Oral Microbiol Immunol 2000: 15: 1–9 Copyright C Munksgaard 2000Printed in Denmark . All rights reserved

ISSN 0902-0055

Mini-review

E. C. S. Chan, R. McLaughlinFaculty of Dentistry, McGill University,Taxonomy and virulence of oralMontreal, Quebec, Canada

spirochetesChan ECS, McLaughlin R. Taxonomy and virulence of oral spirochetes.Oral Microbiol Immunol 2000: 15: 1–9. C Munksgaard, 2000.

All oral spirochetes are classified in the genus Treponema. This genus is in thefamily Spirochaetaceae as in Bergey’s manual of systematic bacteriology. Othergeneric members of the family include Spirochaeta, Cristispira and Borrelia. Thisconventional classification is in accord with phylogenetic analysis of the spirochetesbased on 16S rRNA cataloguing. The oral spirochetes fall naturally within thegrouping of Treponema. Only four species of Treponema have been cultivated andmaintained reliably: Treponema denticola, Treponema pectinovorum, Treponemasocranskii and Treponema vincentii. These species have valid names according tothe rules of nomenclature except for Treponema vincentii, which only has hadeffective publication. The virulence factors of the oral spirochetes updated inthis mini-review have been discussed within the following broad confines:adherence, cytotoxic effects, iron sequestration and locomotion. T. denticolahas been shown to attach to human gingival fibroblasts, basement membraneproteins, as well as other substrates by specific attachment mechanisms. Thebinding of the spirochete to human gingival fibroblasts resulted in cytotoxicityand cell death due to enzymes and other proteins. Binding of the spirochete toerythrocytes was accompanied by agglutination and lysis. Hemolysis releaseshemin, which is sequestered by an outer membrane sheath receptor protein of

Key words: taxonomy; virulence; virulencethe spirochete. The ability to locomote through viscous environments enables factors; oral spirochetes; periodontitisspirochetes to migrate within gingival crevicular fluid and to penetrate sulcular

E. C. S. Chan, Faculty of Dentistry, McGillepithelial linings and gingival connective tissue. The virulence factors of the oralUniversity, 3640 University Street, Montreal,spirochetes proven in vitro underscore the important role they play in the Quebec, Canada H3A 2B2

periodontal disease process. This role has been evaluated in vivo by use of amurine model. Accepted for publication June 30, 1999

Spirochetes were among one of thefirst bacteria in humans documented.While examining a specimen obtainedfrom a person’s teeth, Antonie vanLeeuwenhoek (1632–1723), in his com-munication to the Royal Society (Eng-land), wrote ‘‘I found an unbelievablygreat company of living animalculusaswimming more nimbly than any Ihad seen up to this time. The biggestsort (whereof there were a greatplenty) bent their body into curves go-ing forward...’’ This probably describedthe first microscopic observation of aspirochete (26).

All spirochetes share gross pheno-typic characteristics. They are helicallyshaped bacteria, but differ structurally

from other helical bacteria, such as Spi-rulina and Spirillum species, by exhibit-ing an unusual morphology. The typicalultrastructure consists of an outersheath (outer membrane or outer mem-brane sheath), protoplasmic cylinderand periplasmic flagella (axial fila-ments, axial fibrils, or endoflagella) in-serted at subterminal locations, andlocated between the outer sheath andthe protoplasmic cylinder. The per-iplasmic flagella wind around the proto-plasmic cylinder and overlap and inter-digitate in the middle of the cylinder.The morphology of a spirochete froman oral source is shown in Fig. 1. It isgenerally accepted that the periplasmicflagella are involved in the characteristic

rotational and flexing movements aswell as translational motility of thespirochetes as observed under darkfieldor phase-contrast microscopy. Spiro-chetes also have the unique ability tolocomote in highly viscous environ-ments that render other prokaryotesimmobile (34, 66). Depending on thespirochete species, the number of fla-gella varies from 2 to hundreds per cell(26). Recently, an extracellular polysac-charide layer has been visualized in theoral spirochete Treponema denticola(68). Other unifying characteristics ofspirochetes are resistance to the anti-biotic rifampin (37) and, excepting theleptospiras, the presence of ornithine inthe cell wall peptidoglycan (30).

2 Chan & McLaughlin

Fig. 1. Morphology of an oral spirochete as seen under the transmission electron microscopeafter negative staining. PF, periplasmic flagella; OM, outer membrane; PC, protoplasmic cylin-der. Scale barΩ500 nm. Courtesy of Robert K. Nauman, University of Maryland, Baltimore.

Association of spirochetes withperiodontal disease

Spirochete diseases are not similar intheir mode of transmission and hostrange, but they share some importantcharacteristics, such as a chronic andepisodic nature and the key role of thehost immune response in mediating thedestructive effects of the bacterial infec-tion (65). Periodontal disease sharessimilar characteristics with the well-known spirochete diseases, for example,syphilis caused by Treponema pallidumand Lyme disease caused by Borreliaburgdorferi. The disease is initiated byan overgrowth of the bacterial popula-tion in the gingival crevice creating con-ditions favoring growth of proteolyticgram-negative anaerobes. Such bacteriaconstitute the complex microbiota ofthe subgingival plaque. The resultinginflammatory response appears to bedue to the action of several bacterialfactors and leads to tissue destruction,loss of attachment at the junctional epi-thelium between the gingiva and thetooth, and alveolar bone resorption.

The disease progresses with the forma-tion of a characteristic deep periodontalpocket between the tooth and the gin-giva coupled with a host inflammatoryresponse as a consequence of bacterialinsult (17, 75). The end result, in the ab-sence of prophylaxis and treatment, isthe loss of the tooth.

More than 300 bacterial species havebeen isolated from the subgingivalplaque of patients with severe peri-odontitis (47, 74). Spirochetes can rep-resent up to 50% of the detectable biotain subgingival plaque from patientswith acute necrotizing ulcerative gingi-vitis and chronic adult periodontitis (2,39), whereas they comprise less than 1%in healthy sites (38, 39). The associationbetween periodontal disease and oralspirochetes has now been shown to bemore than circumstantial. Simonson etal. (72) used a monoclonal antibodyspecific for T. denticola to present thefirst quantitative evidence of a positiverelationship between a spirochete spe-cies and severe periodontitis.

This review has the objective of ra-tionalizing the state of taxonomy of the

oral spirochetes (treponemes) of peri-odontal disease and to update brieflytheir putative virulence factors.

Taxonomy of oral spirochetes

The meaning, or definition, of tax-onomy as used in this review includesthe classification (arrangement), no-menclature (naming) and identification(description and characterization) ofmicroorganisms, specifically the oraltreponemes (54). The reader is re-minded that there is no official classifi-cation of bacteria, but that there is avalid nomenclature (names validly pub-lished as a result of conformity with theRules of Nomenclature) (15). The newstarting date for bacteriological no-menclature is 1 January 1980, when theApproved Lists of Bacterial Nameswere published in the InternationalJournal of Systematic Bacteriology (73).The International Code of Nomencla-ture of Bacteria (1990 Revision) is theaccepted authority of bacterial no-menclature, and it is an official publi-cation of the International Committeeon Systematic Bacteriology (36).

Only four species of oral spirocheteshave been cultured widely and reliablyby many laboratories. These are T.denticola, Treponema pectinovorum,Treponema socranskii and Treponemavincentii. Although ultrastructural evi-dence indicates that there are at least adozen morphotypes of oral spirochetes,some of which may play a role in thepathogenesis of periodontal infections(7). In more recent studies, an unclassi-fied, as-yet-uncultivable spirochete, aso-called pathogen-related oral trepon-eme (cross-reacts with monoclonal anti-bodies raised against T. pallidum) hasbeen found in periodontal diseaselesions (58, 59). Other studies describedthe isolation and characterization ofadditional oral spirochetes includingTreponema medium (80), Treponemamaltophilum (84) and Treponema amyl-ovorum (85). But these three new iso-lates are not listed in the catalogue ofthe American Type Culture Collection(ATCC) and are not available. Speciesof oral treponemes with accession num-bers and available from the ATCC cata-logue (www.atcc.org) are only the fol-lowing species: T. denticola, T. pec-tinovorum, T. socranskii and T. vin-centii.

Bergey’s manual of systematic bacter-iology, volume 1, lists only the followingthree oral spirochete species: T. dentico-

Taxonomy and virulence of oral spirochetes 3

la, T. vincentii and Treponema scoli-odontum (35). The prokaryotes, 2ndedn., lists two additional species: T. soc-ranskii and T. pectinovorum (46). TheList of Bacterial Names with Standingin Nomenclature (http://www-sv.cict.fr/bacterio/search.html) includes someoral spirochetes. The appearance of aname on this list simply means that thename has been validly published ac-cording to the rules of nomenclatureand is therefore valid (15). The oralspirochetes listed are: Treponema amyl-ovorum, T. denticola, T. maltophilum, T.medium, T. pectinovorum and T. socran-skii. Note that T. vincentii is not a validname even though it has had effectivepublication through the years. Simi-larly, T. scoliodontum is not a validlypublished name. It is apparent thatBergey’s manual published in 1984 issketchy in its treatment of the oralspirochetes. In any case, one importantgoal of the Manual is to assist in theidentification of bacteria, and anothergoal is to indicate the relationships thatexist between the various kinds of bac-teria. However, the latter goal has notbeen realized due to paucity of work inmolecular biology in many taxa. Thusin the present edition, traditionalcharacteristics instead have been usedextensively in arranging bacterial re-lationships.

Comparison of traditionalcharacteristics of cultivable andavailable oral spirochetes

The four species of spirochetes thathave been cultivated reliably and avail-able from the ATCC are the following:T. denticola, T. pectinovorum, T. socran-skii and T. vincentii. A comparison oftheir traditional characteristics is sum-marized in Table 1, modified fromUmemoto (80). The collection of suchdata for taxonomic arrangements alongconventional microbiological methodshas been hampered in several ways inthe case of oral spirochetes: these tre-ponemes are difficult to cultivate anddifferentiate (3), many species are slow-growing and variability exists amongstrains of a single species in physiologi-cal response to nutrient substrates (84).

Since it is acknowledged that there isdifficulty in collecting conventionalcharacterization data (such as sugarfermentation, metabolic end-productanalysis, etc.), some novel approacheshave been introduced recently to differ-entiate the group arrangements in place

Table 1. Differential characteristics of human oral Treponema species

Characteristic T. denticola T. pectinovorum T. socranskii T. vincentii

Cell length (mm) 6–16 7–15 6–15 5–16Cell width (mm) 0.15–0.20 0.28–0.30 0.16–0.18 0.20–0.25No. of periplasmic flagella 2–3 2 2 4–6Fermentation of:

Glucose ª ª π ªFructose ª ª π ªLactose ª ª ª ªMaltose ª ª π ªMannitol ª ª NR ªMannose ª ª π ªGalactose ª ª π ªStarch ª ª NR ªSucrose ª ª π ªRibose ª ª π ªXylose ª ª π ª

Hydrolysis of:Esculin π ª ª ªb

Gelatin π ª ªb ªProduction of:

Ammonia π π π πIndole ªb ª ª πHydrogen sulfide π ª π π

Fatty acids produced A, l, s, p, f A, F, p, l A, L, S, f A, B, l, sGπC content (mol%) 37–38 39 50–52 44

NR, not reported.b Some strains were positive and some were negative.A, acetic acid; B, n-butyric acid; F and f, fumaric acid; L and l, lactic acid; S and s, succinicacid; p, propionic acid. Uppercase letters indicate major fatty acids and lowercase lettersindicate minor fatty acids.

in volumes like Bergey’s manual. Onemethod is the use of capillary zone elec-trophoresis, separating metabolic prod-ucts from a liquid medium. Electro-pherograms were obtained after capil-lary zone electrophoresis analysis thatdifferentiated between T. denticola, T.pectinovorum and T. vincentii as well asstrains of T. socranskii. This techniqueshowed high resolution and good repro-ducibility and may serve as a valuabletool in the chemotaxonomy of oral tre-ponemes (3).

Another technique advocated forrapid identification of cultivable oraltreponemes is restriction fragment-length polymorphism (RFLP) analysisof 16S ribosomal RNA genes amplifiedby polymerase chain reaction (PCR).Using this technique, Sato & Kuramitsu(64) generated restriction profiles of ref-erence strains of oral treponemes in-cluding T. denticola, T. socranskii, T.vincentii, T. pectinovorum and T. me-dium as well as strains isolated from hu-man periodontal pockets. They foundthat this technique was a rapid andsimple method to differentiate com-pletely these species.

One method that probably is not asrapid as the recent techniques described

above but has been used by many oralmicrobiology workers in confirmingidentification of treponeme isolates hasbeen DNA-DNA hybridization. For in-stance, Chan et al. (6) used the tech-nique to determine the DNA homo-logies of five freshly isolated species oforal treponemes from periodontalpocket with that of reference strains ofT. denticola, T. vincentii and T. socran-skii. Clearcut identification of the un-known isolates were obtained. Umemo-to et al. (80) used DNA-DNA hybridi-zation to distinguish T. medium fromother Treponema species (T. denticola,T. vincentii, T. socranskii, Treponemaphagedenis and T. pallidum). Other con-tributions in this area were by Olsen etal. (50) and Fukumoto et al. (20).

Ribosomal RNA analysis: anothertool for the taxonomy of spirochetes

The simple morphology of most mi-crobes provides few clues for theiridentification, and physiological traitsare often ambiguous (49). This is espe-cially so in the case of the spirochetes.Some workers have advocated that themost fundamental and straightforwardway to classify and relate organisms is

4 Chan & McLaughlin

by appropriate nucleic acid sequencecomparisons (49). Studies comparing c-type cytochromes, globins and othercommon proteins have been rewardingamong higher eukaryotes (21), but sinceprokaryotic and eukaryotic microbesare so phylogenetically and biochem-ically diverse, identification of homo-logous proteins can be difficult (13).rRNA analysis is used for mappingphylogenetic relationships for severalreasons (76):

O The rRNAs, as key elements of theprotein-synthesizing machinery, arefunctionally and evolutionarilyhomologous in all organisms.

O The rRNAs are ancient moleculesand are extremely conserved in over-all structure. Thus, the homologousrRNAs are readily identifiable bytheir sizes.

O Nucleotide sequences are also con-served. Some sequence stretches areinvariant across the primary king-doms, while others vary. The con-served sequences and secondarystructure elements allow the align-ment of variable sequences so thatonly homologous nucleotides are em-ployed in any phylogenetic analysis.

O The rRNAs provide sufficient se-quence information to permit statis-tically significant comparison.

O The rRNA genes seem to lack arti-facts of lateral transfer between con-temporaneous organisms. Thus, re-lationships between rRNAs reflectevolutionary relationships of the or-ganisms.

The 16S rRNAs have been used formost rRNA-based phylogenetic char-acterizations. This 16S rRNA gene is ofappropriate size for broad phylogeneticanalyses and contains sufficient varyingnucleotide positions (49).

Phylogenetic analysis of thespirochetes

By comparison of the sequences of the16S rRNA or the genes coding for the16S rRNA (rDNA), the spirochetes aredivided into two major phylogeneticgroupings (12, 27, 52). The first group-ing, which corresponds to the familySpirochaetaceae, contains species of thegenera Treponema, Spirochaeta, Borrel-ia, Serpulina (and Brachyspira) and Bre-vinema. The second grouping, whichcorresponds to the family Leptospira-ceae, contains species of the genera Lep-

tospira and Leptonema. To date all oralanaerobic spirochetes are members ofthe genus Treponema.

The spirochetes are one of the fewmajor bacterial groups whose naturalphylogenetic relationships are evident atthe level of gross phenotypic character-istics (82, 83). The phylogenetic struc-ture determined from 16S rRNA cata-loguing is in approximate agreementwith the accepted classical taxonomyfor spirochetes (35), in which the orderSpirochaetales is divided into two fam-ilies, Spirochaetaceae and Leptospira-ceae, with the family Spirochaetaceaecomprising the genera Treponema (in-cluding Serpula hyodysenteriae and re-lated species), Spirochaeta, Borrelia andCristispira and the family Leptospira-ceae encompassing the genera Leptospi-ra, and Leptonema.

The 16S rRNA approach is especiallyuseful for the study of oral spirochetes,since most of the members present in adeep periodontal pocket are as yet un-cultivable. The phylogenetic identity oforganisms that cannot be cultivated canbe determined by sequencing of cloned16S rRNA genes that are amplified di-rectly from a specific sample (51). Choiet al. (7) collected subgingival plaquesamples (probing depth, .9 mm) froma 29-year-old white female patient withsevere destructive periodontitis. The16S rRNA genes were PCR amplifiedand a 16S rRNA gene library was con-structed. To screen the library, colonyhybridization was done using a trepon-eme specific probe. A total of 95 recom-binants carrying treponeme-specific 16SrRNA sequences were identified. Four-teen clones were contaminated, andseven clones were chimeras (determinedby DNA sequencing) so they were notincluded in the final analysis. A 500-base partial sequence was obtained forthe 81 clones. The clones represented 20new species of Treponemes. One clonemay represent a new genus related moreclosely to leptospira than to the trepon-emes. This finding implies that previousused methods for isolation, cultivationand identification have grossly under-estimated the diversity of spirochetespresent in the oral cavity (7).

Similar experiments were performedon pathogen-related oral spirochetes,which were found in great numbers inthe majority of gingival plaques frompatients with necrotizing ulcerating gin-givitis and severe periodontitis. With asequence similarity of 96.4%, the mostclosely related cultivable treponeme was

T. vincentii. It was concluded by theseresearchers that pathogen-related oralspirochetes constitute a heterogeneouspopulation of treponemes comprisingT. vincentii and T. vincentii–related or-ganisms (8).

By using 16S rRNA gene sequences,a phylogenetic tree for spirochetes wasconstructed for many cultivable species(Fig. 2) (53). The phylogenetic relation-ship of the oral spirochetes T. denticolaand T. pectinovorum to other spiro-chetes are shown in the figure. Evi-dently, the oral spirochetes also fallwithin the same genus Treponema as inclassical taxonomy.

Virulence of oral spirochetes

We are indebted to the availability oftwo recent and valuable contributionson the putative virulence factors of oralspirochetes facilitating the writing ofthis mini-review. In a review article onspirochetes in oral infections, Dahle etal. (11) surveyed the putative virulencefactors of oral spirochetes. In addition,the virulence factors of T. denticola (thebest characterized cultivable oral spiro-chete) were well reviewed, especiallywith reference to particular emphasison highly expressed membrane compo-nents that mediate its adherence, pro-teolytic activity, and cytotoxic effects,by Fenno & McBride (17). The readeris requested to refer to these reviews forfiner details of some topics raised by

Fig. 2. Phylogenetic tree of spirochetes. Thedendrogram was constructed from 1410 basecomparisons. The scale bar represents a 10%difference in nucleotide sequence as deter-mined by measurement of the lengths ofhorizontal lines connecting two species.Source: Paster et al. (53).

Taxonomy and virulence of oral spirochetes 5

these authors since they will not be dis-cussed at length here.

Adherence

The primary niche for oral spirochetesis in the gingival crevice. In order tocause disease, the spirochetes must beable to attach to a substrate, multiplyand express virulence factors. If thebacteria are not able to attach to a sub-strate then they will be washed away bythe gingival crevice fluid. Treponemadenticola has been shown to attach tohuman gingival fibroblasts under bothaerobic and anaerobic conditions. Itwas suggested that the mechanism forbinding to the fibroblasts was lectin-me-diated, with affinity for galactose andmannose on the human gingival fibro-blast surface (81).

It has been shown that most T.denticola strains adhere well to extra-cellular proteins such as fibronectin,which are synthesized by human gingi-val fibroblasts and bind to the plasmamembrane of the fibroblast. In the pres-ence of anti-human fibronectin anti-bodies, adherence to human gingivalfibroblasts was reduced but not fully in-hibited (14).

T. denticola has also been shown toadhere to basement membrane proteinslaminin, fibronectin and type IV colla-gen as well as type I collagen, gelatinand fibrinogen. Enzyme-linked immu-nosorbent assay tests showed increasedattachments to all proteins comparedwith bovine serum albumin. The bind-ing to laminin was most prominent. In-formation about the nature of attach-ment was gathered by preincubating thespirochetes or the substrate proteinswith potential inhibitors of attachment.The sulfhydryl reagent p-chloromercur-ybenzoic acid showed the strongest ef-fect, reducing the attachment of T.denticola cells by 80–90% for each pro-tein. If T. denticola was heat-treated at70æC for 10 min, the attachment to lam-inin was reduced by over 70% and theattachment to fibrinogen reduced 40–50%. The attachment to laminin andfibrinogen was reduced by 50%. Only a20% reduction in the attachment togelatin occurred in the presence ofmixed glycosidase. It appears that T.denticola binds to different kinds ofproteins by using specific attachmentmechanisms in which the binding in-volved protein-SH groups and/orcarbohydrate residues (25).

The major surface protein of T.

denticola is a 53-kDa protein. Mostlikely outer surface proteins are in-volved in adhesion. Outer envelopepreparations of T. denticola were separ-ated by sodium dodecyl sulfate–poly-acrylamide gel electrophroresis (SDS-PAGE) and transferred to nitrocellulose.After exposure to many mammalianproteins it was determined that laminin,fibrinogen and fibronectin bound to the53-kDa protein. Other T. denticola pro-teins ranging from 75 to 95 kDa boundsome of the mammalian proteins. How-ever, since these proteins are not foundsurface-exposed in the outer sheath of T.denticola, they were not considered im-portant in adherence (24).

In some strains of T. denticola theouter membrane-associated chymotryp-sin-like protease complex is highly ex-pressed. This complex is localized to theouter membrane sheath. On SDS-PAGE, the chymotrypsin-like proteasecomplex (when not heated) migrates asa proteolytic active doublet with a mol-ecular mass of 95 kDa. If the complex isheated, the complex separates into threefragments. The largest fragment is 72kDa, and the two smaller fragmentssizes have been reported as 27 and 23,39 and 32 or 43 and 38 kDa (17). Thechymotrypsin-like protease complex isbelieved to be involved in T. denticolaadherence to epithelial cells by becom-ing associated with 53-kDa major outermembrane protein. The 53-kDa proteinis thought to integrate with the plasmamembrane of the host cell and functionin the transport of T. denticola surfacecomponents into the cells (78). Thecytotoxicity of the chymotrypsin-likeprotease complex will be discussedbelow.

T. denticola has been shown to bindto hyaluronan. Hyaluronan is a high-molecular-weight polysaccharide com-posed of repeating units of glucuronicacid and N-acetyl--glucosamine. Thispolysaccharide is very abundant in epi-thelium and soft tissue. The stratifiedepithelium of the oral mucosa containshyaluronan, which forms a highly hy-drated gel maintaining the intercellularspace to allow the diffusion of nutri-ents. The ability of T. denticola to bindto this molecule may give the bacteriaan ecological advantage since hyalu-ronidases probably act to break downthe polysaccharide. The bacteria arethought to bind to hyaluronan via thechymotrypsin-like protease complex.Since phenylmethysulfonyl fluoride,periodate oxidation, heat and p-chloro-

mercurybenzoic acid inhibited bindingand also inhibited protease activity,it is believed that protease is involv-ed in binding. As well, in binding as-says, purified chymotrypsin-like pro-tease complex bound to hyaluronan (23).

Cytotoxic effects

Adherence of oral spirochetes in thegingival crevice is not itself a patho-genic process. It is only when the cyto-pathic effects of the adherence andcolonization of the bacteria come aboutthat initial colonization can be con-sidered a virulence factor. Therefore, ifthe bacteria benevolently adhere to asite resulting in no damage to host cells,colonization is not a virulence factor.Ellen et al. (14) have reported the cyto-pathic response of human gingivalfibroblasts upon incubation with T.denticola. The effects seen in the humangingival fibroblasts are the following:the plasma membrane blebs, folds andis grossly rounded, F-actin rearrange-ment into a perinuclear array, cell de-tachment from the substratum and re-duced cell proliferation and cell death(14).

Grenier (22) has shown that T.denticola is able to agglutinate and lysered blood cells. This process involvestwo steps. The first step is bacterialbinding to the erythrocyte using a -glucosamine-like-containing compo-nent and the second step results in dam-age to the erythrocyte membrane (22).A 46-kDa hemolysin produced by T.denticola has been studied. The gene re-sponsible for encoding this protein isthe hly gene. The entire hly gene wascloned and transformed into Escherich-ia coli. Both hemolysis and hemoxid-ation was seen when the E. coli cellscontaining the cloned hemolysin wasgrown on sheep blood agar plates.Interestingly, the deduced amino acidsequence from the hly gene was nothomologous to the sequence of otherhemolysins in the protein databases (9).

A second enzyme isolated from T.denticola that was able to cause hemo-lysis of sheep erythrocytes is a chymo-trypsin-like protease. The molecularmass of this protein, which is encodedby the prtB gene, is 30.4 kDa. Becausethis enzyme is affected by the proteaseinhibitors phenylmethylsulfonyl fluor-ide, diisopropylfluorophosphate and N-tosyl--phenyalanine chloromethyl ke-tone, it was concluded that it acted as achymotrypsin-like protease. This pro-

6 Chan & McLaughlin

tein does not hydrolyze the proteinsfibronectin, type IV collagen, laminin,etc., which would be found in the peri-odontal pocket (1).

The chymotrypsin-like protease com-plex has been shown to have potentcytotoxic effects on epithelial cells.Membrane blebbing of epithelial cellswas seen after cells were treated with T.denticola for 2 h. Similar blebbing wasseen when cells were treated for 2 h with20 mg/ ml of chymotrypsin-like proteasecomplex. The purified protein was alsoable to degrade endogenous pericellularfibronectin in epithelial cells and fibro-blasts. This may inhibit cell adhesionand locomotion of migrating cells. Ar-resting adhesion to fibronectin has beenshown to cause apoptosis of epithelialcells (78). Purified chymotrypsin-likeprotease complex can hydrolyse fi-brinogen, transferrin, a1-antitrypsin,IgA, IgG, gelatin, serum albumin, andlaminin. It can also inactivate substanceP by attacking the Phe-8-Gly-9 bond,convert angiotensin I into angiotensinII and then break down angiotensin IIinto tetrapeptides (44).

The 53-kDa major surface protein,along with functioning as a porin andan adhesin, also exhibits cytotoxic ef-fects. The mechanism by which cytotox-icity is induced is not certain, but pore-forming activity is involved. Translo-cation of bacterial porin-like structuresto eukaryotic cell membranes have beendocumented for several bacteria, in-cluding Salmonella typhimurium, Neis-seria gonorrhoeae and Porphyromonasgingivalis (16). Major surface proteinwas found cytotoxic to epithelial cellsand erythrocytes (16, 17).

Iron sequestration

An important step in the colonizationof the periodontal pocket is the acqui-sition of iron. Scott et al. showed thatT. denticola bound Congo red and he-min via a 47-kDa outer membrane pro-tein (69). Further study revealed that T.denticola had no siderophore activityand did not transport [3H]-hemin intothe cytoplasm. The model for iron ap-propriation is as follows: phospholipaseC and other cell-associated and extra-cellular factors cause hemolysis oferythrocytes, hemin then becomes liber-ated and is trapped by a 47-kDa outermembrane sheath receptor protein. Inaddition, T. denticola can utilize lacto-ferrin, an iron binding protein found insaliva, through the expression of 17-

and 43-kDa outer membrane sheath re-ceptors (67).

Locomotion

The locomotory ability of pathogenicspirochetes is one of the factors associ-ated with their virulence (4, 56). Suchan ability enables pathogenic spiro-chetes to negotiate viscous fluids on themucosal surface and in the inter- andintracellular spaces of the host body.Specifically, in periodontal disease,spirochetes have been found betweencells in the junctional epithelium thatare normally tightly joined (63), and inadjacent epithelium and connective aswell as on the surface of the alveolarbone tissue (5, 19, 59, 61, 62). Rivere etal. (57) have demonstrated that thereare spirochetes present in dental plaquethat are capable of penetrating and mi-grating through the mouse abdominalwall in an in vitro model of invasiveness.

Basic studies on the inherent capa-bility of oral spirochetes such as T.denticola to locomote translationally inviscous fluids have been carried out.The results indicate that oral spiro-chetes are able to locomote through vis-cous environments. However, optimallocomotion was shown to be correlatedwith well-defined viscosity values ofthe medium. The ability to locomotethrough viscous environments may con-fer ecological advantages, such as theability to migrate within gingival crevic-ular fluid and to penetrate sulcular epi-thelial linings and gingival connectivetissue (33, 55, 60). Thus optimal loco-motion of oral spirochetes depends onviscosity, and their behaviour is similarto that exhibited by other pathogenicspirochetes (32, 56). In the same way,oral spirochetal viscosity-dependentlocomotion may also be a factor in thecapacity of these microorganisms to in-itiate and to sustain the progression ofperiodontal disease.

A murine model of virulencecharacteristics of oral treponemes

Several in vitro studies have revealedthat the oral treponemes also elaboratea variety of proteolytic enzymes (45),hemolysins (10, 22), esterases (48, 77),collagenase (41), iminopeptidases (42),phospholipase C (71), and hyaluronicacid and chondroitin sulfate–degradingenzymes (18, 70). Whether these en-zymes function in vivo in soft tissue orbone destruction noted in inflammatory

periodontal disease is unclear. To cla-rify this, the oral spirochetes T. dentico-la, T. socranskii, T. vincentii and T. pec-tinovorum were injected subcutaneouslyinto mice to investigate their virulencecharacteristics. With all species therewas a dose-response lesion formation.In the case of T. denticola, within 48 hthere was gross evidence of hyperemiaand diffuse edema at the site of inocu-lation. By day 4, there was gross evi-dence of abscess formation, and the ab-scess reached maximum size by days 5to 7. The abscesses contained a thick,purulent, greyish-white caseous ma-terial, which remained until necrosis ofthe dermal layers at the center of ab-scess occurred. Note that neither heat-killed nor formalin-killed cells werecapable of inducing lesions with charac-teristics similar to those induced by vi-able bacterial infection. T. vincentii pro-duced abscesses that were significantlylarger than those noted for other oraltreponemes (31). It has been suggestedthat T. vincentii is associated with amore tissue-invasive disease affectingthe periodontium and that this micro-organism can be detected deep withinthe periodontal tissues (29).

Trypsin-like protease activity wasdemonstrated in strains of T. denticolaand T. pectinovorum, whereas enzymeactivity was devoid in strains of T. vin-centii and T. socranskii. To determinethe effects of trypsin-like protease onthe formation of soft tissue lesions, T.denticola and T. pectinovorum cells werepreincubated with inhibitors of this en-zyme, which completely eliminated theactivity of this enzyme. Interestingly,there was no effect on abscess forma-tion in mice. This would suggest thatthe trypsin-like protease activity of theoral treponemes differs from that foundin Porphyromonas gingivalis and ap-pears to have a negligible functionalrole in this measure of virulence in vivo(31).

Proteases are considered significantvirulence factors in periodontal disease.Several proteases or peptidases of T.denticola have been described, and theirpathogenic effects have been character-ized (40, 44, 43, 79). In a more recentstudy by Ishihara et al. (28) a dentilisin-deficient mutant (K1) was constructedin T. denticola. The gene, prtP, codingfor the protease was inactivated via al-lelic exchange mutagenesis. In the mu-tant no chymotrypsin-like protease ac-tivity was detected. The organization ofthe outer sheath was also affected. The

Taxonomy and virulence of oral spirochetes 7

major surface protein of 53 kDa wasobserved in protein preparations fromboth the wild type and the K1 mutant,indicating that major surface proteinwas expressed in both cases. However,in the mutant the ability of the organ-ization of the high-molecular-mass oli-gomeric protein decreased. To evaluatethe effects of the surface structure alter-ations displayed by mutant K1 on thevirulence of the microorganism, thewild-type and mutant strains were in-jected subcutaneously into the posteriordorsolateral surface of two groups ofmice. The lesion areas of the group in-fected with the prtP mutant weresmaller than those of the group injectedwith the wild type over a 3- to 14-dayperiod after infection.

Conclusion

Oral spirochetes, like all other mi-crobial periodontal pathogens, havebeen implicated in the periodontal dis-ease process by virtue of their numeri-cal predominance in inflamed affectedsites. This numerical association hasbeen reinforced by laboratory-provendemonstration of various virulence fac-tors encompassing adherence, cytotox-icity, iron sequestration and loco-motion. Conventional bacterial tax-onomy has speciated the treponemesinto a limited number of species (lessthan a dozen) but only four are avail-able and reliably maintained by variouslaboratories. These include T. denticola,T. pectinovorum, T. socranskii and T.vincentii; the first three are nomenclat-urally valid names. Oral spirochetolog-ists are fortunate in that conventionalclassification of the treponemes is in ac-cordance with phylogenetic analysis ofthe spirochetes based on 16S rRNAcataloguing; they fall into a naturalgrouping in the phylogenetic tree ofspirochetes.

Epilogue and acknowledgments

The selective use of references by theauthors was constrained by the limi-tation of space in a mini-review. Theomission of any investigators has noimplication on their significant contri-butions to the field of oral spirochetol-ogy. Studies reported from our labora-tory were supported by the Medical Re-search Council of Canada. The costs in-curred in writing this mini-review weresupported financially by Ian Hend-erson.

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