Restriction Fragment Length Polymorphism Analysis of the Fimbrillin Locus, fimA, of Porphyromonas gingivalis B.G. LOOS and D.W. DYER1 Department of Oral Biology, School ofDental Medicine, State University ofNew York at Buffalo, Buffalo, New York 14214; and 'Department of Microbiology, School ofMedicine, State University ofNew York at Buffalo, Buffalo, New York 14214

With hybridization probes derived from the fimbrial locus of Porphyromonas gingivalis strain 381, fimA 3811 restriction fragment length polymorphisms (RFLPs) were examined at the fimbrillin locus in 39 human and animal strains ofthis species. The 39 strains were subdivided into nine RFLP groups (I-IX) after genomic digests were probed with the internal coding sequence of the fimA381 gene. Thirty-three strains showed one or more AluI fragments of moderate-to-high homology (2 77%) with the internal coding sequence of fimA381. These strains were distributed into the first seven RFLP groups, based solely on the size of the major hybridizing AluI fragment. Five human strains (RFLP Group VIII) had only oneAlul fragment that hybridized very poorly with this probe. One animal strain did not have homology at all (RFLP Group IX). When allAlul fragments that hybridized withfimA381 were analyzed, RFLP groups I-VIII were further differentiated into 25 distinct RFLP patterns. Hybridizations were also performed with the internal coding sequence of fimA381 to probe PstI genomic digests of selected strains that appeared to have lesser homology with fimA381. These hybridizations were performed to determine the level and location of the region of poor homology within the fimA genes of these strains. The results suggested that fimbrial coding sequences are more commonly conserved between these strains in the 5'-region of the fimA locus (. 92% sequence homology). However, the five human strains ofRFLP Group VIII had only one PstI fragment that hybridized very poorly with a probe derived from fimA381 coding sequence, and this sequence homology (only . 66%) was located in the central or 3-end of the fimA gene. The 5'-region of the fimA allele in Group VIII strains did not have any detectable sequence homology. In contrast, the Group VIII strains were highly homologous with the sequences flanking the fimA38, gene. This indicates that these strains do possess a fimA allele at the same chromosomal location as fimA381. J Dent Res (71)5:1173-1181, May, 1992

Introduction. Porphyromonas gingivalis, formerly Bacteroides gingivalis, is a Gram-negative obligate anaerobic coccobacillus (Shah and Collins, 1988). This species is frequently isolated from subgingival lesions in periodontal disease (Dzink et al., 1988; Rodenburg et al., 1990) and from root canal infections (van Winkelhoffet al., 1985; Haapasalo et al., 1986). The organism is rarely found in subgingival sites in periodontally healthy humans or in patients with gingivitis (Zambon et al., 1981; Christersson et al., 1989). Although the exact etiology of periodontal disease is unclear, these observations suggest that P. gingivalis is an important oral pathogen. Several potential virulence factors have been described for P. gingivalis, and the ability of this organism to cause spreading infections in some rodents has been documented (reviewed by Mayrand and Holt, 1988). Adherence of P. gingivalis to dental plaque or gingival tissue surfaces is probably an initial step in the pathogenesis ofperiodontitis. P. gingivalis has been shown to adhere to oral epithelial cells (Slots Received for publication September 9, 1991 Accepted for publication December 2, 1991 This workwas supportedbyU.S. Public Health Service Grants DE07034, DE08240, and DE04898 from the National Institute of Dental Research.

and Gibbons, 1978), to other oral bacteria (Goulborne and Ellen, 1991; Stinsonetal., 1991), to red blood cells (Okudaetal., 1981), and to collagen complexes (Naito and Gibbons, 1988). By analogy with other bacteria, fimbriae on the surface of P. gingivalis may be involved in certain of these adherence phenomena. Isogai et al. (1988) showed that a monoclonal antibody directed against the fimbrillin of P. gingivalis strain 381 blocked adherence to buccal epithelial cells. Similarly, it was demonstrated that fimbriae play a direct role in the adhesion of P. gingivalis toActinomyces viscosus (Goulborne and Ellen, 1991). Lee et al. (1991a) presented evidence which indicated that P. gingivalis adherence to saliva-coated hydroxyapatite was mediated by fimbriae. It has recently been shown that mutants of P. gingivalis strain 381 that lack fimbriae do not bind effectively to saliva-coated hydroxyapatite (Malek and Dyer, unpublished). This indicates that P. gingivalis fimbriae may be important for adherence to the pellicle-coated tooth surface. These fim- mutants hemagglutinate normally and co-adhere effectively with Streptococcus sanguis G9B, which suggests that fimbriae may not be important for these other adherence events. P. gingivalis fimbriae are highly immunogenic. Gnotobiotic rats, immunized with either whole P. gingivalis cells or with partially purified fimbriae, develop high anti-fimbrial antibody titers (Klausen et al., 1991). Immunization of these rats with fimbriae purified from P. gingivalis strain 381 blocks the progression of experimental periodontal disease caused by the homologous strain (Klausen et al., 1991). This suggests that purified fimbriae may be a useful immunogen for vaccination to block or reduce the severity of periodontal disease. However, the degree of conservation of epitopes on fimbriae among the strains in the natural P. gingivalis population is currently unknown. In another study, Lee et al. (1991b) demonstrated size and antigenic heterogeneity of the fimbrial subunit in a survey of 24 P. gingivalis strains. Based on the sequence of the first 20 amino acids at the N-terminus, these investigators identified four fimbrial groups (Lee et al., 1991b). However, they were unable to purify fimbriae from P. gingivalis strains W50, W83, and AJW5 (Lee et al., 1991b). Moreover, these strains were not immunoreactive with fimbrial antisera (Lee et al., 1991b). Thus, the purpose of the present study was to examine fimbrial heterogeneity at the genetic level. The fimA gene encoding the major fimbrial subunit protein of P. gingivalis strain 381 has been cloned and the nucleotide sequence determined (Dickinson et al., 1988). The availability of this clone provided the necessary reagents for a survey, by restriction fragment length polymorphism (RFLP) analysis, of a collection of 39 P. gingivalis strains. RFLP analysis is a powerful tool for the investigation of small differences among closely related chromosomal segments in different bacterial strains. RFLP patterns can be used to group strains and have been used to follow individual isolates in epidemiological surveys. The variation in location ofrestriction sites within a gene locus among a collection of strains can be used as a measure of DNA sequence heterogeneity, and the distribution of enzyme sites is often indicative of conserved and variable regions in homologous loci. Finally, RFLP analysis can indicate the existence of multiple gene copies. In the present study, purified DNA fragments from the cloned fimA 381 locus were used as probes for analysis of RFLPs in and around the fimbrial locus. The objectives of the present study were: (i) to investigate whether all

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LOOS & DYER

Strain Human Porphyromonas gingivalis 381* 9-14K-1** 17-5** 19A4 23A4

34-4* 295-1 817H

A7A1-28** AJW1

TABLE ORIGINS AND SOURCES OF STRAINS Origin (site, disease status) Source subgingival, periodontitis ,,

A ,, ,, ,, ,, ,,

SUNYAB collection' J.J. Zambon, Buffalo, NY L. Wolff, Minneapolis, MN D. Mayrand, Quebec City, Canada

L. Wolff, Minneapolis, MN J. Carlsson, Umea, Sweden S. Edwardsson, Malmo, Sweden J.J. Zambon, Pima Indian Res., AZ A.J. vanWinkelhoff, Buffalo, NY

AJW2 AJW4** AJW5

,,

BH18/10**

G.H.W. Bowden, Winnipeg, Canada ,. ,, B.G. Loos, Pima Indian Res., AZ ,. ,, Sunstar Inc., Osaka, Japan

,, ,,

BH6/26 CLN16-6-4 CLN17-6-1 ESO-75 ESO-127 ESO-132 FAY19M-1** HG564 HG565 JKG6 JKG7**

,, J.J. Zambon, Buffalo, NY ,, A.J.vanWinkelhoff, Amsterdam, The Netherlands ,. , W. Loesche, Ann Arbor, MI

OMG406 OMG434

,, G. Dahl6n, Kenya, Africa ,. ,, D. Mayrand, Quebec City, Canada , J.J. Zambon, Buffalo, NY ,, M. Lantz, Birmingham, AL abscess, periodontitis A.J. vanWinkelhoff, Amsterdam, The Netherlands saliva, periodontitis A.J. vanWinkelhoff, Buffalo, NY sinus tract, root canal infection J.A. Herweijer, Buffalo, NY root canal, root canal infection ,, root canal, root canal infection G. Sundqvist, Umeat, Sweden unknown, "clin. specimen" SUNYAB collection

RB22D-1** THUR28BM-2** W12** HG445** AJW3 4ZR6-13 EM-3** JBB-c W50** Animal Porphyromonas gingivalis

Chat2 (cat) Chien5B (dog)** TG1 (sheep) Bacteroides salivosus 157 (cat)

supragingival, gingivitis ,. subgingival, periodontitis

D. Mayrand, Quebec City, Canada .. K. Frisken, Dunedin, New Zealand

abscess, oral infection

D. Love, Sydney, Australia

Bacteroides macacae

33141 (monkey) subgingival, induced periodontitis American Type Culture Collection a Individual who isolated this strain and geographic location of isolation. bStrain collection from the Department of Oral Biology, State University of New York at Buffalo. * Non-invasive or ** invasive strain in mouse abscess model (Neiders et al., 1989; Chen et al., 1991; P.B. Chen, unpublished data). Downloaded from jdr.sagepub.com at UNIVERSITE DE MONTREAL on June 26, 2015 For personal use only. No other uses without permission.

ANALYSIS OF fimA OF P. gingivalis Vol. 71 No.FNLP 5

1175

strains in the present collection have a fimA allele at the same chromosomal location; (ii) to determine whether there is a common RFLP pattern; (iii) to correlate RFLP patterns or groups of patterns with the four fimbrial types proposed by Lee et ac. (1991b), (iv) to investigate the degree of homology ofthe diffrentf nmA genes with (v) to search for homologous finmA DNA regions within the firnA,; P. gingwalis chromosome located outside the firmA locus; (VI) to determine the usefulness of RFLP analysis with finiA for epide biological purposes; and (vii) to determine whether Bacteroides nacacae and B. salivosas have firmA homologous sequences These latter black-pigmented, asaccharolytic, anaerobic coccobacilli were not previously tested by Dickinson et at. (1988).

Materials and methods. Unless otherwise stated, all chemical reagents were purchased from Sigma Chemical Co., St. Louis, MO. Bacteria and culture conditions. -The 39 strains ofR Pgngiwalis

examined in this study are listed in the Table. Each human isolate was obtained from an individual dental patient with either periodontitis or an infected root canal. Each strain was speciated bythe investigator supplyingthe strain. Upon receipt, the bacterial cultures were tested for purity, production of non-fluorescent, black-pigmented colonies on blood agar plates (Slots and Reynolds 1982), and for an API ZYM profile characteristic of R gingivalis (Analytab Products, Plainview, NY) (Slots, 1981 Laughon et at., 1982). Several A gingivats strains isolated rom animals were included in this study, as well as a strain of Bacteroide macacae and a strain of B. salivosus The virulence of several strains has been measured in a mouse abscess model of ivvasive infection (Neiders et at. 1989; Chen et at., 1991; P.B. Chen, unpublished data). Invasive or non-invasive strains are identified with asterisks in the Table All strains were obtained from frozen or lyophilized stocks and maintained on enriched blood agar plates as described by Loos et at. (1990). Cultures were incubated at 37C in an anaerobic chamber (Forma Scientific Marietta, OH) in an atmosphere of 5% CO 10% and 85% N . H2, Southern blots Total genumic DNA was isolated as desenbed previously (Loos et at., 1990). This isolation method did not separate genomic DNA fom potentially endemic plasmids. However, P. gingivalis plasmids were not identified in two independent

250

SI

II

I.

4

S

studies (Dickinson et a.t, 1987; Sako et aL, 1988), which used some of the same strains examined in the present study. The restriction

bpa M

HP a

A TH Is

a

a

Pp ]a

HAT

A A

HP

la,,,a~

H3-

Sa;

Fig 2-Souther blots ofAuI digest hybridized with Probe I (panels A and B). Strain 381 (lane ) yielded Ala! fragments of 747, 225, and 90 bp indicatedd at the left the Fig.). Panel A (four h of exposure) shows a non5y hybrdizingwlngeA alfmntrtncachstrain, varyinginsizfrefm about 890 bp down to 500 bp. Panel B is a longer exposure (48 h) and shows the smaller Alul fagments. Strain lane 1, 381 lane 2, dKG6, lane 3, 0M0406; lane 4, AJWI lane 5, AJW3; lane 6 AJW4; lane 7, CLN16-6-4 lane 8, NAl28;lane 9 THUR28BM-2;lare 10 34-4 lane 11, 23A4 lane 12 BH6 26; lane 13 0MG434, lane 14, Chen5B; lane 15 JBBc; and lane 16, CLN176-1. Roman numerals idicate the RELP group to which each strain was assigned (see Fig. 3).

HK2

Probe I

S

Probe 2

I11

Probe 3

S

P p I I III

Probe 4

III

SK1

Fig. I Trobes used in this study. Restriction enzymes are identified by the ollowing abbreviations 8 Sacl HP, HinPI, A, Alau; T, Tql; H, Hincld and P, PtI. The cross-hatched line indicates unsequenced DNA, while the solid lines represent DNA whose sequence is known (Dickinson et at., 1988). The double line indicates the open leading fame offirtA 3 aid the arrow indicates direction of tanscription. Probes 1-4 ar indicated below the original clone. It should be noted that no information is currently available regarding the fimA flanking sequences. Recent preliminary data have demonstrated that the first 200 bp ofthe upstream unsequenced flanking region are part of the distal portion of a 1-2-kb gene that encodes a 63-kDa protein of unknown function in anE. coli expression system (Yoshimura and Takahashi, 1991). The downstream flankingsequence does not seem to contain any coding sequence (Yoshimura and Takahashi, 1991). Downloaded from jdr.sagepub.com at UNIVERSITE DE MONTREAL on June 26, 2015 For personal use only. No other uses without permission.

1176

J Dent Res May 1992

LOOS & DYER

RFLP GROUP I III

RFLP PATTERN

STRAIN

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Fig. 3-Summary of data on RFLP analysis. This is a computer-drawn Fig. depicting RFLP patterns for all strains ofP. gingivalis. The patterns were derived from multiple Southern blots of AluI digests that were hybridized with Probe 1 (fimAA381 internal coding sequence). The major AluI fragment is depicted as a larger, filled band, and was used to group the strains (RFLP Groups I-IX). Solid bands indicate strong homology, and cross-hatched bands indicate weak homology. MinorAlul fragments are indicated by smaller unfilled bands. Very weak bands were observed in strains of RFLP Group VIII, indicated by horizontal lines. The sheep P. gingivalis strain in Group IX had no detectable homology with Probe 1.

endonucleasesAluI and PstI were used under the conditions recommended by the manufacturer (Promega Corp., Madison, WI) for digestion of the genomic DNA preparations to completion. DNA fragments were separated on 1.2% (AluI digests) or 0.7% (PstJ digests) agarose gels in a Tris-acetate-EDTA (TAE, 40 mmol/L Tris, 66 mmolJL glacial acetic acid, 1 mmol/L EDTA) buffer containing 0.5 pg/mL ethidium bromide (Maniatis et al., 1982). Gel electrophoresis was performed in 12 x 20 x 0.7-cm gels at 2 V/cm overnight. Electrophoresis was followed by photography under UV illumination. To ensure that equal amounts of DNA (approximately 1 jig) were

loaded per lane,

aliquots

of completed

restriction-digest

reac-

tions were used for trial agarose gels until each DNA fingerprint appeared of the same intensity on the photograph. Commercially obtained lambda bacteriophage HindIII andEcoRI restriction fragments were used as size markers (Boehringer Mannheim Biochemicals, Indianapolis, IN). After electrophoresis, the DNA fragments generated by the PstI digestion were transferred to 0.45-pm-pore nylon membranes (Zetabind, CUNO, Life Sciences Division, Meriden, CT). Prior to transfer, the DNA was depurinated and denatured in the agarose gel, as described by Medveczky et al. (1987). The DNA fragments were then transferred to the membrane by means of a vacuum blot apparatus (VacuBlot, American Bionetics, Hayward, CA.; cur-

rently available as MilliBlot-V, Millipore, Bedford, MA) (Medveczky et al., 1987). A vacuum of 8.5 x 104 Pa was applied for 30 min, and

fresh neutralizing buffer was used forthe transfer. The membranes were air-dried and subsequently baked in an oven at 800C for two h for immobilization of the DNA. In preliminary experiments, the smallAluI fragments ofgenomic P. gingivalis DNA digests were retained more effectively on nylon membranes with a 0.1-pm pore size (Nytran, Schleicher & Schuell, Inc., Keene, NH). Therefore, the AluI digests were analyzed after Southern transfer to Nytran membranes. Briefly, DNA fragments were blotted by capillary transfer (Maniatis et al., 1982) for 20 h in alkaline buffer (0.4 mol/L NaOH in 0.6 mol/L NaCl). The membranes were further processed according to the protocols from the manufacturer (Schleicher and Schuell, 1987). Preparation of DNA probes. -DNA probes consisted of purified DNA fragments from the cloned fimA381 locus in pUC13Bg12.1 propagated in the Escherichia coli host JM83 (Dickinson et al., 1988). Plasmid pUC 13Bgl2.1 DNA was extracted and purified by lysis with alkali followed by cesium chloride-ethidium bromide gradient centrifugation (Maniatis et al., 1982). The plasmid was then cleaved with restriction enzymes (Promega Corp.), and specific fragments were purified from agarose gels by electro-elution (Maniatis et al., 1982) in MWCO 6000-8000 dialysis tubing (Spectrum Medical In-

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V61V.No 71 N. 5

1177 ANALYSLS OF fimA OF P ir rivali7

Fig. 4Southern blot ofAlul dige ts hybrdized with Prob1 (p ls A and B) or v Prh 2 (panel C) Strain 381 (lane 1) yielded AMl framents lef fte g.). nel A (eight h of ros ) shFws Xt RFLP GroupVJI ai (laes 15) did no bybrJi a appreciably with Probe 1 Panel B is a longer exposure (48 h) d shows weakly bybridzingAll fragments for RFLP Group VIII strains. Panel C (eight s panel C show h of exposure) shos the same SontFern blot a panels A and B ftera second Fybridizain with Probe 2 h RLP Group VIII str in strongly hybridizig AlM fragments f about kb and weaker hybridizing fra rents o about 225 bp The arrow in panel(md cates an artifact this radiolucent spot is located between lanes ard was not observed on simiar blots. Strains lane 1, 381; lane 2, W50 lane 3, W12, lane 4 9 14K1; lan 5, AJW5 and iane 6, HG564. dustries, Inc., Los Angeles CA) filled with TAL buffer e elued turned probe to the pr-hyoridizanon solution 0 cpnvmLj the blots DNA was then purified from the TAE buffer by chromatography on a were incubated at 65 C for 16h. The filters we'e washed first at 65 C with 500 mL of a high-salt NACS PREPAC column under conditions recommended by the buffer (4X SSC, 0.1% S)D Subsequet wash steps were performed manufacture (GIBCO BRL, Gaithersburg MD Probe DNA fragments were radiolabeled with ot3P-dCTPby nick at 65 C with various salt concentrations to control the stringency of translation as described by the manufacturer (Promega Corp. The probe hyb 'dization to target DNA sequences Lowstringency P labeled DNA wias separated from unincurporated d32wdCTP by washes wer perftnmed vith 4X SSC (01% SDS,) moderatestrinchromatography on a 25 x 05-cm column of G 50 Sephadex equili gency washes with 1X SSC (0 1% SDS) and high stringency washes bratedwithcolumnbuffer(0IXSSC 0.1%SDS I mmo EDTA20X with 0 1X SSC (0. 1% SDS. Washed filers were dried and sujected SSC contained 3 moL NaCI and 0.33 mof sodium citrate [pH 7.01.) to autoradiography. EAstmates of levels ofhomology It was anticipated that a low These probes had specific activities of3-10 x 10 cpm/gof DNA. H ybridizationsofslulgnomic dgestsThe Nytrnmemranes stringency, Probe I containing the internal coding sequence of were pre-hybridized and hybrdized according to te prtocs d- firA4 woudd hyhndize to riA sequences from a1 srains, ern scribed by the manufacturer (Schleicher and Schuel 1987) exept hen he was considerable sequence vaaton. However at high that 10% dextran sulfate and 05% SDS were included in these strngercy this probe would hybridize only to/fA sequences with a solutions. Labeled, heat-denatured probe was added at 10 cpmper high degree f homology to fimA,,. For ach of the stringency mL of hybr'dization solution After 22 h of incubation i a making washes he T (melting temperature) was calculated according to waterbath at 42 C, the blots were removed from the sealed plastic the following formula (Meinkoth and Wahl 1984)' bags and successive washes were performed as recommended by the 1 81.51C + 16.6 log + 041 (% G + C 0S1hO -61 (% fo'mamide) manufacturer (Schleicher and Schuell, 1987). The final wash was TM perfumed in IX SSPE 0.5% SDS fr 30 in at 65C (IX SSPE contains 0.18 mol/L NaCI, 10 mmoL NaPO 1and I rmol/L EDTA where M the ionic strength (in mol/L) of the washing solution, [pH 7.77)* The washed filters were blotted for removal of excess % G + C = base composition of the probe (44.8% for the 855-bp buffer sealed in Saran Wrap, and exposed to Kodak XAR5 film from encli fragment [Probel), and n = shortest chain in the hybrid fur to h at -70 C with an intensifing screen duplex (the average length of Probe 1 after nick translation is Hy~bridizations of Nst genomic digsts.--The Zetabmnd memrc assumed to be 500 bp [Promega Corp.]). No foriamide was used brakes were washed for two h at 65 C in 500 mL of a O 1X SSC and in any of the washing solutions. Phe T fotr Probe 1 annealing to 0.5% 5DS solution. The blots were then hybridized in a blot-process* target sequences in the hybridizations to the AluI digests (1X ing system (OmmBlot, American Bionetics Inc. Hayward, CA cur SSPE, 0.5% SDS stringency wash) was calculated to be 87.6 C. rently availed as MilbBlot-BP Millipore Bedford, MA). The blots The T decreases by IC wi1t every 1% ofbp which is mismatched were incubated in a pre-hybridization solution of 2XDenhardts (5OX (Meinkoth and Wahl, 1984). Thus, it was calculated that auto Denhardts contains 1% Ficol, 1% polyvinylpyrrobdone, and 1% radiographic signals detected in these blots would correspond to bovine serum albumin), 4X SSC, 0.1% SDS 10% dextran sulfate and at least 77% sequence homology between Probe I and target 100 gg/nL denatured, sonicated salmon sperm DNA at 65 C in a sequences. Similarly, hybridizations of Probe 1 to PstI digests had shaking waterbath for two h. After addition of labeled, hea-dena- TM values of 98.9C (low stringency) 88.9 C (moderate strin-

f747, 225, ad 90 bp indicatedd

-

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LOOS &DYII a19 WnfR J Dent Res A14 1992

scription-translation system from E roli, These investigators sug-

gested that the first 200 bp of the upstream, unsequenced flanking

region contain the 3 end of a 1.2-kb gene that encodes a 63-kDa protein

of unknown function, while the downstream flanking sequence did not encode any proteins that could be identified in those experiments (Yoshimura and Takahashi, 199 1). Probes 3 and 4 were also generated from the purified 2.5-kb Sadl frag~ ment (Fig. 1). Probe 3 was the I kb SaclPatIt fragment that included an upstream flanking sequence and the first 257 bp of the S coding sequence. Probe 4 was the 1.5-kb PstI Sacl fragment that included the central region and 3-end of the coding sequence (total, 768 bp) and downstream flanking sequences. AMIJRFL~swithin the finmAcod ing sequence.-In strain 381, an AuIA digest will generate three frag~ments containing the finiA coding sequence (Figs. I and 2) This in~ eludes the 5'- andd3' ends ofthe gene (747 bp and 225 bp, respectively) and a small 90-hp internal frag-

Fig. 5- Southern blots of Pst diaests hybridized within Probe I. Strain 381 (lanes I and 19) yielded PitI fragments of 5.9 and 4.4 kB (indicated at the night of the Fig.) Panel A (23 h of&xposure) shows ateat one strongly hybridizing fragmrent for strains in lanes 2 13 after a amoderate strirngeney wash. For each strain, the PatI fragmernt with hornology to ifhe r regionaof fimA31is indicated with an asterisk. The fragmert idert fled vwith ar arrov in panel A had weak hromology with Probe 1, as seen by dirminiutio orr disappearance after a high-stringency wash, shown in panel B. Strains: lane 1,3S1,lane2,ESO 127,lare3,AJiW2;lane4,EM-3;lane 5,AJW3;lane 6, AJW4, iane 7, BH6/26; lane a, uMG4J4s lane 9u JBB e lane 10o ESu #5; lane 11, Chien5B, lane 12, EAY19M-1, lane 13, 23A4; lane 14,9-14K41lanae 15, AJW5; lane 16, 11564; lane 17, W50, lane 18, TG1l and lane 19 381.

gency), and 73.21C (high stringency). These TM values corre~ sponded to maximum homology levels of 66'e, 76%, and 92%, respectively.

Results. Probes used in thins study. -A 2.5-kb Sad fragment was generated from plasmid pLTC13BgI2.I (Fig. 1). This Sacl fragment was the original fragment of the firnA locus isnlated in. pTIClABgl2.1 (Dickinson et at., 1988). Probe 1 was an 855-bp Hinell fragment that included most of the internal fitnA coding sequence (Fig. 1). This probe was generated by sequential dgstions of the purified Sadl fragment, first with Hincli, followed by digestion with TaqI. This protocol was chosen since the digestion withifrndllyielded the 855-bp Hincll fragment. as well as an upstream 860-bp Sacl-Hiricll and downstream 830-p Hincll-SacI fragment. Digestion of these latter fragments with TaqI reduced the sizes of these fragments so that the 855 bp Hincll fragment (Probe 1) could be easily purified. From these results, it can be deduced that the unsequenced flank-, ing sequences contained TaqI sites, although the location of these sites was not identified. Probe 2 was the 2.5-kb Sadl fragment that included the fimrbrial coding sequence as well as flanking sequences of about 800 bp on each side of the open reading frame (Fig. 1). Recently, Yoshimura and Takahashi (1991) obtained preliminary data on the flanking sequences of fimzA3., using a coupled tran-

Fig. 6--Southern blot of PstI

digests hybridized with Probe 1 and washed at low stringency. Strain 381 (lane 6) yielded PitI fragments of 599and 444kb(indi heated at the right of the Fig.). Weakly hybridizing fragments, are seer in lanes 1-4. The frag~ ment that hybridized weakly to Probe 1 for these strains corresponded to the central region of fir, A ,as was determined with Prob 3 and 4 (data not shown) Molecular weight (in kb) is indicated at the left of the Fig. Strains: lane 1, 9-14K 1,

lane 2,AJWS5 lane 3, HG564; lane 4, W50; lane 5, TG1; and lane 6, 381. ment. of 39 human and animalP. gingwvalis strains had Thirty-three sequences whien vere at least 77%e nomologous with Probe I after a iX SSPE, 0.5% SDS stringency wash at 651C. The majority of th-ese strains had at least one strongly hybridizingAlul fragment in the range of 500 900 bp. Fig. 2A is a representative aurora diograph (four h of exposure) of a Southern blot with 16 strains, including the homologous strain 381. Fig. 2A and similar auto.-

radiogiraphs iuot shuwii demonstrated a miajur fragiient for each strain. The sizes of the majorAlul fragments for each strain were calculated. Based on the similarities or dissimilarities of the size of the major AlM fragment for each strain that hybridized with Probe 1, seven RFLP groups were identified among the 33 strains These data were compiled and are summarized for comparison in Fig. 3, which is a computer-drawsn representation of the Southern blot analyses. Isolates AJW3 and AJW4 had additional fragments at approximately 1 and 1.2 kb that hybridized with Probe I (Fig. 2A, lanes 5 and 6). Probe I hybridized weakly to some strains. For example, strain AJW3 (Fig. 2A, lane 5) did not have as strong an autoradiographic signal as did the other strains shown in tbis Southern hlot. Weak hvhridization with Probe I is depicted in Fig. 3 by the cross-hatching of the major Alu fragment. These fragments could have up to 23% bp mismatches with Probe 1. The approximate 20 fod difference in signal intensity on the autoradiographs between the 747 bp, on the one hand, and 225 bp and 9Obp. on the other, for strain 381 (Fig. 2A) was explained by two factors, First, small DNA fragments (200 300 bp) are retained relatively poorly by membranes (Meinkoth and Wahl, 1984); an attempt was made to circumvent this problem by the use of a nylon membrane (Nytran) with a pore size of 0.1 pm (Schleicher and Schuell, 1987). Second, the rate and extent of the formation of stable hybrids between molecules of Probe l and the much smaller 225-bp and 90-hp fragments are reduced due to poor accessibility of these small target fragments, to long probe molecules (Meinkoth and Wahl, 1984). The average length of Probe I after nick translation is estimated at SOO bp (Promega Corp.). The use of dextran sulfate

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RFLP ANALYSIS OF fimA OF P. gingivalis

Vol. 71 No. 5

in the hybridization mixture during these experiments reduced this problem (Meinkoth and Wahl, 1984). Thus, after additional exposure (48 h) of these Southern blots, the predicted 225-bp and 90-bp AluI fragments of the homologous strain 381 were clearly revealed (Fig. 2B). The majority of the 33 P. gingivalis strains in Groups I-VII also had multiple AluI fragments in this region. Sixteen strains had fragments indistinguishable in size from the 225-bp fragment of strain 381, and eight strains had a 90-bp fragment similar to strain 381 (Fig. 2B and data not shown; summary data compiled in Fig. 3). However, only one strain (HG565) had an AluI RFLP pattern indistinguishable from that of 381 (data not shown). This indicates that the RFLP pattern for strain 381 is not well-conserved in the members ofthis species. Only RFLP pattern 15 (Group V) was encountered more than twice, but the occurrence in only four strains also does not suggest that this RFLP pattern is conserved. When each major and minor fragment was included in the analysis, 21 RFLP patterns were identified among the 33 strains of RFLP Groups I-VII (Fig. 2 [A, B] and data not shown; summarized in Fig. 3). fimA coding sequence divergence in RFLP Groups VIII and IX.Initially, no appreciable hybridization was seen ofProbe 1 to theAluIdigested DNAs of the human strains 9-14K-1, AJW5, HG564, W12, and W50 (Fig. 4A) and the sheep isolate TG1 (data not shown). After prolonged exposure (48 h) of the autoradiographs, very faint bands (relative to strain 381) were observed ofabout 1 kb for strains 9-14K1, AJW5, HG564, W12, and W50 (Fig. 4B). This indicates that these strains have homology only at the extreme limits ofdetection (77%) in these hybridizations. The sheep strain TG1 still showed no hybridization signal after prolonged exposure (data not shown). The five human strains (9-14K-1, AJW5, HG564, W12, and W50) were assigned to RFLP Group VIII, based on the poor homology with Probe 1 (summarized in Fig. 3). The sheep P. gingivalis strain TG1 was assigned to Group IX (no homology to Probe 1). In addition, the animal Bacteroides species (B. macacae and B. salivosus) did not hybridize with Probe 1 (data not shown). Homology with sequences flanking fimA381.-In contrast to the results with Probe 1, Group VIII strains hybridized as strongly with Probe 2 as did the homologous strain 381 (Fig. 4C). All other human P. gingivalis strains included in this study also hybridized strongly with Probe 2 (data not shown). Sheep strain TG1 had one AluI fragment that hybridized weakly with Probe 2 (data not shown). These data suggest that sequences flanking fimA are conserved in strains that have poor homology withfimA coding sequence. Probe 2 did not hybridize to the animal Bacteroides species (data not shown). Homology to fimA38, and location ofcoding sequence divergence in RFLP Groups I-VII -The regions of homology between the fimA381 gene and the fimA alleles of other P. gingivalis strains were determined. For these experiments, PstI genomic digests were used to generate larger fragments of the fimA region in 17 strains of P. gingivalis, most ofwhich demonstrated weak homology to Probe 1 in previous experiments (see summarized data in Fig. 3). There are two PstI sites within the coding sequence ofthe fimA gene (Fig. 1), and this enzyme cleaves the fimA381 locus into three fragments, one of which (18 bp) is too small to be visualized on agarose gels and Southern blots (Dickinson et al., 1988). Before proceeding with this homology experiment, consecutive hybridizations were performed with Probes 3 and 4 to PstI digests ofthe selected strains to identify which ofthe twoPstl fragments corresponded to the 5'- and 3'- ends of thefimA locus. Probe 3 hybridized strongly to a singlePstI fragment ofall selected human P. gingivalis strains, including strains ofRFLP Group VIII, and to two fragments in strain AJW4 (data not shown). Probe 3 also hybridizedweaklyto a singlePstI fragment of strain TG1 (data not shown). Hybridization of these same blots with Probe 4 identified a second homologous PstI fragment in each strain, except strains AJW4 and TG1 (data not shown). This suggests that strain TG1 (RFLP Group IX) is substantially divergent in the flanking 381

sequences.

Strain TG1

may

have

only

weak homology to the

up-

1179

stream flanking regions of fimA381 or may be missing internal PstI sites. Thus, for strain 381, the 4.4-kb PstI fragment includes the 5'region of the gene, while the 5.9-kb PstI fragment includes the 3'portion ofthefimA381 locus. The PstI fragment ofeach strain that was homologous to the 5-region of fimA381 locus is marked with an asterisk in Fig. 5A. At moderate stringency, 12 of 17 strains hybridized to the internal coding sequence of fimA381 (Probe 1), comparably with or slightly weaker than did strain 381 (Fig. 5A, lanes 2-13). After a highstringency wash, a diminution or disappearance was observed of one ofthe PstI fragments of the strains ESO-127, EM-3, AJW3, BH6/26, OMG434, Chien5B, FAY19M-1, and 23A4 (Fig. 5B, lanes 2, 4, 5, 7, 8, and 13; the disappearing fragments are labeled with arrows in Fig. 5A). Thus, it can be seen from Fig. 5 that strains Chien5B (lane 11) and FAY19M-1 (lane 12) had less hybridization to the 5'-region ofthe fimA genes after the blot was washed at high stringency. From TM calculations, it can be estimated that these two latter strains may have up to 24% bp sequence divergence in the 5-region, while the central/3'-region has > 92% homology. By contrast, the region of lower homology (minimal 76%) between fimA381 and strains ESO127, EM-3, AJW3, BH6/26, OMG434, and 23A4 corresponded to the central/3'-region ofthe fimbrial gene of strain 381 (Fig. 5, lanes 2,4,5, 7, 8, and 13). The 5'-region in these strains probably had more than 92% homology with Probe 1. Note that the 4.4-kb PstI fragment of strain 381 was homologous with only 187 bp of Probe 1, while the 5.9kb PstI fragment was homologous with 650 bp of Probe 1. This explains the slight difference in signal strength between the two PstI fragments (Fig. 5). As observedpreviously (Fig. 4), RFLP Groups VIII and IX strains hybridized extremely poorly with Probe 1 (Fig. 5, lanes 14-18). Extreme fimA sequence divergence in RFLP Groups VIlland IX.For determination of the region of the fimA from strains in RFLP Group VIII with the poor homology tofimA381 (see Fig. 4B, lanes 2-6), genomic PstI digests of these strains were hybridized with Probe 1 (internal coding sequence), and the Southern blots were subjected to low-stringency washes. RFLP Group VIII strains showed low homology to Probe 1 under these stringency conditions (Fig. 6, lanes 1-4). For each Group VIII strain, the fragment that hybridized weakly with Probe 1 corresponded to the centrall3'-region of fimA381 (as determined above with Probes 3 and 4). The relative weakness of these fragments suggested that the level of sequence homology was close to 66%. These data indicated that the fimA locus of Group VIII strains diverged fromfimA381 throughout thefimA coding region, but most markedly in the 5-region of the gene (> 34% sequence divergence). Strain TG1 (RFLP Group IX) was substantially divergent in both the firnA coding (Fig. 6, lane 5) and flanking sequences (see above). The additional faint PstI fragments for strain 381 in Fig. 6 (lane 6) are probably the results of partial digestions and degraded DNA fragments that hybridized with Probe 1 under these lowstringency conditions.

Discussion. All P. gingivalis strains testedin the present study proved to possess a finzA allele, as judged by high levels of sequence homology with DNA surrounding thefimA gene. However, extensive RFLP heterogeneity in thefimA locus existed (results are summarized in Fig. 3). This diversity is reminiscent of the extensive genetic heterogeneity previously observed by whole genomic DNA fingerprinting of P. gingivalis (Loos et al., 1990). The strains were distributed into a total of nine RFLP groups, but 25 uniqueAlul RFLP patterns in this collection were identified. This large number of RFLP patterns is also consistent with the heterogeneity for the fimbrillin monomer reported by Lee et al. (1991b). These authors proposed that P. gingivalis strains could be differentiated into four fimbrillin groups based on the amino acid sequence ofthe first 20 N-terminal residues of the fimbrillin monomer. Eighteen strains from that study were

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LOOS & DYER

included in the present study. No general correlations between strain grouping based on RFLP analysis and N-terminal amino acid sequence were found. In the present study, it was demonstrated that fimbrial coding sequences were more commonly variable between strains at the centrall3'-region of the locus. The lack of correlation between RFLP patterns and amino acid sequence groupings may be explained by the fact that the AluI enzyme used for genomic digests has recognition sites in the upstream non-coding sequence and in the more variable 3-regions of the fimA locus, but not in the conserved 5-region of the gene (Fig. 1). Furthermore, there was no correlation observed between RFLP patterns and the invasive properties of selected strains in a mouse abscess model. The majority of strains that have been investigated were invasive when injected subcutaneously into mice (Neiders et al., 1989; P.B. Chen, unpublished data) (marked in the Table). These invasive strains were distributed over RFLP Groups IV, V, VI, and VIII. Thus, the poor homology to fimA381 of strains in Group VIII is probably not unique to invasive strains. Strains 381 (Group IV) and 34-4 (Group V) were the only strains identified as non-invasive in this collection (Neiders et al., 1989; P.B. Chen, unpublished data). Therefore, no conclusion could be reached regarding the relationship of non-invasiveness and RFLP grouping. Fifteen strains demonstrated poor homology to the internal coding sequence of fimA381 (Probe 1) (summarized in Fig. 3). The regions of sequence divergence ofthefimA381 coding sequence to the fimA locus were examined in selected P. gingivalis strains. Six strains-ESO-127, EM-3, AJW3, BH6/26, OMG434, and 23A4 (Fig. 5, lanes 2, 4, 5, 7, 8, and 13k-had poor homology with the 3-region of the fimA381 gene (estimated homology, 76%), but appeared to be highly homologous with fimA381 at the 5'-end ofthe gene. Only two strains, Chien5B and FAY19M-1 (Fig. 5, lanes 11 and 12), appeared to diverge from fimA381 coding sequence in the 5'-region of the fimA gene. These data suggest that fimbrial sequence is more commonly conserved between strains at the 5'-region of the fimA gene. This is consistent with the high degree of amino acid conservation at the Nterminus for FimA isolated from many strains (Lee et al., 1991b). Human strains from RFLP Group VIII had fimA RFLP patterns markedly different from the basic patterns observed in Groups I through VII (compare Figs. 2 and 4). Group VIII strains had a low degree of homology to the internal coding sequence of the fimA381. However, Group VIII strains strongly hybridized with a probe that included sequences flanking fimA381 (Fig. 4C). This indicates that these strains do carry a fimA allele, which probably resides in the same chromosomal location as fimA381. However, there is significant sequence divergence (more than 34%) between the coding regions of the fimA loci of Group VIII strains and fimA381. This is consistent with the lack of reactivity of the strains 9-14K-1, AJW5, HG564, and W50 with fimbrial antisera and monoclonal antifimbrial antibodies (Lee et al., 1991b). The sequence divergence seems most significant in the 5-region of the fimA locus. This suggestion is consistent with the data of Lee et al. (1991b). These authors reported that FimA from strains 9-14K-1 and HG564 differed substantially from that of strain 381 at the N-terminus (in eight of 20 amino acids). Structural conservation in the C-terminus region of FimA suggests that important functional portions of the protein are located in this region of FimA, such as adhesive properties, or interaction with fimbrial-associated proteins. Preliminary data suggest that C-terminal regions of the protein are important for binding to saliva-coated hydroxyapatite (Lee et al., 1991a). Extensive RFLP heterogeneity could be associated with chromosomal DNA re-arrangements that cause antigenic or phase variation. An immune response directed against P. gingivalis fimbriae during periodontal infection could make antigenic or phase variation an advantageous event. Antigenic variation could proceed through recombination or gene conversion, as has been shown in the pathogenic Neisseriae (Meyer and van Putten, 1989). This will produce heterogeneity in RFLP patterns (Segal et al.,

1986). However, multiple fim alleles within a single strain were not observed, which suggests that gene conversion events do not take place. Moreover, although P. gingivalis fimbriae are antigenically diverse between strains (Lee et al., 1991b), intra-strain antigenic variation has never been observed (J.-Y. Lee, personal communication). Phase variation for E. coli type I fimbriae is associated with the inversion of a small promoter-containing 314bp DNA segment (Eisenstein, 1988). Such an inversion could also be associated with a change in RFLP pattern if a restriction enzyme with a recognition site within this region is selected. Although DNA inversions upstream from the fimA gene could cause RFLP heterogeneity, phase variation for P. gingivalis fimbriae has not been observed (J.-Y. Lee, personal communication). Therefore, circumstantial evidence suggests thatfimA diversity is most likely generated through mutation and genetic exchange between P. gingivalis strains, rather than through specialized mechanisms of genetic variation. This is similar to explanations for the observed heterogeneity in RFLP patterns inpap pili-related DNA sequences in uropathogenic E. coli. Genetic variation in the pap genes is thought to be generated through interchromosomal recombination events and/or horizontal transfer of selected sequences (Plos et al., 1989; Arthur et al., 1990). However, the potential variation in fimA has important implications in the

pathogenesis of periodontal disease associated with P. gingivalis. For example, an immune response directed against the FimA protein of a P. gingivalis strain associated with an episode of periodontal disease might not protect against subsequent infection with a new strain of the organism. Previously, it was shown that the cloned fimA381 locus did not have any detectable homology to other Porphyromonas species (P. endodontalis and P. asaccharolyticus) or to the black-pigmented Prevotella species, P. intermedia, P. corporis, P. melaninogenica, P. denticola, andP. loescheii (Dickinsonetal., 1988). In the present study, it was also found that Bacteroides macacae (strain ATCC 33141) and B. salivosus (strain 157) had no homology with Probes 1 or 2, even under low-stringency conditions. In contrast, DNA from P. gingivalis isolated from a cat and a dog was highly homologous to Probes 1 and 2 and could be placed into RFLP groups containing human strains. However, PstI digestions of DNA from these strains yielded two much smaller hybridizing fragments (1and 3-kb), which were not observed in any of the human strains. A PstI fragment of the sheep strain TG1 hybridized with the internal coding sequence of fimA381 (Probe 1) only under lowstringency conditions. Therefore, if additional animal strains demonstrate similar results, RFLP analysis at the fimA locus could be helpful fordifferentiation ofhuman and animalP. gingivalis. Although a limited number (39) ofP. gingivalis strains were tested in the present study, all human strains had homology with flanking regions ofthefimA381 locus. This suggests that flanking regions are more suitable than fimA coding sequences for use as probes in ecological and diagnostic studies involving P. gingivalis. Regions of DNA flanking the fimA locus are common among, and unique to all, human and most domestic animal P. gingivalis strains and differentiate these strains from other black-pigmented Bacteroides. Therefore, these regions of flanking sequence could serve as DNA probes for the detection of P. gingivalis in dental plaque samples. RFLP analysis is often used for molecular epidemiology (Eisenstein, 1990). However, RFLPanalysis ofthefimA locusis notas sensitive as DNA fingerprinting for distinguishing between individual P. gingivalis strains. For example, the RFLP patterns of strains 381 and HG565 were indistinguishable, but these strains were distinctly different by DNA fingerprinting (Loos et al., 1990). In general, strains with the same RFLP patterns proved to be genetically different by DNA fingerprinting, with the exception of strains A7A128 and CLN16-6-4. For ecological and epidemiological purposes, RFLP analysis ofP. gingivalis at thefimA region will not discriminate between strains as effectively as will DNA fingerprinting.

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RFLP ANALYSIS OF fimA OF P. gingivalis

Vol. 71 No. 5

Acknowledgments. We thank Dr. D.P. Dickinson for his generous gift of the recombinant E. coli strain JM83 containing pUC13Bg12. 1. Thanks are also due to the investigators listed in the Table for generously providing many ofthe strains used in this study. Dr. A.J. Lesse performed the DNA sequence analysis of the fimA locus with the GCG Package (version 7) from Genetics Computer Group, Inc., Madison, WI. Finally, Mrs. Rickie Duffy is acknowledged for expert artwork. 381

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