plasmidminiscreen analyses (5) performed on 40 ran-domly selected recombinant clones from theclone bank confirmed that clones possessed anumber average insert size of about 12 kb, eventhough a few clones did possess relatively smallinserts (i.e., below 5 kb). The presence of largecloned inserts was also supported by observingthe agarose gel mobility of total clones plasmidDNA restricted by PstI digestion; inasmuch asintact PstI sites flank the cloned inserts, veryhigh molecular weight linear insert moleculescan be observed under these conditions, al-though the presence of internal PstI sites inmany of the large inserts causes underestimationand obscures accurate assessments.

Detection of E. coli clones expressing T. palli-dum antigens. Figure 2 shows examples of re-sults obtained using the RICB assay for thedetection of E. coli clones expressing T. palli-dum antigens. These RICB+ clones were neverobserved at frequencies of greater than 2 per1,000 clones tested (f = 0.1 to 0.2%), whichseemed appropriate by our crude estimations.Furthermore, because it has been our experi-ence and the experience of others that consider-able degrees of both infidelity and peculiar "out-side edge" artifacts exist in this and comparablesystems (M. V. Norgard, unpublished observa-tions), great caution was observed in employingand interpreting results of the RICB assay. Con-sequently secondary rescreenings (Fig. 3) were

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always necessary to confirm preliminary posi-tive RICB reactivities. As positive controls,sonicated suspensions of T. pallidum (seeabove) were always applied to each filter. Theinclusion of negative controls were unnecessaryin the primary RICB screening procedure, butwere included in secondary rescreening RICBassays (Fig. 3) for clarity of interpretation. Also,the lysis steps employing lysozyme and TritonX-100 treatments appeared important, sincevery few clones, if any, could definitively bedetected in the absence of this strategy. This isin contrast to a similar published report (51).Thus far, several RICB+ clones expressing T.

pallidum antigens have been isolated. Figure 3shows the RICB reactivities of three repre-sentative clones, designated RICB2-1, RICB2-2,and RICB2-3. Note the greater reactivity ofRICB2-1 (lane 2) when applied to the outsideedge (lane 6) of the test strip. The positive T.pallidum control (lane 1) yields a very strongsignal, and the host RR1 (pBR322) cells (lane 4)show a barely detectable background signal, asexpected. Relative to these positive and nega-tive controls, the reactivities of the three clonesshown in Fig. 3 (lanes 2, 3, 5, and 6) areextremely strong considering the potential mo-lecular stress involved in the synthesis and pos-sible secretion of these foreign antigens.

Partial characterization of recombinant anti-gens by RIP analysis. RIP followed by SDS-PAGE analyses were used to partially character-ize recombinant treponemal antigens. Thestrength of the RICB reactivity demonstrated by

Tpa

3

Tp

Tp

.2

FIG. 2. RICB assay for the initial detection of T.pallidum antigen-expressing recombinant DNAclones. Tp, 5 x 10 sonicated T. pallidum cells.Arrows indicate positive reactivities for clones ex-pressing T. pallidum antigens.

Tp 1

NC*

C+j

2 cr 3 I

1 2 3 4 5 6FIG. 3. Confirmation of RICB reactivity using a

secondary RICB assay. Lower numbers on figuredenote lane position on the filter strip. Lane 1, 5 x 105sonicated T. pallidum cells (Tp); lanes 2 and 6, cloneRICB2-1; lane 3, clone RICB2-2; lane 4, E. coli RR1(pBR322) used as a negative control; lane 5, cloneRICB2-3. Note the relatively high degree of RICBreactivity relative to the positive Tp control.

clone RICB2-1 in Fig. 2 and 3 suggested thatfurther antigen amplification in minicells (32) ormaxicells (48) was not necessary for initial char-acterization and isolation studies. Consequent-ly, both E. coli RR1 (pBR322) and clone RICB2-1 were radiolabeled with [35S]methionine asdescribed above and used as solubilized antigensin RIP analyses (Fig. 4). NRS was ineffective inimmunoprecipitating antigens from the labeledclone RICB2-1 (lane 1), as was IRS in immuno-precipitating components from the RR1(pBR322) host (lane 2). However, when IRS(preabsorbed with both E. coli and T. phage-denis biotype Reiter antigens) was used in anRIP with the labeled clone RICB2-1 (lane 3), astrong 35S signal was observed in the 44,000molecular weight region of the SDS-PAGE geltrack. Other background bands observed in lane3 were also found to be present in lanes 1 and 2 ifthe gel was exposed to the X-ray film for alonger period of time. Further control experi-ments using PBS in place of serum in the RIPalso revealed the same banding pattern; thesebands were therefore attributed to nonspecificadsorption to the protein A-bearing S. aureuscells. The reason for the apparent increasednonspecific binding observed by clone RICB2-1components when exposed to IRS is not clearbut was reproducible. Nevertheless, the appear-ance of the 44,000 molecular weight recombi-nant antigen was definitive, even though a veryfaint band of close but apparently different mo-lecular weight could sometimes be observed in

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Clone 322 CloneNRS IRS IRS MW

200K

-92.5 K

46K-0--44K Ag

-m~ 30K

1 2 3

-: 12.3K

4

FIG. 4. RIP and SDS-PAGE analysis of cloneRICB2-1. Lane 1, preabsorbed NRS used to immuno-precipitate solubilized RICB2-1; lane 2, preabsorbed(E. coli and T. phagedenis) IRS used to immunopreci-pitate solubilized E. coli RR1 (pBR322); lane 3, preab-sorbed IRS used to immunoprecipitate solubilizedRICB2-1, showing the presence of the 44,000 antigen(Ag); lane 4, purified protein molecular weight (MW)markers.

synthesized from purified pBR322 plasmidDNA. Lane 3 shows the gene translation prod-ucts of purified plasmid DNA from cloneRICB2-1 (designated here as RICB+-1), withthe discrete presence of the 44,000 molecularweight gene translation product. Interestingly,an additional 77,000 molecular weight product isalso apparently synthesized by this clone but hasnever been immunoprecipitable by any serum orcontrol agent used in RIP assays. As expected,there is also the distinct disappearance of themajor beta-lactamase product normally found inthe pBR322 control lane, inasmuch as the beta-lactamase gene has been interrupted at the PstIsite by the cloning strategy employed. Indepen-dent assays from separately purified plasmidDNA preparations gave identical results. In con-trast to the nonimmunoprecipitable 77,000 mo-lecular weight gene product, the 44,000 molecu-lar weight antigen synthesized in vitro wasimmunoprecipitable by IRS but not NRS uponRIP analysis. Furthermore, if IRS used in theRIP assays against either in vitro synthesizedpBR322 or RICB2-1 gene translation productswas not extensively preabsorbed with E. coliantigens, both NRS and IRS were capable of

MW *:- C

M ;

negative control samples. The amount of therecombinant antigen synthesized, however, byclone RICB2-1 was striking, and even somewhatsurprising. Additionally, most of the early diffi-culties in these analyses were the result of veryhigh amounts of naturally occurring E. coli anti-bodies found in all rabbits tested. Extensivepreabsorptions with E. coli antigens (see above)practically eliminated such difficulties, leavingonly the more minor problem of nonspecificbinding of E. coli components protein A-bearingS. aureus cells, which could theoretically also bedecreased by performing preabsorptions withintact protein A-bearing S. aureus cells.

Analysis of recombinant antigens by in vitrotranscription/translation assays. To further elimi-nate the possibility of peculiar artifacts, it wasevident that purified plasmid DNA from cloneRICB2-1 added to E. coli extracts of an in vitro(cell-free) transcription/translation assay systemshould reveal the presence of the 44,000 molecu-lar weight gene translation product after SDS-PAGE. Figure 5 shows the results of radiola-beled gene translation products synthesizedfrom purified plasmid DNAs. Lane 1 containsmolecular weight markers. Lane 2 shows thenormal complement of gene translation products

92.5 K -rn-

46 K -4 44K Ag

30 K :-

12.3K__

FIG. 5. In vitro-synthesized gene translation prod-ucts from clone RICB2-1. MW, Protein molecularweight markers; pBR322, gene translation productsfrom pBR322 plasmid vector with no inserts; RICB+-1,gene translation products from clone RICB2-1. Themajor beta-lactamase band of pBR322 is absent in theRICB+-1 lane, with the concomitant appearance of anapproximately 23,000 band, believed to be a PstI site-truncated partial beta-lactamase protein. Ag, Antigen.

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immunoprecipitating an apparent 35,000 molec-ular weight beta-lactamase (intact) product (ac-tual molecular weight is about 31,430 [28,900 +2,530] which includes the 23 amino acid signalsequence (3, 52]) from pBR322 DNA, presum-ably due to the presence of naturally occurringcommon (3) P-lactamase antibodies found inanimal serum. The use of this nonpreabsorbedNRS and IRS in RIP assays proved propitious,however, since this same antiserum also precip-itated a 23,000 molecular weight gene translationproduct only from clone RICB2-1 and not frompBR322 (data not shown); this was believed tobe a PstI site-truncated beta-lactamase partialprotein, which should theoretically have a mo-lecular weight of about 22,440, considering thatthe PstI site occurs at amino acids 181 to 182(nucleotide 3608 in pBR322) plus the added 23amino acid signal (52, 53). An analogous bandcorresponding to 23,000 can also be observed inlane 3 (RICB+-1) of Fig. 5 which is not found inthe pBR322 control lane.

Size of the cloned insert from clone RICB2-1.Results of the in vitro transcription/translationassays showed that a total of approximately121,000 d of protein (44,000 + 77,000) was en-coded by the insert in clone RICB2-1. Estimatesbased on the average molecular weight of anamino acid suggested that the cloned insert ofclone RICB2-1 should be at least 3.30 kb insize. Careful PstI restriction enzyme mapping toexcise the insert from the pBR322 plasmid vec-tor (followed by 1 or 2.5% agarose gel electro-phoresis) revealed the insert to possess threeinternal PstI sites comprising a total length of3.70 kb. Fragments were composed of 2.15,1.18, 0.20, and 0.17 kb (major fragments of 2.15and 1.18 kb shown in Fig. 6). Thus, the clonedinsert contained adequate theoretical DNA cod-ing capacity for synthesis of the treponemalproteins.

Transformability of the RICB+ phenotype byRICB2-1 plasmid DNA. When purified RICB2-1plasmid DNA was used as donor DNA for thetransformation of E. coli RR1, virtually 100% ofthe clones arising on selective medium werefound to be reactive by RICB testing and there-fore indistinguishable from clone RICB2-1 in theRICB assay. Thus, the possibility of selecting ananomolous mutant host clone from within theclone bank with increased reactivity in the RICBassay was eliminated.

DISCUSSIONThis report describes the construction of a T.

pallidum DNA clone bank in E. coli by employ-ing a useful cloning strategy to promote expres-sion of the cloned inserts. Particular emphasishas been placed on the detection of those genesencoding relevant antigens of the organism that

123

-pBR322(Pst I)

-.-2.15 Kb

--1.8 Kb

FIG. 6. Agarose gel (1%) electrophoresis of Pstl-restricted plasmid DNA from clone RICB2-1. Lane 1,lambda-HindIII molecular weight markers; lane 2,pBR322-AluI molecular weight markers; lane 3, PstI-restricted plasmid DNA from clone RICB2-1. Lane 3shows the linear pBR322 plasmid vector at the properlocation and the presence of two major PstI fragments(2.15 and 1.18 kb). Two other PstI fragments of sizes0.20 and 0.17 kb were not resolved on this particulargel. The T. pallidum DNA insert of clone RICB2-1 hasa total of three internal PstI sites, for a total size of3.70 kb.

may play a role in the pathogenesis, immunolo-gy, and serodiagnosis of syphilis.

Other investigators undertaking the cloning ofT. pallidum antigen genes have employed eithercloning into bacteriophage lambda (59) or intothe BamHI site of pBR322 (51). Cloning intobacteriophage ultimately requires tedious sub-cloning experiments, and the pBR322-BamHIsite provides only moderate strength in the tetra-cycline resistance promoter region to aid in theexpression of cloned genes without functionalendogenous promoters. For potential ease in theamplification and cultivation of cloned genesand their translation products, and because ofthe relatively low gene complexity of the T.pallidum genome (35) as compared to eucaryoticsystems, we wished to exploit the pBR322 plas-mid system in a different manner. We chose astrategy with several more apparent reasons foran increased likelihood of successful geneticexpression. Cloning into the PstI site of pBR322provides a relatively strong promotor in thebeta-lactamase gene that may function to allowfor the synthesis of read-through translatablegene fusion products in the event that endoge-nous treponemal promoters are not operative inE. coli. Additionally, a previous report showedthat gene fusion products arising as the result ofcloning into the PstI site of pBR322 can besecreted by the E. coli host due to the presenceof the 23 amino acid P-lactamase signal se-quence at the N terminus of the cloned and

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translated gene fusion protein product (58); thisapproach could result in enhancing the detectionof the translated product and its availability foreventual isolation. Furthermore, it has beenobserved that dG-dC -tails that flank clonedinsert genes can occasionally provide some typeof "false" promotor region in E. coli systems(R. F. Manning, J. Stoltenborg, C. Miyamoto,H.-F. Kung, J. Chu, and M. V. Norgard, Abstr.Annu. Meet. Am. Soc. Microbiol. 1983, H114,p. 124). For these reasons, our strategy was toclone restriction enzyme-generated randomfragments (31) of the T. pallidum Nicholsgenome into the PstI site of pBR322 by the dG-dC tailing method (43), with the resulting newlygenerated PstI sites flanking the cloned insertsused to easily map and excise the cloned DNAinserts.Based on the apparent size and complexity of

the T. pallidum Nichols genome (9.05 x 109)(35), only 5,115 clones having a mass averagesize of 13 kb would be required to fulfill aprobability of P = 0.99 that any one particulargene of interest would be successfully cloned(11). Considering that there is possibly only onechance in six that any particular gene is posi-tioned in the proper phase and orientation forexpression, the net complexity of the describedclone bank of over 72,000 clone colonies ade-quately satisfies the theoretical criteria (30,690clones) with over a 2-fold bank redundancy andover a 10-fold gene redundancy necessary topromote successful gene cloning and expres-sion. The additional bank of over 53,000 cloneson hand also adds an additional large margin ofsafety. Furthermore, the more than twofoldclone bank redundancy is necessary inasmuchas the actual number average size of the T.pallidum DNA inserts in the bank should be lessthan the mass average size determined either byagarose gel electrophoresis (Fig. 1) or sucrosedensity gradient centrifugation. A previouslydescribed plasmid-derived T. pallidum clonebank (51) may not meet this criterion necessaryto enhance the successful expression of any onecloned gene; colony blot hybridizations per-formed on that bank revealed that only 11.2%(224 of 2,000 clones tested) of a 40,000 clonesbank exhibited strongly hybridizable-sized T.pallidum DNA inserts, making the bank of ques-tionable size complexity.The strategy of employing both primary and

secondary radioimmuno-colony blot assays wasuseful. Positive clones detected by primaryRICB assays were detected at a frequency of 0.1to 0.2% but did not always exhibit positivesignals in the secondary screening test. Howev-er, clones with positive reactivities in secondaryor other follow-up RICB assays have alwayscontinued to yield positive reactivities upon

multiple reclonings and rescreenings. Further-more, we have demonstrated that purified plas-mid DNA from RICB-reactive colonies is capa-ble of transforming the normal host strain toRICB reactivity at a frequency of essentially100%. Thus, the efficacy the RICB assay em-ployed has been demonstrated, and the positivereactivity detected in this assay has been shownto be plasmid mediated and therefore transform-able to other normal recipient strains.One puzzling problem has been the fact that

some clones consistently positive in the RICBassay do not necessarily yield immunoprecipi-table products on SDS-PAGE gels after RIPassays are performed on radiolabeled RICB re-active clone cells. In this case, the use of theRICB assay appears essentially more sensitivethan an RIP assay, at least with respect to initialscreening and detection. Although it can bedemonstrated that the RICB reactivity remainsstable and transferrable via plasmid-specifictransformation to a normal E. coli host strain,isolation of some of these cloned antigens can beproblematic. This may reflect denaturation ofthe cloned antigen product by the detergentsolubilization system employed in the RIP; fur-ther investigations on these clones are neces-sary. The identification and isolation of thecloned antigens from such "antigen-labile"clones have not yet been attempted using eitherminicell (32) or maxicell (48) systems, but suchsystems are also under investigation.The frequency of detection for antigen-ex-

pressing clones (0.1 to 0.2%) in the RICB assaydescribed here is in relative agreement with thefrequency of 0.13% (1 unique clone per 750tested) described by Walfield et al. (59); it is incontrast, however, to almost a 5- to 10-foldhigher frequency obtained in similar filter-bind-ing assays reported by Stamm et al. (50, 51). Theapparent significantly higher degree of successdemonstrated by Stamm et al. may be the resultof the manner in which certain recombinantDNA clones were preselected before being sub-jected to the filter binding assay (50, 51). Thereport of Walfield et al. (59) describes recombi-nant antigens of 46,000, 43,000, 38,000, 24,000,23,000, 20,000 and 18,500 daltons, of which boththe 46,000 and 43,000 antigens are close inapparent molecular weights to the 44,000 antigenof clone RICB2-1 described here. The observedgene linkage pattern, however, for these anti-gens was not evident in this study, but cloneRICB2-1 contains a significantly smaller insert(3.70 kb) as compared to the 16 kb insert inthe respective phage clone. A previous report byStamm et al. (50) describes recombinant trepo-nemal antigens (74,000, 38,000, 34,000, 32,000and 25,000 daltons) dissimilar in apparent molec-ular weight to the 44,000 antigen of clone

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RICB2-1, but little importance can be assignedto these findings at this time.Although several RICB-positive clones are

currently under analysis, clone RICB2-1 hasbeen best characterized. This clone possesses aT. pallidum DNA insert of 3.70 kb and encodesfor the synthesis of at least two proteins, a77,000 protein and a 44,000 protein antigen.Only the 44,000 protein antigen is immunopreci-pitable by IRS in RIP assays employing solubi-lized clone cells or using in vitro synthesizedgene translation products. These data suggestthat there exists no genetic linkage of encodedantigens with respect to at least one side of the44,000 antigen gene.

Although the 44,000 antigen can be routinelyimmunoprecipitated by using IRS, only two offour samples of late latent human syphiliticserum have been capable of immunoprecipitat-ing the 44,000 antigen. The significance of thispreliminary observation cannot be overempha-sized. Hanff et al. (21), utilizing SDS-PAGEWestern blotting techniques, detected IgG anti-body against approximately 22 specific T. palli-dum polypeptide antigens in the sera of patientswith secondary and early latent syphilis. Thesepolypeptides ranged in molecular weight from115,000 to 15,500. In contrast, IgG antibodyagainst only 10 and 11 of these antigens wasdetectable in sera from patients with late latentand late cardiovascular syphilis, respectively.Furthermore, Hanff et al. (P. A. Hanff, N. H.Bishop, J. N. Miller, and M. A. Lovett, J. Im-munol., in press), employing similar methodolo-gy, were also able to detect IgG antibody to 22specific T. pallidum polypeptides with similarmolecular weights ranging from 14,400 to 94,000in sera from rabbits experimentally infected withT. pallidum for 3 months, at a time when immuni-ty to reinfection had developed. Of equal signifi-cance was the observation that IgG antibody tothese antigens persisted in the sera of animalswith latency but that were solidly resistant toreinfection at 4.5, 13.5, and 17 months postin-fection. Inasmuch as T. pallidum-infected rab-bits fail to develop the manifestations of latesyphilis observed in humans, it is exciting tospeculate a potential correlation between loss ofantibody in human late latent and late syphilis,and disease progression as postulated by Hanffand his co-workers (21; Hanff et al., in press).Indeed, the failure to demonstrate reactivity oftwo latent syphilitic sera against the 44,000cloned antigen in this study could be a reflectionof a breakdown in endogenous and/or exogenousresistance.

In light of its potential importance, severaladditional striking characteristics of the 44,000antigen are noteworthy. First, it was unneces-sary to amplify the expression of the 44,000

antigen in E. coli by employing either a minicellor maxicell system. It appears that a large quan-tity of this antigen may be synthesized in the E.coli host and can be easily immunoprecipitatedby the methods described. Second, the highdegree of RICB reactivity and ease in detectionof the 44,000 antigen demonstrated for cloneRICB2-1 may also be a reflection of efficientantigen secretion by the E. coli host. This mayinclude either secretion into and trapping in theperiplasmic space, or insertion into the outermembrane of E. coli. Experiments describedherein, however, neither prove nor disprovethese possibilities as yet.The experiment describing the observation of

a 23,000-dalton protein that could be immuno-precipitated from clone RICB2-1 by either NRSor IRS suggested that a 1-lactamase, PstI site-truncated partial protein was being synthesized.These experiments may be the first proof that E.coli is indeed capable of utilizing endogenous T.pallidum operator-promotor regions, although arole for the dG-dC tail in promotion cannot becompletely ruled out. On this basis, our currentworking hypothesis with respect to synthesis ofthe 44,000 antigen cloned into the PstI site ofpBR322 by the cloning strategy employed is asfollows. It appears that synthesis of the N-terminal P-lactamase leader sequence and struc-tural protein occurs in normal fashion untilreaching the PstI site, whereupon this 23,000partial protein is truncated as it enters the dG-dCtail. Either the 77,000 or the 44,000 proteinantigen synthesis is then initiated, most likelythrough use of an endogenous promoter, fol-lowed by termination and then promotion of thesecond gene, until both proteins are made andthe distal end of the 13-lactamase gene (fused tothe T. pallidum DNA insert) is reached. Theorder of synthesis for the 77,000 protein and the44,000 antigen genes remains uncertain. Howev-er, recent RIP experiments in our laboratory,which have been independently confirmed byother investigators using Western blotting (29;S. A. Lukehart, personal communication), indi-cate the presence of a 47,000 or 48,000 immuno-dominant antigen present in T. pallidum andprobably other pathogenic treponemes (S. A.Baker-Zander and S. A. Lukehart, Abstr. Annu.Meet. Am. Soc. Microbiol. 1983, E31, p. 81). Ifso, then it is possible that the 44,000 antigendescribed here may be a partial 47,000 to 48,000antigen normally found in T. pallidum or othertreponemal pathogens. The fact that antigens ofT. phagedenis biotype Reiter were incapable ofabsorbing out anti-44,000 cloned antigen anti-bodies from IRS further supports this possibili-ty. Consequently, our working hypothesis maybe extended to synthesis of first the 77,000protein, followed by synthesis of the 44,000

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antigen gene, which may be partially incompletewith respect to about 3,000 to 4,000 daltons ofstructural protein. It was disturbing to observe,however, that monoclonal antibodies in our lab-oratory (47) reactive against the 47,000 antigenhave not been capable of immunoprecipitatingthe 44,000 antigen protein from either E. coliclone RICB2-1 or from in vitro transcription/translation assay products of the clone. It re-mains possible, however, that the loss of theadditional protein from this cloned antigen or alack of proper posttranslational modification(i.e., glycosylation) may be sufficient in alteringthe antigenic determinants against which theanti-47,000 monoclonal antibodies may be di-rected.Although much additional work needs to be

performed on clone RICB2-1 and other RICB-reactive clones described in this report, thisstudy and the work of others (50, 51, 59) haveaccumulated ample information to suggest thatthe overall approach for the possible study of thepathogenesis, immunology, and serodiagnosis ofT. pallidum infection using the cloned antigenstrategy remains a feasible one. These approach-es, combined with the potential power of em-ploying monoclonal antibodies (47), reflect someof the most exciting approaches to T. pallidumresearch since the discovery of the etiologicalagent. Although the possibility of in vitro culti-vation (13, 14, 44) lends equally exciting possi-bilities, this has not yet advanced to a practicalstage. Consequently, we are hopeful that molec-ular biological approaches to the study of thisorganism will be fruitful. Indeed, the forthcom-ing purification of the 44,000 recombinant anti-gen described here may provide intriguing newinsights into the nature of the antigens or im-munogens of this complex human pathogen thathave remained so elusive.

ACKNOWLEDGMENTSWe thank H.-F. Kung for performing transcription/transla-

tion assays and supplying assay mixtures, D. A. Hart forguidance with rabbit IgG purifications, E. J. Hansen forinstruction on RIP assays, E. S. Vitetta for helpful discussionson various immunoassay approaches, L. V. Stamm for earlydiscussions on the feasibility of using a modified Henningassay, and C. F. Frisch for discovering the two small PstIfragments of clone RICB2-1. The technical assistance of R. E.Shanks was appreciated. We are indebted to D. Tucker for theheroic and excellent typing effort.

This work was partially supported by World Health Organi-zation grant V3-181-64, Welch Foundation grant 1-940, PublicHealth Service grant AI-16692 from the National Institute ofAllergy and Infectious Diseases to M.V.N., and World HealthOrganization agreement V3-181-26 and Public Health Servicegrant AI-12601 from the National Institute of Allergy andInfectious Diseases to J.N.M.

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Cloning Expression of Treponema Antigen Genes Escherichia · 436 NORGARD ANDMILLER purification, and physicochemical characteriza-tion cannot be overemphasized. Inaneffort to circumventthe