biology of treponema pallidum: correlation of functional ... · biology of treponema pallidum:...

Biology of T. pallidum 37

Biology of Treponema pallidum: Correlation ofFunctional Activities With Genome Sequence Data

*For correspondence. Email [email protected];Tel. (713) 500-5338; Fax. (713) 500-0730.

J. Mol. Microbiol. Biotechnol. (2001) 3(1): 37-62.

© 2001 Horizon Scientific Press

JMMB Review

Steven J. Norris1,2 *, David L. Cox3,and George M. Weinstock2

1Department of Pathology and Laboratory Medicine,University of Texas-Houston Medical School, P.O. Box20708, Houston, Texas 77225, USA2Department of Microbiology and Molecular Genetics,University of Texas-Houston Medical School, Houston,Texas 77225, USA3Division of AIDS, STD and TB Laboratory Research,Centers for Disease Control and Prevention, Atlanta, GA30333, USA

Abstract

Aspects of the biology of T. pallidum subsp. pallidum,the agent of syphilis, are examined in the context of acentury of experimental studies and the recentlydetermined genome sequence. T. pallidum and a groupof closely related pathogenic spirochetes have evolvedto become highly invasive, persistent pathogens withlittle toxigenic activity and an inability to surviveoutside the mammalian host. Analysis of the genomesequence confirms morphologic studies indicating thelack of lipopolysaccharide and lipid biosynthesismechanisms, as well as a paucity of outer membraneprotein candidates. The metabolic capabilities andadaptability of T. pallidum are minimal, and this relativedeficiency is reflected by the absence of manypathways, including the tricarboxylic acid cycle,components of oxidative phosphorylation, and mostbiosynthetic pathways. Although multiplication of T.pallidum has been obtained in a tissue culture system,continuous in vitro culture has not been achieved. Thebalance of oxygen utilization and toxicity is key to thesurvival and growth of T. pallidum, and the genomesequence reveals a similarity to lactic acid bacteriathat may be useful in understanding this relationship.The identification of relatively few genes potentiallyinvolved in pathogenesis reflects our lack ofunderstanding of invasive pathogens relative totoxigenic organisms. The genome sequence willprovide useful raw data for additional functionalstudies on the structure, metabolism, andpathogenesis of this enigmatic organism.

Introduction

Syphilis was deemed one of the three great plagues of theearly 20th century (along with cancer and tuberculosis)because of its high prevalence and difficulty in treatment.

The demonstration of experimental transmission of syphilisto chimpanzees (Metchnikoff and Roux, 1906) was followedsoon thereafter by the identification of a pale, spiral-shapedorganism called Spirochaeta pallida by Schaudinn andHoffman (Schaudinn and Hoffman, 1905). With thedevelopment of experimental models and identification ofthe infectious agent, hopes were high that research on thisenigmatic organism and the scourge of syphilis wouldproceed with the same rapidity as with the other pathogensdiscovered and characterized during this golden era ofmicrobiology.

The organism, since renamed Treponema pallidumsubsp. pallidum, did not cooperate. Despite intensive effortsby some of the best minds in science, this seemingly simplebacterium yielded information begrudgingly and in smallincrements. Ninety-five years after its discovery, T. pallidumstill has not been cultured continuously in vitro, althoughprogress has been made in the achievement of in vitromultiplication. Investigators have had to rely on inoculationof rabbits and other animals both for the propagation of T.pallidum for study and as models of human infection. Theinability to produce large quantities of the spirochete in apure state, let alone to perform colony isolation, mutationalanalysis, and other standard bacteriologic procedures, hashampered progress greatly.

Prior to the 1970’s, less was known about T. pallidumthan about virtually any other pathogenic bacterium. Theapplication of electron microscopy, protein gelelectrophoresis, biochemical techniques, molecular biologyprocedures, and genome sequencing have led to dramaticincreases in our understanding of the morphology, in vivoand in vitro growth characteristics and requirements,metabolism, and antimicrobial susceptibility of T. pallidum.

The complete sequence of T. pallidum subsp. pallidum(Nichols) genome was determined in 1998 (Fraser et al.,1998), shortly after the complete genome sequence of theBorrelia burgdorferi B31 (one of a group of closely relatedLyme disease spirochetes) was reported in 1997 (Fraseret al., 1997). As with other genome sequences, the T.pallidum sequence provided a complete ‘parts list’ for theorganism, revealing not only what the genetic capabilitiesof the spirochete are, but also what is missing. However,gene function can only be predicted from sequence dataand must be verified experimentally. This review representsa compendium of the experimental data available on thebiology of T. pallidum and its correlation with what ispredicted from the genome sequence.

Various aspects of the biology of T. pallidum have beenreviewed previously, and in many cases the olderreferences provide a more complete representation. Turnerand Hollander (Turner and Hollander, 1957) and Willcoxand Guthe (Willcox and Guthe, 1966) provided excellentoverviews of T. pallidum physiology and animal infectivity.Two books edited by Johnson (Johnson, 1976) and Schelland Musher (Schell and Musher, 1983) and a review by

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38 Norris et al.

Sell and Norris (Sell and Norris, 1983) also provide generalinformation. Summaries of the molecular and antigenicproperties of T. pallidum were published by Strugnell et al.(Strugnell et al., 1990) and Norris et al. (Norris and Group,1993). The molecular architecture of the outer membraneof T. pallidum has been an area of controversy in recentyears, and has been reviewed by Blanco et al. (Blanco etal., 1997) and Radolf (Radolf, 1994; Radolf, 1995). A smallbut informative book published by the U.S. Public HealthService (United States Public Health Service, 1967) in 1964still provides the best overview of clinical aspects ofsyphilitic infection. The immunology of T. pallidum infectionwill not be addressed here, but has been reviewedelsewhere (Pavia et al., 1978; Schell and Musher, 1983;Sell and Norris, 1983; Norris, 1988; Radolf, 1994; Blanco

et al., 1997). Several short reviews have been publishedon aspects of the T. pallidum genome sequence (Norris etal., 1998; Weinstock et al., 1998; Radolf et al., 1999; Norrisand Weinstock, 2000; Weinstock et al., 2000; Weinstocket al., 2000), as well as a detailed comparison of thepredicted gene products of T. pallidum and B. burgdorferi(Subramanian et al., 2000).

Pathobiology of T. pallidum

Syphilis is a multistage disease characterized by localized,disseminated, and chronic forms of infection (United StatesPublic Health Service, 1967; Musher, 1987; Larsen et al.,1999). Inoculation occurs through direct contact of skin ormucous membranes with infectious lesions during sexualactivity. In the primary stage, T. pallidum multiplies locallyat the site of inoculation, resulting in the characteristicprimary lesion (chancre) within days to weeks of exposure.Dissemination occurs during the primary stage, resultingin systemic infection. Approximately 30% of patientsdevelop the multiple skin lesions and proteanmanifestations characteristic of secondary syphilis. Primaryand secondary lesions contain large numbers of viable T.pallidum and as such are the principal source oftransmission. Latency is characterized by serologicreactivity in the absence of other detectable manifestations,and may last the lifetime of the individual. Roughly one-third of untreated syphilis patients develop tertiary syphilis,consisting of gummatous syphilis, cardiovascular syphilis,or neurosyphilis. T. pallidum can also be transmittedtransplacentally from mother to fetus, resulting in congenitalsyphilis. Congenital infection may result in stillbirth, earlyfulminant infection during infancy, or chronic disease withbone, tooth, corneal, and neural manifestations.

Bacterial pathogens can be characterized by theirinvasive and toxigenic properties. T. pallidum is a highlyinvasive organism with limited toxigenic capabilities. Theinvasive properties of the spirochete are demonstrated inFigure 1. Dissemination and persistent colonization oftissues results from the organism’s characteristic cork-screw motility, hematologic spread, adherence andpenetration of epithelial cell layers and other host barriers,and ability to evade the immune response. Most of thepathology appears to be due to the inflammatory reactionto infection, which damages the surrounding tissue (Selland Norris, 1983; Norris, 1988). With few exceptions, our

Table 1. Characteristics of Pathogenic Treponema

Agent Disease Distribution Climate Pathogenicity in Humans Pathogenicity in Animals

Treponema pallidum Venereal syphilis Worldwide Temperate Highly invasive. Local and systemic Rabbits - highly susceptible to skin and testicularsubsp. pallidum infection, with early, latent, late and infection. Hamsters - lymph node infection, but no

congenital forms skin lesions. Guinea pigs - relatively resistantTreponema pallidum Endemic syphilis North Africa; Desert and Moderately invasive. Early, latent, Rabbits, hamsters, and guinea pigs susceptible tosubsp. endemicum (bejel) Near East; Temperate and late stages infection.

South EuropeTreponema pallidum Yaws Central Africa; Tropical and Moderately invasive. Skin and bone Chronic skin lesions in hamsters. Rabbits andsubsp. pertenue South America; Desert lesions. Congenital disease rare. guinea pigs also susceptible.

IndonesiaTreponema carateum Pinta Central and Tropical Not invasive. Local dermal lesions, Not infectious to laboratory animals.

South America no systemic involvement.Treponema Venereal Not applicable Not applicable Not infectious to humans Genital lesions in rabbitsparaluiscuniculi spirochetosis of

rabbits

a Compiled from references (Turner and Hollander, 1957; Willcox and Guthe, 1966; Sell and Norris, 1983).

Figure 1. The invasive nature of T. pallidum, as demonstrated bytransmission electron microscopy of rabbit testicular tissue followinginoculation with T. pallidum subsp. pallidum Nichols. A longitudinal sectionof a treponeme traveling between host cells is present in the lower part ofthe micrograph. Several cross-sections of a spirochete going through ahost cell are visible in the upper portion of the photomicrograph.


understanding of invasive mechanisms are extremelylimited relative to the extensive knowledge of the action ofbacterial toxins on host cells and tissues. Also, proteinsinvolved in invasion (e.g. InlA and ActA of Listeriamonocytogenes) tend not to be conserved across a broadrange of bacterial species, as is often the case with bacterialtoxins (such as the RTX family). Some T. pallidum geneproducts predicted from the genome sequence havesequence similarity to known bacterial virulence factors(Weinstock et al., 1998); however, as with other aspectsof its biology, functional studies will be necessary to providea clearer picture of the organism’s pathogenic mechanisms.

Classification of T. pallidum

Properties of the pathogenic Treponema species andsubspecies are listed in Table 1. The Treponema causingvenereal syphilis, endemic syphilis, and yaws werereclassified in 1984 (Smibert, 1984) to reflect new DNA-DNA hybridization information (reviewed by Fieldsteel,1983). The pathogenic Treponema are virtuallyindistinguishable in terms of DNA homology, morphology,protein content and physiology. None has been cultivatedcontinuously in vitro. No genetic information is availablefor Treponema carateum (which can not be cultured evenby animal inoculation), whereas Treponemaparaluiscuniculi causes venereal spirochetosis in rabbitsbut does not cause disease in humans (Graves andDownes, 1981). Therefore these infectious agents haveretained their status as separate species. Until the recentadvent of comparisons based on PCR and other methods(see below), the only reliable criterion for differentiatingthese organisms was their pattern of pathogenesis inhumans and in experimentally infected animals.

Miao, Fieldsteel and coworkers used DNA-DNAhybridization to determine the degree of homology betweensyphilis isolates (exemplified by the Nichols strain of T.pallidum subsp. pallidum) and other spirochetes (Fieldsteel,1983; Stanton et al., 1991). Based on DNA-DNAhybridization and estimates of G + C content, all syphilis,yaws and endemic syphilis isolates were closely related(>95% homology). On the other hand, DNA from theseorganisms exhibited <5% cross-hybridization with DNAfrom easily cultivable treponemes, including Treponemaphagedenis, Treponema refringens, and “Treponema”(now Brachyspira) hyodysenteriae; their respective G + Ccontents were also quite different. The 16s ribosomal RNAsequence of T. pallidum exhibits significant identity withrRNA sequences of other Treponema species, Spirochaetazuelzerae, and Spirochaeta stenostrepta (Paster et al.,1991); in addition, Treponema species and certainSpirochaeta species both contain cytoplasmic filaments,an unusual intracytoplasmic structure not found in otherspirochetes (see below). These findings suggest that aclose evolutionary grouping of Treponema and someSpirochaeta species may exist, and may justifyreclassification of some organisms.

Sequence analysis, restriction fragment lengthpolymorphism (RFLP), and monoclonal antibody reactivityhave been examined as means for distinguishing thepathogenic treponemes. In comparing T. pallidum subsp.pallidum and T. pallidum subsp. pertenue gene sequences

for the 190 kDa 4D antigen, Noordhoek et al. (Noordhoeket al., 1990) found that only a single nucleotide out of 936was different; even this single base pair difference wasnot consistent among isolates of each subspecies. Inaddition, monoclonal antibodies against T. pallidum subsp.pallidum Nichols antigens were equally reactive with T.pallidum subsp. pertenue proteins (Noordhoek et al., 1990),providing further evidence that these subspecies are veryclosely related.

More recently, Centurion-Lara et al. (Centurion-Laraet al., 1998) described the occurrence of sequence andRFLP differences in the regions flanking tpp15 (TP0171),encoding a prominent 15 kDa lipoprotein. RFLP analysiscould be used to distinguish T. pallidum subsp. pallidumstrains from yaws and endemic syphilis isolates as well asfrom T. paraluiscuniculi. Cameron et al. (Cameron et al.,1999) described the consistent occurrence of a singlenucleotide difference between T. pallidum subsp. pallidumisolates and subsp. pertenue and endemicum isolates inthe 579 bp gene encoding glycerophosphodiesterphosphodiesterase (gpd; TP0257). In contrast, 6 nucleotidedifferences were found in gpd of T. paraluiscuniculi, 5 ofwhich were silent substitutions. This result verifies that T.paraluiscuniculi has diverged to some extent from thehuman treponemal pathogens, and also indicates thestrong selective pressure for the conservation of thisenzyme involved in phospholipid metabolism. The surface-exposed protein candidate Tp92 exhibits a similar patternof conservation within 7 T. pallidum subsp. pallidumisolates, with progressively greater heterogeneity observedin subsp. pertenue and T. paraluiscuniculi isolates(Cameron et al., 2000).

The T. pallidum repeat (tpr) genes (Fraser et al., 1998;Stamm et al., 1998; Centurion-Lara et al., 1999) appear torepresent the regions of greatest genetic divergence in anotherwise highly conserved genome, exhibiting bothinterstrain and intrastrain differences in venereal syphilisisolates. By sequence analysis of PCR products, tprDexhibits at least two alleles within T. pallidum subsp.pallidum (Centurion-Lara et al., 2000), and tprK has beenshown to have multiple alleles even within individual syphilisisolates (Centurion-Lara et al., 2000). Whole genome PCRanalysis has revealed additional strain differences at tprloci (E. Sodergren, unpublished data).

A molecular subtyping system for T. pallidum subsp.pallidum recently described by Pillay et al. (1998)demonstrates the existence of genetic heterogeneity in alarge number of strains and patient specimens. This systemutilizes a combination of size heterogeneity within the arp(acidic repeat protein) gene and RFLP patterns of threemembers of the tpr gene family (tpr E, G and J). The arpgene contains a series of 60 bp repeats; these repeatscode for a highly acidic amino acid sequence. Thus far, 15arp repeat sizes (4-16, 18-20, 21, 24) and 13 tpr gene RFLPtypes (designated a to m) have been identified (Pillay etal., 1998; Pillay et al., personal communication; Sutton etal., submitted). For example, T. pallidum subsp. pallidum(Nichols) falls into group 14d; it has 14 repeats in the arpgene and displays RFLP pattern d for the 3 tpr genes. Bycombining the tpr and arp typing methods 43 differentsubtypes have been observed among 229 specimens(genital ulcers or blood) from the United States,

40 Norris et al.

Madagascar, and South Africa. The 14d subtype was mostcommon and was found in 58 (25.3%) of the 229specimens. Furthermore, both of these genes or sets ofgenes are present in T. pallidum susp. pertenue andendemicum. The limited number of samples of these twosubspecies examined indicated lower heterogeneity thanin subsp. pallidum especially in the arp gene. Theheterogeneity in these genes suggests that additionaldivergence may be present in other genes.

Genome Analysis

In 1991, Walker et al. (Walker et al., 1991) used pulsedfield electrophoresis techniques to characterize the genomeof T. pallidum. The genome was shown to consist of asingle, circular chromosome approximately 1,000,000 basepairs (one megabase) in size, much smaller than theprevious estimate of 13.7 megabases based on DNA-DNAhybridization kinetics (Fieldsteel, 1983). T. pallidum thuswas shown to possess one of the smallest genomes withinthe prokaryotes (similar to Mycoplasma and Borreliaspecies) (Krawiec and Riley, 1990).

The genome sequence of T. pallidum subsp. pallidum(Nichols) was determined in 1998 (Fraser et al., 1998) usingnow well-established random, shot-gun cloning andsequence assembly techniques. The genome consists ofa single circular chromosome of 1,138,006 base pairs (+possible undetected sequencing errors). The G + C contentis 52.8%, much higher than the 39% value obtained withthe B. burgdorferi genome (Fraser et al., 1997). A total of1041 open reading frames (ORFs) were identified,accounting for 92.9% of the total genomic DNA. Based onprotein similarity searches, predicted biological roles couldbe assigned to 55% of the ORFs; 17% matchedhypothetical proteins of unknown function in otherorganisms, and the remainder (28%) did not havesignificant similarity to other known sequences. This ratiois similar to those obtained with other bacterial genomes.Surprisingly, only 476 (46%) of the predicted proteinproducts had orthologs in B. burgdorferi, and most of these(76%) were proteins with a predicted biological function(e.g. housekeeping functions). Thus relatively few (~5%)of the T. pallidum genes appear to be ‘spirochete-specific’,i.e. common to spirochetes but not found in other bacteria.This result indicates significant divergence among theSpirochaetacae. Other aspects of the T. pallidum genomewill be considered in the remaining sections of this review.

Morphology

The spirochetes, members of the order Spirochaetales,are characterized by a morphologically unique, helical orwave-like cell body (Figure 2). When imaged in twodimensions by darkfield microscopy or transmissionelectron microscopy, the helical cells of T. pallidum have aregular, wave-like appearance (Hovind-Hougen, 1983). Thecells of T. pallidum range from 5 to 15 µm in length, asmeasured along the axis of the helix, and are typically 0.16to 0.20 µm in diameter. The sinusoidal profile of theorganism has a wavelength and amplitude of approximately1.1 µm and 0.4 µm, respectively.

The cross-sectional ultrastructure of T. pallidum

superficially resembles that of gram-negative bacteria inthe concentric arrangement of the outer membrane (OM),periplasmic space, peptidoglycan layer, inner (cytoplasmic)membrane (CM) and protoplasmic cylinder (Figure 2). Thismorphologic similarity is deceptive, however, since it is nowclear that the composition of the T. pallidum OM differsmarkedly from that of gram-negative bacteria (see OuterMembrane below). The OM delimits the outer cellularsurface of the organism, and the CM surrounds theprotoplasmic cylinder. Unlike gram-negative bacteria, theorganelles of motility of T. pallidum lie entirely within theperiplasmic space, between the CM and the OM. Theseorganelles, termed periplasmic flagella or endoflagella,insert in groups of two to four subterminally at either endof the cell cylinder of T. pallidum; the insertion points arearranged in a row parallel to the long axis of the helix. Theperiplasmic flagella wrap around the helical protoplasmic

Figure 2.Morphology of T. pallidum subsp. pallidum Nichols by electronmicroscopy. Panel (a) is a scanning electron micrograph, (b) is a preparationof T. pallidum negatively stained with ammonium molybdylate, and (c-f)show ultrathin sections. Note the overall helical shape (panel a) and theinsertion (I) of several periplasmic flagella (PF) or endoflagella at each endof the cells (panel b). In ultrathin sections, the outer membrane (OM),cytoplasmic membrane (CM), cytoplasmic filaments (CF) and PFs areevident. Bars = 1 µm (panel a) and 0.1 µm (panels b-f). Reprinted withpermission from (Larsen et al., 1999).


cylinder and interdigitate near the center of the organism.The presence of an electron dense layer closely apposedto the CM has been identified by thin-section electronmicroscopy (Hovind-Hougen, 1983), although this layer,which by inference corresponds to the peptidoglycan cellwall, is not always clearly visible. Biochemical evidenceindicates that T. pallidum contains typical spirochetalpeptidoglycan (Radolf et al., 1989). The presence of theOM, CM, and periplasmic flagella, like helicity, ischaracteristic of all spirochetes; differences in othermorphological parameters (including cell diameter andlength and the number and length of periplasmic flagella)exist among various members of this diverse group ofbacteria.

Outer MembraneThe OM forms the cellular surface in most intimate contactwith the host and is also a structure with which manycomponents important for bacterial virulence areassociated. As a result, there has been great interest incharacterizing the OM of T. pallidum. A perspective hasarisen in the field of treponemal research, acquired fromwork performed in several laboratories over a number ofyears, that the surface of T. pallidum exhibits a puzzlinglack of antigenicity. The kinetics of in vitro killing of T.pallidum by specific antibody and complement areprolonged. A minimum of 4 hours incubation with high titerantiserum and active complement are necessary beforeany killing of treponemes occurs; typically 16 h arenecessary to achieve 100% killing (Nelson and Mayer,1949; Miller et al., 1963; Bishop and Miller, 1976). Thisobservation is paralleled by the finding that the surface ofT. pallidum seems to require perturbation by chemical orimmunological means before the binding of antibody tothe organism can be detected by standardimmunocytochemical techniques (Metzger and J., 1964;Houvind-Hougen et al., 1979; Penn, 1981; Fehniger et al.,1986; Radolf et al., 1986).

Because T. pallidum is propagated in the rabbit testis,the possibility has been explored that the relative antigenicinertness of the organism may result from uniform bindingof host molecules to the treponemal surface. It has beenreported that treponemes harvested from testes havefibronectin, albumin, and other host molecules associatedwith their surface (Fitzgerald et al., 1975; Fitzgerald andJohnson, 1979; Peterson et al., 1983). These studies havestimulated speculation that T. pallidum utilizes a form ofantigen masking as a mechanism of evading the hostimmune response, as has been hypothesized for theschistosomes (Bloom, 1979).

Ultrastructural analyses indicate that T. pallidum maypossess a simple and elegant property that limits theantigenicity of its surface. Freeze-fracture studies haveshown that OM of the organism is populated by aremarkably low concentration of transmembrane proteins,approximately 100-fold fewer than in other spirochetes andin gram-negative bacteria (Radolf et al., 1989; Walker etal., 1989; Walker et al., 1991). Both the concave (outerleaflet of the lipid bilayer) and convex (inner leaflet of thelipid bilayer) OM fracture faces of T. pallidum contain few,sparsely distributed intramembranous particles, indicatingthe presence of few integral transmembrane proteins

(Radolf et al., 1989; Walker et al., 1989). In contrast, theintramembranous particles of the OM concave fracture faceof E. coli form a two-dimensional array that essentiallycovers the entire surface of the membrane leaflet (compare10,000 particles/µm2 for E.coli to 90 particles/µm2 for T.pallidum) (Lugtenberg and Van Alphen, 1983; Radolf etal., 1989; Walker et al., 1989). In a modified immobilizationassay it was shown that although antibody binds to thesurface of T. pallidum within one hour in the absence ofactive complement, a potentiation incubation period of 8 his required before the addition of active complement willmediate killing of the organism (Blanco et al., 1990).Freeze-fracture of organisms incubated for 16 h in heat-inactivated immune rabbit serum revealed that thetransmembrane proteins of the OM, which are normallywidely separated, had formed aggregates (Blanco et al.,1990). The combined results of the modified immobilizationassay and freeze-fracture of treponemes incubated for 16h in immune rabbit serum indicate that cross-linking of theOM proteins by antibody is the rate-limiting step in antibody-dependent, complement-mediated in vitro killing of T.pallidum. The slow kinetics of this step are consistent withthe model that proposes that the OM of T. pallidum ispopulated by a low concentration of proteins.

Outer Membrane PurificationThe purification of T. pallidum outer membranes andidentification of associated proteins has been problematic((Radolf et al., 1995; Blanco et al., 1997). The OM of T.pallidum does not contain lipopolysaccharide (LPS) (Hardyand Levin, 1983; Johnson, 1983; Bailey et al., 1985; Pennet al., 1985; Radolf and Norgard, 1988), a molecule foundin great abundance in the outer leaflet of the OM of gram-negative bacteria; these studies are confirmed by the lackof genes encoding LPS biosynthesis enzymes in thegenome (Fraser et al., 1998). LPS imparts a higher densityto the OM of gram-negative bacteria, allowing separationof the OM from the CM in sucrose density gradients. Thelack of LPS in the OM of T. pallidum probably explains thedifficulty in applying this simple technique to separation ofthe treponemal CM and OM (Norris and Group, 1993).Moreover, ‘gold standard’ markers for the outer membrane(such as the Omp proteins of E. coli) are lacking, anddetection of outer membrane proteins in cellular fractionsis predictably difficult due to their relative paucity.

Blanco et al. (Blanco et al., 1994) used a combinationof Ficoll purification of T. pallidum, octadecyl rhodamine Bchloride labeling of intact organisms, and selective releaseof OM vesicles with 0.05 M citrate buffer, pH 3.2, to purifyT. pallidum and T. vincentii OMs. Ficoll purification removedcontaminating host proteins, whereas incorporation of thehydrophobic rhodamine compound into the surface of intacttreponemes permitted monitoring of OM purification. Blancoet al. (Blanco et al., 1994) found that the acidic citrate bufferreleased vesicles that lacked cytoplasmic membranemarkers (such as the 47 kDa lipoprotein), but containedseveral polypeptides deemed treponemal rare outermembrane proteins or Tromps.

Two of the proteins identified by this means, Tromp1and Tromp2, have been characterized. Tromp1, also knownas TroA (Hardham et al., 1997), is a 30.4 kDa protein witha hydrophobic leader sequence; there is contradictory

42 Norris et al.

evidence regarding whether this leader is cleaved or servesas a hydrophobic anchor (Akins et al., 1997; Blanco et al.,1999). Native Tromp1 purified by nondenaturing isoelectricfocusing has porin activity as determined by conductancemeasurements in a lipid bilayer model (Blanco et al., 1996),leading to the identification of Tromp1 as an integral OMprotein. However, other evidence is consistent with Tromp1being a periplasmic binding protein for an ATP-bindingcassette (ABC) transport system. Tromp1/TroA hassequence similarity to other known periplasmic bindingproteins and is encoded by the first gene in an operon withgenes for an ATP-binding protein (TroB), two hydrophobicmembrane proteins (TroC and TroD), and a manganese-dependent regulatory protein (TroR) resembling theCorynebacterium diphtheriae regulator DtxR (Hardham etal., 1997; Posey et al., 1999). The last gene in the operonencodes phosphoglycerate mutase, a glycolytic pathwayenzyme that in many organisms requires magnesium as acofactor. Posey et al. (Posey et al., 1999) demonstratedthat TroR binds to the tro operator in a Mn++-dependentfashion, and like DtxR acts as a cation-dependent repressorprotein.

The three dimensional structure of a recombinant formof TroA has been determined (Deka et al., 1999; Lee et al.,1999). It exhibits a structure nearly identical to those ofperiplasmic binding proteins, and contains a captive Zn++

ion in the crystal structure. Interestingly, mutation of thetroA homolog scaA in the Streptococcus gordonii scaCBAoperon results in poor growth in low [Mn++]; this operon isalso inhibited by high concentrations of Zn++ (Kolenbranderet al., 1998). Thus there is convincing evidence that TroAis a periplasmic protein involved in the capture of Mn++ orother metal cations for transport across the cytoplasmicmembrane. However, recent data (Zhang et al., 1999)suggest that the recombinant protein exists in a differentconformation than its native treponemal counterpart, andthat trimer formation and porin activity can be recoveredby denaturation and renaturation of recombinant Tromp1/TroA. Thus the structural location and function of Tromp1/TroA remain controversial.

Tromp2, characterized by Champion et al. (Championet al., 1997) has a predicted signal peptidase I leadersequence that if cleaved would yield a mature protein of24.8 kDa. The structure is predicted to have ninetransmembrane beta-pleated sheet segments, consistentwith an integral membrane protein. Unlike Tromp1,membrane conductance experiments did not provide clearevidence that Tromp2 has porin activity. The native andrecombinant forms partition to the detergent phase in TritonX-114 partitioning experiments, and recombinant Tromp2was associated with the outer membrane and binds anti-Tromp2 antibodies on the surface of E. coli. Thereforeevidence to date indicates that Tromp2 is a surface-exposed integral membrane protein. It should be notedthat Tromp2 has sequence similarity (26% identity, 40%similarity) with 131 amino acids of FlaA, the T. pallidumendoflagellar sheath protein (see below).

Radolf et al. (Radolf et al., 1995) utilized an alternativeapproach to OM purification, combining plasmolysis withisopycnic sucrose density gradient sedimentation.Treatment of T. pallidum with a 20% sucrose solutionfollowed by adjustment to 45% sucrose dissociated the

OM from the remainder of the cell, permitting separationon a bottom-loaded, 5% to 50% sucrose gradient. Underthese conditions, OM fragments sedimented at a densityof 1.05 g/cm3 as compared to 1.14 g/cm3 for theprotoplasmic cylinders. Analysis of the lipid content of theresulting OM fraction indicated that the majority of thecardiolipin was associated with the inner membrane (CM).However, some cardiolipin was present in the OM fraction,which may explain the observed opsonic activity of anti-VDRL antigen antibodies (Baker-Zander et al., 1993).Analysis of the protein content of these OM preparationsrevealed the presence of orthologs of thioredoxin (Tlp),peptidyl prolyl cis-trans isomerase (PpiB), andglycerophosphodiester phosphodiesterase (GlpQ), as wellas the following previously described proteins: treponemalmembrane protein A (TmpA), basic membrane protein(Bmp), Tromp1/TroA, T. pallidum lysine rich repeat protein(TpLRR), and 17 kDa lipoprotein Tpp17 (Shevchenko etal., 1997). Many of these proteins had previously beenshown to have a periplasmic location in T. pallidum or otherorganisms. The authors conclude that some of these outermembrane candidates may represent periplasmic or CMcontaminants. Without a definitive measure of OMlocalization, distinguishing contaminants from true OMconstituents is a vexing problem.

Other approaches to identifying OM proteins haveincluded surface labeling of intact organisms (reviewed by(Norris and Group, 1993; Radolf, 1995)) and constructionof phoA fusions to identify exported proteins (Blanco et al.,1991; Hardham, 1995; Stamm et al., 1998). The firstmethod has proven to be insufficient due to the fragility ofthe OM and exposure of subsurface components duringprocessing. The second identifies any protein that istransported across the CM, and thus is also not specificfor OM proteins. Surface immunofluorescence using T.pallidum embedded in agarose microbeads (Cox et al.,1995) has been used to verify the subsurface location ofmany proteins, but thus far has not resulted in the detectionof surface-localized proteins. Although surface exposedproteins (Osps A-C) in the OM of B. burgdorferi (Cox et al.,1996) have been labeled using this microbead technology,it is still unclear if the assay is sensitive enough to detectthe much less abundant (as revealed by freeze-fractureEM ) integral protein(s) of the T. pallidum OM. Finally,opsonic activity of T. pallidum-specific and monospecificantisera and the protective effects of immunization havebeen employed as means of identifying surface-exposedproteins, including GlpQ (Stebeck et al., 1997; Cameronet al., 1998), TrpK (Centurion-Lara et al., 1999), and Tp92(Cameron et al., 2000). However, Shevchenko et al.(Shevchenko et al., 1999) provided evidence that GlpQwas entirely subsurface in location, and were unable toreproduce the opsonic or protective activities of this protein.

The tpr Gene FamilyThe tpr gene family was discovered by four independentmeans: TnPhoA mutagenesis (Stamm et al., 1998),genome sequence analysis (Fraser et al., 1998),subtraction hybridization and differential immunologicscreening (Centurion-Lara et al., 1999). This family of 12paralogous genes (tprA through tprL) is found as individualgenes or gene clusters throughout the genome, and some


loci are accompanied by additional paralogous genefamilies (tpr-associated genes, such as taaA through taaD).The predicted Tpr proteins can be subdivided into 3 familiesand vary in size from 55.5 kDa to 81.5 kDa. The sequenceidentity of overlapping regions of the predicted Tpr productsranges from 29.2 to 100 percent. In addition, they are similarto the major sheath protein (Msp) of T. denticola (36.7 to42.8 percent identity). Msp is a 58 kDa protein that isthought to form a regular hexagonal array on the surfaceof T. denticola (Haapasalo et al., 1992; Fenno et al., 1996),although its surface localization and arrangement havebeen questioned (Caimano et al., 1999).

Thus far, the detection of Tpr protein expression in T.pallidum has not been reported, and preliminary studiesindicate that they either are expressed in low quantities orare relatively non-reactive in standard western blottechniques (S. J. N., unpublished). Centurion-Lara et al.(1999) reported that rabbit antiserum raised against arecombinant form of the TprK variable domain enhancedthe phagocytosis of T. pallidum by rabbit peritonealmacrophages; this result suggests that TprK or cross-reactive proteins are expressed on the surface of T.pallidum and thus serve as opsonic targets. Moreover,rabbits immunized with recombinant forms of TprK andTprF exhibited decreased lesion severity, indicating thatanti-Tpr immune responses provide some degree ofprotection against syphilitic infection (Centurion-Lara et al.,1999; Weinstock et al., 2000). Based on reversetranscription PCR, each of the tpr genes appear to beexpressed in the Nichols strain (RT-PCR) (J. M. Hardham,M. P. McLeod, E. Sodergren, J. K. Howell, G. M. Weinstock,and S. J. Norris, unpublished data), but semi-quantitativeRT-PCR suggests that tprK and tprH transcripts areexpressed at higher levels than those of other tpr genes(Centurion-Lara et al., 1999). Although heterogeneity intpr genes exists both between and within T. pallidum strainsand subspecies (see Classification of T. pallidum), to datethere is no evidence for rapid recombination that could giverise to antigenic variation.

Periplasmic Flagella and MotilityThe gross architecture of the periplasmic flagellum of T.pallidum is similar to that of other bacterial flagella, in thatthree structural components, the basal body, the hook, andthe filament, have been identified (Hovind-Hougen, 1972;Hovind-Hougen, 1983). The genes corresponding to wellconserved basal body and hook proteins are present inlarge operons in the T. pallidum genome sequence (Fraseret al., 1998), and mutation of these genes in otherspirochetes results in loss of motility (Charon et al., 1992;Limberger et al., 1994; Li et al., 1996; Ruby et al., 1997;Limberger et al., 1999). The T. pallidum flagellar filamenthas been the subject of intense study. Upon release fromthe cell under appropriate conditions, the filament retainsa helical shape (Hovind-Hougen, 1972; Hovind-Hougen,1983). The filament is composed of an inner core (10 nmdiameter), and an outer sheath (core + outer sheath, 17nm diameter) (Hovind-Hougen, 1983).

The polypeptides comprising the core and sheathstructures have been characterized in great detail. Theperiplasmic flagellar filament of T. pallidum is composedof four major flagellin subunits, in contrast to the single

flagellin found in most bacteria (Penn et al., 1985;Cockayne et al., 1987; Blanco et al., 1988; Norris et al.,1988). The flagellins are subdivided into two distinctclasses, as originally defined by two-dimensionalelectrophoresis, Western blotting and N-terminal aminoacid sequence analysis (Norris et al., 1988). The class Aflagellin FlaA is a 37-kDa polypeptide that forms the sheathof the periplasmic flagellum, while the core of the filamentis composed of three class B flagellin molecules withrelative molecular masses (Mrs) of 34,500 (FlaB1), 33,000(FlaB2) and 30,000 (FlaB3) (Penn et al., 1985; Cockayneet al., 1987; Blanco et al., 1988; Norris et al., 1988). Thegenes that encode the flagellins of T. pallidum have beencloned and sequenced (Isaacs et al., 1989; Pallesen andHindersson, 1989; Champion et al., 1990; Isaacs andRadolf, 1990). The class B flagellin genes share significantsequence homology with one another and with theubiquitous flagellin sequence found in both gram-negativeand gram-positive bacteria (Norris et al., 1988; Pallesenand Hindersson, 1989; Champion et al., 1990). The genesflaB1 and flaB3 were found to be separated by 277 basepairs; these genes may be transcribed as polycistronicmessage from a single promoter located upstream of flaB1(Champion et al., 1990).

The importance of the periplasmic flagella derives fromtheir function in cell locomotion and their highly antigenicnature. Motility is an important virulence factor for T.pallidum, promoting tissue invasion and dissemination. Therole of the periplasmic flagella in motility has been wellestablished in the cultivatable spirochetes (Canale-Parola,1978; Bromley and Charon, 1979; Paster and Canale-Parola, 1980; Charon et al., 1984; Goldstein and Charon,1988; Kimsey and Spielman, 1990), although themechanism is not entirely understood. The corkscrewmotion characteristic of spirochetes allows them to translatethrough media of relatively high viscosity (Canale-Parola,1978; Kimsey and Spielman, 1990). Like other spirochetes,treponemes spin about their longitudinal axis as the resultof rotation of the endoflagella (Canale-Parola, 1978;Bromley and Charon, 1979; Paster and Canale-Parola,1980; Charon et al., 1984; Goldstein and Charon, 1988;Kimsey and Spielman, 1990). In nonviscous media, thismotion leads to vigorous rotation but no translationalmovement. The real character of treponemal motility isobserved in tissue or highly viscous medium, where theorganisms can swim at a rate of up to 19 micrometers persecond (Kimsey and Spielman, 1990; Charon et al., 1992;Ruby and Charon, 1998). T. pallidum can rapidly reversedirection in response to obstacles and, because of itsflexibility, change its course through bending. ObservingT. pallidum in a bit of tissue under a darkfield microscopeprovides a convincing demonstration of its invasivecapabilities.

Periplasmic flagella stimulate a strong, early antibodyresponse that persists throughout infection (Hanff et al.,1982; Pedersen et al., 1982; Pedersen et al., 1982; Hanffet al., 1983). The activity of antiserum to the periplasmicflagella in the standard immobilization assay and the lackof activity of this antiserum in the modified immobilizationassay suggest that the periplasmic flagella may beimportant second-hit targets in the humoral immuneresponse (Blanco et al., 1990). The presence of pathogen-

44 Norris et al.

specific epitopes (Norris and Group, 1993) in the centralregion of FlaB proteins may enable the development ofmore specific serodiagnostic tests for syphilis, similar torecombinant flagellar antigens examined for serodiagnosisof Lyme disease (Gomes-Solecki et al., 2000).

PeptidoglycanBiochemical characterization of the residual sacculus leftafter exhaustive Triton X-114 extraction of T. pallidumdemonstrated the presence of muramic acid, a signatureamino sugar of peptidoglycan (Radolf et al., 1989).Ornithine, which commonly constitutes the sole diaminoacid found in spirochetal peptidoglycan, was also identified,whereas diaminopimelic acid was not present (Radolf etal., 1989). These findings are consistent with thoseobtained in studies of peptidoglycan from other spirochetesand confirm the presence of a peptidoglycan layer asinferred from thin-section electron micrographs (Hovind-Hougen, 1983).

Cytoplasmic MembraneThe CM of T. pallidum, as revealed by freeze-fracturestudies, is ultrastructurally similar to the CM of typical gram-negative bacteria (Lugtenberg and Van Alphen, 1983;Radolf et al., 1989; Walker et al., 1989). The inner leafletof the CM of E. coli and other gram-negative bacteriatypically contains a high concentration of intramembranousparticles. The corresponding CM fracture face of T. pallidumis also densely populated with intramembranous particles,in sharp contrast to either fracture face of the OM of theorganism (Radolf et al., 1989; Walker et al., 1989).

T. pallidum has been shown to contain a set oflipoproteins that are both abundant and highlyimmunogenic (Chamberlain et al., 1989; Chamberlain etal., 1989; Schouls et al., 1989; Purcell et al., 1990; Swancuttet al., 1990; Schouls et al., 1991; Yelton et al., 1991; Norrisand Group, 1993). The lipoproteins were originally identifiedas molecules that partition into the detergent phasefollowing solubilization of T. pallidum with the non-ionicdetergent Triton X-114, a characteristic of hydrophobicmembrane proteins (Chamberlain et al., 1989). Many ofthese lipoproteins, including Tpp47, Tpp17, Tpp15, andperhaps GlpQ (Shevchenko et al., 1999) appear to bepredominantly associated with the periplasmic leaflet ofthe CM, based on the nonreactivity of antisera against theseproteins in intact organisms and the occurrence of reactivityupon treatment of T. pallidum with concentrations of TritonX-100 that disrupt the outer membrane (Cox et al., 1995).In addition, antiserum to the Triton-X114 soluble proteins(principally lipoproteins) did not kill T. pallidum in themodified immobilization assay; under identical conditions,immune rabbit serum has treponemicidal activity (Blancoet al., 1990).

Penicillin-binding proteins, which are involved in thetranspeptidation reactions of peptidoglycan synthesis, arecommonly found in the CM of gram-negative bacteria(Waxman and Strominger, 1983). Penicillin-binding proteinshave also been identified as constituents of T. pallidum(Cunningham et al., 1987; Radolf et al., 1989). Afterextraction of T. pallidum with detergents under conditionsthat solubilize the OM but leave the CM morphologicallyintact, the penicillin-binding proteins remain with the

protoplasmic cylinders (Cunningham et al., 1987; Radolfet al., 1989). The hydrophobic nature of the penicillin-binding proteins is indicated by their retention in thedetergent phase after complete membrane solubilizationand phase-partitioning with Triton X-114 (Radolf et al.,1989). Weigel et al. (Weigel et al., 1994) demonstratedthat the major 47 kDa lipoprotein of T. pallidum (Tp47) is apenicillin-binding protein with carboxypeptidase activity,despite having no close orthologs. This result suggeststhat other prominent CM-associated lipoproteins of T.pallidum (such as 44.5, 17, and 15 kDa proteins) may alsobe involved in a novel peptidoglycan biosynthesis pathway.

Cytoplasmic FilamentsAn unusual morphological feature of T. pallidum, and mostother members of the genus Treponema and someSpirochaeta species, is the presence of cytoplasmicfilaments that lie just beneath the CM and in directapposition to the periplasmic flagella. Although thesestructures have been called cytoplasmic tubules (Bermudeset al., 1994), electron microscopy of several cultivatabletreponemes (Eipert and Black, 1979; Masuda and Kawata,1989) and T. pallidum (You et al., 1996) indicates that theyare fibrillar in structure. The filaments originate close tothe basal bodies of the periplasmic flagella and in somecases appear to attach to them (Hovind-Hougen and Birch-Andersen, 1971; You et al., 1996); however, more recentstudies with wild type and flagella-deficient mutants of T.phagedenis (Izard et al., 1999) indicate that flagellarcomponents do not appear to be associated with thecytoplasmic filaments and are not required for theirassembly. It is not known whether the filaments extendthe entire length of the cell or end near the middle, as isthe case for the flagella. The filaments have a ribbon-likeappearance with a rectangular cross-section of ~7.0 to 7.5nm by 1 nm. Several cytoplasmic filaments aggregate alongtheir flat surface to form a tightly packed array that is visiblein electron micrographs of cross-sections of treponemes.The major subunit of the cytoplasmic filaments from T.phagedenis and other treponemes appears to be aconserved polypeptide with an Mr of 82,000 to 83,000(Masuda and Kawata, 1989). The gene encoding the majorcytoplasmic filament protein CfpA of T. pallidum subsp.pallidum was identified b y N-terminal sequence analysisof the cytoplasmic filament protein of T. phagedenis andthe corresponding protein of T. pallidum (You et al., 1996).Overproduction of CfpA in E. coli results in formation ofshort, irregular bundles suggestive of partial self-assembly(You et al., 1996). Targeted mutagenesis of cfpA inTreponema denticola results in altered motility of thespirochetes, indicating that the cytoplasmic filaments areinvolved in some way with motility; there are also indicationsthat chromosomal segregation and cell division are affected(J. Izard and R. J. Limberger, unpublished data). Thesestructures are not found in most spirochetes, and as suchare not globally required for spirochetal motility and celldivision processes.

Other Cytoplasmic ConstituentsRibosomes are present as electron dense bodies in thecytoplasm of lead citrate- and uranyl acetate- stained thinsections of T. pallidum (Hovind-Hougen, 1983). The


genome of T. pallidum contains two copies of ribosomalRNA genes in the typical bacterial 16S-23S-5Sarrangement (Fraser et al., 1998); in contrast, the B.burgdorferi genome contains two 23S-5S gene operonsand a separate, single copy of the 16S rRNA gene (Fraseret al., 1997). The T. pallidum genome contains a fullcomplement of genes encoding ribosomal proteinsarranged in large and small operons as well as isolatedgenes.

Other cytoplasmic structures, such as mesosomes,vacuoles, and nuclear regions have been reported basedon examination of thin sections (Hovind-Hougen, 1983).These structures have not been studied in detail and aresubject to the caveat that they are difficult to distinguishfrom preparation-induced artifacts.

Culture of T. pallidum

In Vivo PropagationAt present, none of the strains or subspecies of T. pallidumcan be grown continuously in vitro. Therefore, nearly allstudies of T. pallidum have utilized organisms propagatedin vivo by infection of laboratory animals. The Nichols strainof T. pallidum subsp. pallidum, which was originally isolatedin 1912 from the cerebrospinal fluid of a neurosyphilispatient (Nichols and Hough, 1913) and has retained itsvirulence for humans (Magnuson et al., 1956; Fitzgeraldet al., 1976), is most commonly used strain of T. pallidum.Shortly after the description of T. pallidum by Schaudinnand Hoffman (Schaudinn and Hoffman, 1905), severalinvestigators found that venereal syphilis isolates could bepropagated easily in rabbits. The primary and latent stagesin the rabbit closely resemble the human disease, butsecondary and tertiary manifestations are not observed inrabbits. Guinea pigs and hamsters are less susceptible toT. pallidum subsp. pallidum infection and lesiondevelopment. However, these animals have proven to beuseful models of infection with endemic syphilis(subspecies endemicum) and yaws (subspecies pertenue)isolates. Primates can be infected with T. pallidum subsp.pallidum, as originally described by Metchnikoff and Roux(Metchnikoff and Roux, 1906); however, the variability ofmanifestations (Turner and Hollander, 1957) and high costof these animals render such studies impractical. Mice canbe infected T. pallidum (Magnuson et al., 1949; Klein etal., 1980) but are not effective models of human diseasebecause of the minimal and short-lived pathology observed.

Animal models and procedures for experimentalinfection with T. pallidum strains are described in detailelsewhere. A definitive reference for animal models oftreponemal infections is Biology of the Treponematoses,published by Turner and Hollander in 1957 (Turner andHollander, 1957), and is highly recommended to anyoneinterested in the study of these organisms. The methodsinvolved in the detection and quantitation of T. pallidum bydarkfield microscopy and the inoculation of animals havebeen described by Miller (Miller, 1971).

Based on the injection of successive dilutions of T.pallidum subsp. pallidum (Nichols strain) suspensions,Magnuson et al. (Magnuson and Eagle, 1948) concludedthat a single organism is sufficient to cause infection ofrabbits. The intradermal and intratesticular routes of

inoculation appeared to be equally sensitive indicators ofinfectivity; our own studies are consistent with thisobservation (S.J. Norris, unpublished). In the Sing SingPrison study published in 1956 (Magnuson et al., 1956),intradermal inoculation of human volunteers with theNichols strain of T. pallidum also indicated a high degreeof susceptibility. Prisoners with varied histories of syphilisexposure were inoculated with T. pallidum, observed forlesion development, and subsequently treated with acurative dose of penicillin. The median infectious dose of57 organisms in volunteers with no previous history ofsyphilis, whereas the median infectious dose in rabbitsinfected with the same inoculum was 23 organisms(Magnuson et al., 1956).

The in vivo multiplication rate of T. pallidum has beenestimated by sequential quantitation of T. pallidum ininfected rabbit tissue (Magnuson and Eagle, 1948) and bycorrelating infectious dose with the time of lesiondevelopment (Cumberland and Turner, 1949). By eithermethod, the division rate is extremely slow, with anestimated doubling time of 30 to 33 hours (Magnuson andEagle, 1948; Cumberland and Turner, 1949). These resultscorrespond well with in vitro multiplication rates, asdescribed below.

In Vitro Survival and MultiplicationAttempts to culture T. pallidum in vitro were stimulated byits discovery as the causative agent of syphilis in 1905.Within the next few years, there were several reports ofsuccessful cultivation, but subsequent attempts atconfirmation yielded negative results (reviewed by Kastand Kolmer, 1929 and Turner and Hollander, 1957). Insome instances, nonpathogenic spirochetes were culturedfrom syphilitic tissue and identified as avirulent strains ofT. pallidum, but these isolates were subsequently shownto represent separate species of saprophytic organisms(e.g. Treponema phagedenis Reiter and Treponemadenticola Noguchi).

In 1948, Nelson and coworkers (Nelson, 1948; Nelsonand Steinman, 1948; Nelson and Mayer, 1949) were thefirst investigators to systematically apply quantitativetechniques to the study of factors affecting the retention ofmotility and virulence by T. pallidum subsp. pallidum. Theyfound that bovine serum (or tissue extract), bovine serumalbumin, sodium pyruvate, carbon dioxide, three sulfhydrylcompounds (sodium thioglycollate, cysteine, andglutathione), and incubation at 30°C were beneficial to thesurvival of T. pallidum in vitro. Incubation under theseconditions maintained treponemal motility and some degreeof virulence for 6 to 8 days. The survival provided byNelson’s medium permitted the development of the T.pallidum immobilization (TPI) test, one of the first tests fordetecting anti-T. pallidum antibody in patients’ sera (Nelsonand Diesendruck, 1951). Using a modification of Nelson’smedium, incubation under air, and a paraffin overlaycontaining an oil-soluble antioxidant, Weber (Weber, 1960)was able to maintain treponemal motility for up to 18 days.Kimm et al. (Kimm et al., 1960; Kimm et al., 1962) reportedthat inclusion of glucose and magnesium resulted in a slightextension of survival, whereas Graves et al. (1975) stressedthe importance of a low redox potential.

Two important revelations occurred in the mid-1970’s.

46 Norris et al.

First, T. pallidum was found to utilize oxygen in itsmetabolism (Cox and Barber, 1974; Baseman et al., 1976;Cox, 1983) and to survive for longer periods in amicroaerobic environment as compared to anaerobiosis(reviewed by Jenkin and Sandok, 1983). Subsequentstudies confirmed that T. pallidum is a microaerophile andnot an obligate anaerobe as originally thought. Second, itwas found (or rather rediscovered) that T. pallidum readilyadhered to the surface of mammalian cells in culture, andthat the presence of tissue culture cells prolonged thesurvival of the bacterium (Steinhardt, 1913; Kast andKolmer, 1943; Fitzgerald, 1983). These findings arediscussed in greater detail below in the context ofphysiology and host cell interactions; however, they providean important backdrop for the discussion of themultiplication of T. pallidum in the Fieldsteel culture system.

In Vitro Multiplication of T. pallidum: the Fieldsteel etal. Culture SystemIn 1981, Fieldsteel, Cox, and. Moeckli (Fieldsteel et al.,1981) obtained consistent and significant multiplication ofT. pallidum in a tissue culture system. Previous studies inFieldsteel’s laboratory (Fieldsteel et al., 1979) had utilizedmammalian cell cultures in Leighton tubes incubatedvertically to provide an oxygen gradient. They found thatT. pallidum adhered to the mammalian cells and appearedto multiply in a region of the gradient corresponding to lowto moderate oxygen levels. Adaptation of these conditionsto standard monolayer cultures resulted in the consistentobservation of treponemal multiplication (Fieldsteel et al.,1981), as confirmed in other laboratories (Norris, 1982;Levy, 1984).

A key feature of the Fieldsteel et al. culture system isits reproducibility. Although in vitro culture of T. pallidum

had been reported previously, this was the first system inwhich significant increases were obtained consistently.Several hundred experiments with significant multiplicationhave been carried out by the laboratories involved in thisresearch (Fieldsteel et al., 1981; Fieldsteel et al., 1982;Norris, 1982; Cox et al., 1984; Levy, 1984; Konishi et al.,1986; Norris and Edmondson, 1986; Norris andEdmondson, 1987; Norris and Edmondson, 1988; Rileyand Cox, 1988; Cox et al., 1990). Following a lag period of36 to 48 hours, T. pallidum multiplies with a doubling timeof 35 to 40 hours (Figure 3), consistent with its slowreplication rate in vivo (Magnuson and Eagle, 1948;Cumberland and Turner, 1949). Viability, as measured byboth motility and virulence, remain high during the first 12

Figure 3.Multiplication and motility of Treponema pallidum subsp. pallidum(Nichols) in the Fieldsteel et al. (Fieldsteel et al., 1981) culture system asmodified by Cox et al. (Cox et al., 1990). In this representative experiment,the number of T. pallidum increased ~100 fold over the 17 day period ofincubation, and motility (a measure of treponemal viability) was maintainedat a high level.

Figure 4.Scanning electron micrographs of T. pallidum on the surface ofSF1Ep cottontail rabbit epithelial cells after 9 days (A) and 12 days (B) of invitro culture. These clusters may represent microcolonies resulting fromthe division of a single organism. Bars = 1 µm. Reprinted with permissionfrom (Fieldsteel et al., 1981).


days of culture. The yield of T. pallidum per standard 25cm2 culture is limited to 2 x 108 to 5 x 108 regardless of theinoculum size (within the range of 1 x 106 to 1 x 108). Thisceiling is most likely due to the depletion of one or moregrowth-limiting components by the treponemes, or to theaccumulation of toxic products. Multiplication typicallyceases after 10 to 12 days of in vitro culture. Continuousculture has not as yet been achieved, although promisingsubculture results have been obtained (see Serial Passageof T. pallidum below).

In vitro multiplication of T. pallidum requires thecombination of several key elements, as originallydescribed by Fieldsteel et al. (Fieldsteel et al., 1981) andmodified by Cox et al. (Cox et al., 1990; Cox, 1994) (Table2). Treponemal growth in this system only occurs in thepresence of mammalian cells; Sf1Ep cottontail rabbitepithelial cells and RAB-9 rabbit fibroblasts are particularlyeffective. T. pallidum extracted from infected rabbit tissueis inoculated into glass or plastic tissue culture flaskscontaining tissue culture cells and a modified tissue culturemedium. The treponemes adhere to the mammalian cellsand appear to multiply in microcolonies on the cell surface(Figure 4).

Serum, glucose, and dithiothreitol (an antioxidant) arethe most critical medium components. Only certain lots offetal bovine serum or calf serum support T. pallidum survivaland growth; 20% is the optimal concentration (Fieldsteelet al., 1981; Fieldsteel et al., 1982; Norris and Edmondson,1987). Human serum will also support growth (Norris andEdmondson, 1986). The required component(s) presentin serum are associated with the protein fraction (Norrisand Edmondson, 1986) and are thought to be lipids boundto serum proteins, which are also required by otherspirochetes, including easily cultivable Treponema,Borrelia, and Leptospira (Smibert, 1973; Ellinghausen,1976; Smibert, 1976; Van Horn and Smibert, 1983).

Another important component of the Fieldsteel et al.culture system is a 1:30 dilution of testis extract (TEx). Asoriginally described (Fieldsteel et al., 1981), TEx wasprepared by centrifugation or filtration of the infected testisextract used for culture inoculation. Without this component,growth is 30% to 50% less than in cultures with TEx. Theextract can also be obtained from uninfected rat, hamster,or rabbit testes (Fieldsteel et al., 1982). Heat inactivation

of TEx (56°C for 30 min.) improves treponemalmultiplication by 50 to 75 percent (D. L. Cox, unpublished),presumably by inactivating complement or othercomponents extracted from the tissue. A 1:60 dilution ofextracts of rabbit brain or flexor muscle can substitute forTEx in the culture system (D. L. Cox, unpublished). Bothof these tissues contain high concentrations of three potentantioxidants, carnosine, homocarnosine, and anserine(Kohen et al., 1988). However, the exact nature or activitiesof the stimulatory components present in tissue extractshave not as yet been determined.

It is now well established that T. pallidum is amicroaerophilic organism. The oxygen concentration rangethat is best for survival and multiplication is 1.5 to 5 percent(Figure 5) (Cover et al., 1982; Fieldsteel et al., 1982; Coxet al., 1990), although higher concentrations can be usedif additional antioxidants are present (Cox et al., 1990).Temperature is also critical, with optimal growth occurringat 33°C to 35°C (Fieldsteel et al., 1982).

Cox et al. (Cox et al., 1990) improved the averageculture yield by approximately two-fold by severalmodifications of the incubation conditions, including theaddition of oxygen radical scavengers (Table 2).Incremental increases in growth and survival resulting fromrefinements of the culture system could lead to continuousculture, just as multiplication in primary cultures wasachieved by the additive effects of several key factors.

Extended survival and growth of venereal syphilisstrains other than the Nichols strain have also beenobtained (Cox et al., 1984; Kohen et al., 1988), althoughless multiplication was observed. Little or no multiplicationwas obtained with yaws and endemic syphilis isolatesunder the ‘standard’ Fieldsteel et al. conditions (Cox et al.,1984).

The restricted growth obtained in vitro as comparedto in infected tissue may reflect differences in the tissueculture environment and the in vivo milieu (Norris andEdmondson, 1987). Tissue conditions are relativelyhomeostatic, whereas the in vitro environment changessignificantly and may eventually produce toxic conditionssuch as low pH or high redox potential. Depletion ofessential nutrients that are supplied continuously in vivowould result in starvation, limiting the number ofreplications. However, simple medium replacement

Table 2. Key Components of the Fieldsteel et al. (Fieldsteel et al., 1981) Culture System

1. Tissue Culture Sf1Ep cottontail rabbit epithelial cells2. Medium A modified form of Eagle’s MEM containing 20% heat-inactivated fetal bovine serum, 0.66 mM dithiothreitol, and testicular extract3. Atmosphere Microaerobic (1% to 3% O2), 5% CO2, balance N24. Temperature 33°C to 35°C

Cox et al. (Cox et al., 1990; Cox, 1994) Modifications

1. Tissue Culture Sf1Ep cottontail rabbit epithelial cells orRAB9 rabbit fibroblast cellsStatic monolayer or microcarrier bead (107) cultures

2. Medium T. pallidum Culture Medium (TpCM) - modified BRMM with reduced amounts of essential (50%) and nonessential (25%) aminoacids and vitamins (25%). Addition of oxygen radical scavengers superoxide dismutase (25 U/ml), catalase (10 U/ml), CoCl2(21 nM), cocarboxlyase (4.3 nM), histidine (0.23 mM), mannitol (0.55 mM). Heat inactivation of testis extract.

3. Atmosphere 3 to 4 percent O2, 5% C02, balance N24. Temperature 34°C to 35°C5. Results of Two-fold increase in total growth; treponemes remain highly motile for 17 to19 days; cytopathic effects observed in mammalian

Modifications cell cultures with >100 T. pallidum per cell; limited success with serial passage (see Figure 6).

48 Norris et al.

prolongs multiplication and survival only slightly (S. J.Norris, unpublished). The tissue cells may producenutrients or detoxifying compounds or activities (e.g.oxygen radical scavengers), thereby providing the requiredmicroenvironment for growth. Irreversible changes (suchas DNA breakage (Steiner et al., 1984)) must graduallyaccumulate so that the organisms eventually lose theirability to replicate (i.e. their replicative potential), even underpermissive conditions as exemplified by reinoculation intorabbits. Creation of a tissue-like environment (extremelyhigh cell density and homeostatic conditions) using capillaryperfusion cultures or tissue explant techniques (Steinhardt,1913) may provide the conditions required for continuousmultiplication, but such cultures are technically difficult tomaintain (S.J.N., unpublished).

Alternate Methods for CultivationTwo additional modifications of the Fieldsteel et al.cultivation system were developed. First, Riley and Cox(Riley and Cox, 1988) described a suspension culturesystem using Sf1Ep cells grown on microcarrier beads.Suspension cultures present several advantages overconventional static monolayer cultures. 1) Nondisruptivepassage of infected cells can be accomplished easily with

suspension cultures. 2) Beads carrying other cell typescan be added to the suspensions to more closely simulatetissue conditions. 3) Culture conditions (i.e. pH, glucose,other nutrients, etc.) can be more easily monitored andadjusted in suspension flasks than in conventional tissueculture flasks by using a chemostatic system. 4) They canbe readily adapted to large scale production of treponemes.

In the second modification, T. pallidum cultivation hasbeen adapted to 6- and 24-well microtiter plates (D. L. Cox,unpublished). For 24 well plates, the optimal volume ofTpCM medium is 1.25 to 1.5 ml; for 6 well plates, 5 ml isused. The optimal Sf1Ep cell concentration is 4 x 104 for24 well plates and 2.5 x 105 for 6 well plates. Microtiterplate cultures are ideal for experiments exploring manyvariables (such as antimicrobial susceptibility testing).

Serial Passage of T. pallidum In VitroThe primary goals of T. pallidum cultivation studies are 1)continuous in vitro passage, 2) eventual replacement ofthe rabbit with in vitro cultures as the source of organismsfor research and diagnostic reagents, and 3) elucidationof the physiologic requirements of T. pallidum and theirrelationship to the pathogenesis of syphilis. It is unlikelythat culture of T. pallidum will ever be a practical approachto the routine laboratory diagnosis of syphilis.

T. pallidum suspensions for passage from one cultureto another can be prepared from culture supernatant, frombacteria dissociated from the mammalian cell monolayersby mechanical disruption or by treatment with trypsin orEDTA (Norris and Edmondson, 1987). Alternatively,microcarrier bead cultures with adherant treponemes canbe transferred intact to fresh cultures (Riley and Cox, 1988).Initial attempts at subculture using the Fieldsteel et al.system resulted in limited multiplication (Norris andEdmondson, 1987; D.L. Cox, unpublished). Culture yieldsdecreased as the age of the primary culture increased; ina study by Norris and Edmondson (Norris and Edmondson,1987), increases of 18.1, 5.7, 3.0, and 0.75 fold wereobtained following subculture on days 3, 6, 9, and 12,respectively. As a result, the maximal combined growthobtained in primary and secondary cultures was only three-fold greater than in primary cultures alone (Norris andEdmondson, 1987). This outcome indicates that T. palliduma progressive loss of replicative potential under theconditions of the ‘standard’ Fieldsteel et al. system.

More promising results were obtained using the Coxet al. modifications (Table 2). A series of 12 experiments

Figure 5. Influence of atmospheric oxygen levels on the in vitro multiplicationof T. pallidum in BRMM medium using the Fieldsteel et al. culture system(closed circles). Addition of the oxygen radical scavengers cobalt chloride,cocarboxylase, mannitol, histidine, superoxide dismutase, and catalase inthe medium TpCM permitted treponemal growth at higher oxygenconcentrations (open circles). Reprinted with permission from Cox et al.,(Cox et al., 1990).

Table 3. Summary of Physiology and Growth Requirements

1. Oxygen utilization and toxicity. T. pallidum is microaerophilic. It requires oxygen for energy production, but is extremely sensitive to reactive oxygen species.2. Energy production. T. pallidum possesses complete glycolysis and hexose monophosphate shunt pathways, but lacks Krebs (tricarboxylic acid) cycle and oxidative phosphorylation

pathways.3. Nutritional requirements.

a. Carbon source - D-glucose, maltose, and mannose are metabolized by and support the growth of T. pallidum, but other sugars apparently can not be utilized.b. Amino acids and vitamins. T. pallidum lacks pathways for biosynthesis of most amino acids and vitamins. It is able to utilize and incorporate exogenously supplied

amino acids.c. Serum. Treponemal survival and growth is dependent upon components present in the protein fraction of serum. Serum apparently provides fatty acids and other activities.

4. Temperature. T. pallidum survival and growth is highly temperature dependent both in vivo and in vitro. The optimal temperature range is 33°C to 35°C.5. Host cell interactions. T. pallidum readily adheres to mammalian cells. Host cells are required for multiplication of T. pallidum in the Fieldsteel et al. culture system, but the reason

for this dependence is not known.6. Other activities.

a. Motility. The motility of T. pallidum promotes dissemination.b. Toxicity. T. pallidum does not appear to produce any potent toxins, but may exert cytopathic effects on mammalian cells when present at extremely high concentrations.c. Capsule. Although it has been postulated that T. pallidum produces a capsule, the mucoid material present in T. pallidum-infected tissue is predominantly host-derived. The

genome lacks recognizable capsule biosynthesis genes.


fresh cultures, the resulting passage grew only 5.2-fold,but >95% of the treponemes were still motile after a totalof 28 days of in vitro culture. The overall increase obtainedwas 2057-fold, equivalent to 11 successive cell divisions.

It must be emphasized that these results have not beenobtained consistently in every experiment. It is not clearlyunderstood why enhanced growth was obtained in someexperiments and not in others. Given the fastidious natureof T. pallidum and our limited knowledge of its growthrequirements, the sporadic success of serial passage isnot unexpected. Further definition and optimization of T.pallidum growth requirements should improve culturestability and reproducibility and hence may lead tocontinuous in vitro passage.

Physiology

A summary of our current knowledge of the physiology andgrowth requirements of T. pallidum is given in Table 4. Amore extensive discussion of each of these topics isprovided below.

Figure 6.Serial passage of T. pallidum subsp. pallidum in tissue culture. Results represent 4 experiments in which significant enhancement of treponemalgrowth was observed in secondary and tertiary cultures; little or no enhancement of growth relative to the primary cultures was obtained in 8 other experiments.Cell cultures consisted of standard monolayers of Sf1Ep cells (Experiments 1 and 2), microcarrier bead suspension cultures of Sf1Ep cells (Experiment 3),or monolayers of RAB-9 cells (Experiment 4). In these experiments, the maximum cumulative fold increase was 136, 445, 1,691, and 2,057, respectively (D.L. Cox, unpublished data).

were performed in which T. pallidum was passaged 2 or 3times in vitro, using either monolayer cultures or suspensioncultures as the source of inoculum (D. L. Cox, unpublished).Significant enhancement of multiplication was obtained infour experiments, as depicted in Figure 6. The first twosubculture studies were performed with standardmonolayer cultures of Sf1Ep cells and resulted incumulative increases of 136- and 445-fold in the numberof T. pallidum, as compared to 26- and 61-fold increasesin primary cultures without subculture. The third experimentused microcarrier suspension cultures of Sf1Ep cells.Microcarrier beads with attached Sf1Ep cells andtreponemes were passed after one week of culture, whena 21-fold increase had already occurred. During the secondpassage, the treponemes multiplied 81-fold over anadditional 12 days, producing a cumulative increase ofnearly 1700-fold. The fourth successful attempt was in staticmonolayer cultures of RAB-9 cells (a rabbit skin fibroblastcell line, ATCC CRL 1414). Three successive passageswere performed; secondary cultures were established after7 days of in vitro culture, and these were subcultured at 18days. When the secondary cultures were transferred to

50 Norris et al.

The Microaerophilic Nature of T. pallidumAlthough long thought to be an obligate anaerobe, it isnow clear that T. pallidum is a microaerophilic organism.Its microaerophilic nature results from the balance betweendependence upon oxygen for energy production on onehand and extreme susceptibility to the toxic effects ofoxygen radicals on the other. In 1974, Cox and Barber(Cox and Barber, 1974) reported that suspensions of T.pallidum extracted from rabbit tissue consumed oxygen ata rate similar to that of aerobic bacteria. This finding wasinitially viewed with skepticism, but several other lines ofevidence now show that the organism requires oxygen forenergy production. First, it utilizes oxygen as a terminalelectron acceptor (Lysko and Cox, 1977; Lysko and Cox,1978; Barbieri and Cox, 1981) (see Energy Productionbelow). Second, anabolic activity, as measured byincorporation of radioactive precursors in protein, RNA, andDNA, is maximal in the presence of low to moderateconcentrations of oxygen (Baseman et al., 1976; Nicholsand Baseman, 1978; Sandok and Jenkin, 1978; Basemanet al., 1979; Norris et al., 1980; Cover et al., 1982). Third,the survival of T. pallidum is prolonged by the presence of1.5 to 5 percent O2, both in cell-free systems (Norris et al.,1978; Jenkin and Sandok, 1983) and in the presence oftissue culture cells (Fitzgerald et al., 1977; Sandok et al.,1978; Fieldsteel et al., 1979). Lastly, multiplication of T.pallidum in the Fieldsteel et al. system is dependent uponthe presence of oxygen (Fieldsteel et al., 1982; Cox et al.,1990). As shown in Figure 5, growth in BRMM medium ismaximal at 3% atmospheric 02 and drops off dramaticallyas oxygen levels are either increased or decreased.

Despite this dependence, T. pallidum is extremelysusceptible to oxygen toxicity and dies within ~4 hours ofexposure to atmospheric levels of O2 (21%) if reducingagents are absent from the medium (Norris et al., 1978;Cover et al., 1982). This sensitivity is apparently due tothe organism’s lack of superoxide dismutase, catalase, andother oxygen radical scavengers, as confirmed by thegenome sequence (Fraser et al., 1998). Superoxidedismutase, which catalyzes the conversion of superoxideanions to hydrogen peroxide and water, has not beendetected in T. pallidum preparations (Austin et al., 1981;Steiner et al., 1984). Austin et al. (Austin et al., 1981) foundthat catalase activity was present in cell free extracts of T.pallidum. However, Steiner et al. (Steiner et al., 1984)showed that the catalase present was a contaminant fromthe rabbit tissue. It had electrophoretic mobility identical tothat of rabbit catalase, and its activity was neutralized bygoat anti-rabbit catalase antiserum. Hence T. pallidumapparently lacks two enzyme activities that are commonlyfound in aerobic organisms, but absent in obligateanaerobes. A deficiency in protective mechanisms againsttoxic products of oxygen most likely accounts for oxygensensitivity.

Recently two independent groups (Jovanovi’c et al.,2000; Lombard et al., 2000) reported the presence of a 26kDa iron-containing protein in T. pallidum (TP0823) withsuperoxide reductase activity. Like superoxide dismutase,superoxide reductase catalyzes the conversion ofsuperoxide anion to peroxide, but requires the presenceof a reducing cofactor such as NAD(P)H. The treponemalenzyme has very similar characteristics to neelaredoxin,

which has typically been found in hyperthermophilic andsulfate reducing bacteria. Thus, its discovery in T. pallidummay indicate that the syphilis spirochete copes withoxidative stress by a primitive mechanism not found in otherpathogenic organisms. As described in greater detail inthe section on metabolism, the genome of T. pallidumencodes homologs (TP0509 and TP0921, respectively) tothe Salmonella typhimurium alkyl hydroperoxide reductaseenzyme system proteins, AhpC and AhpF, which usesNAD(P)H to reduce peroxide to water (Jovanovi’c et al.,2000). This homolog may provide the mechanism forperoxide detoxification, but this possibility has yet to beconfirmed.

The mechanisms of oxygen-dependent killing are notknown. However, DNA damage manifested as single-stranded breaks in the genomic DNA has been notedfollowing exposure of T. pallidum to hydrogen peroxide(Steiner et al., 1984). This damage was repaired very slowlyand inefficiently in comparison to E. coli a repair proficientorganism (Steiner et al., 1984). A factor that could contributeto the poor repair of DNA damage due to oxygen toxicitymay be the high proportion of cysteine (2.4%) in thepredicted amino acid sequence of the important DNA repairenzyme DNA polymerase I (PolA) (Rodes et al., 2000).This level of cysteine is the higher than that of in any PolAsequenced to date and is 7.5 times higher than that foundin E. coli (0.32%). The abnormally high cysteinecomposition could increase the susceptibility of treponemalpolA to inactivation by oxygen and its byproducts (Rodeset al., 2000).

As with anaerobes, oxygen toxicity against T. pallidumcan be neutralized by addition of reducing agents such assulfhydryl compounds. Contrary to popular belief, reducingagents do not remove molecular oxygen from the medium;rather, they react with reactive oxygen intermediates (e.g.hydrogen peroxide, superoxide anion, and hydroxylradical), preventing their reaction with cell components.Dithiothreitol appears to be the most effective in prolongingsurvival, although cysteine, glutathione, and sodiummetabisulfite have also been shown to have protectiveactivity (Nelson, 1948; Nelson and Steinman, 1948; Weber,1960; Fitzgerald et al., 1977; Norris et al., 1978; Sandoket al., 1978; Chalmers and Taylor-Robinson, 1979;Fieldsteel et al., 1979; Fitzgerald et al., 1980; Fitzgerald,1983; Cox et al., 1990). Other oxygen radical scavengers,including cobalt chloride, cocarboxylase, mannitol, andhistidine, also increase survival (Steiner et al., 1983) andreplication of T. pallidum in the Fieldsteel et al. system (Coxet al., 1990). Addition of the enzymes superoxide dismutaseand catalase resulted in a 38% increase in yield in thissystem, although the efficacy of catalase may be limitedby its rapid inactivation (Cox et al., 1990). Overall, Cox etal. (Cox et al., 1990) were able to increase average growthfrom 35-fold to 70-fold by using a medium that contains allof the above antioxidants and enzymes. Peroxidases mayalso have protective activity (Steiner et al., 1983).

Protection of T. pallidum from reactive oxygenintermediates is critical in attempts to achieve continuousin vitro multiplication. Mammalian cells may promote thesurvival and growth of T. pallidum (both in vivo and in vitro)by providing this activity (Steiner et al., 1983).


Temperature DependenceThe survival and growth of the T. pallidum subspeciesexhibit marked temperature dependence, both in vivo andin vitro. During human syphilitic infection, spirochetes canbe found in any area of the body, in some cases causingfulminant infections in internal regions; the most obviousexample is congenital syphilis, where organisms can befound in great numbers in the fetus and placenta. ThereforeT. pallidum subsp. pallidum is capable of survival andgrowth at 37°C. However, multiplication appears to befavored in low temperature areas of the body, most notablythe skin. During secondary syphilis, the disseminatedinfection seems to be most severe (as well as most obvious)in the skin, although internal organs are also affected. Yawsand pinta lesions tend to be located on the distalextremities, which may also be related to temperatureeffects (Hollander, 1981). It has been theorized that thegeographic distribution of the nonvenereal treponematoses(yaws and pinta in tropical regions, endemic syphilis in aridand temperate regions) is influenced by differential effectsof climate on the transmission, growth and pathogenesisof the causative organisms (Hackett, 1963). A moreextreme view (not widely held) is that all human treponemalinfections are caused by the same organism, and variationin manifestations is due to climate and mode oftransmission (Hollander, 1981).

The temperature effect is dramatic in experimentalinfection of rabbits, as reviewed by Turner and Hollander(Turner and Hollander, 1957). Because of the higher bodytemperature, fulminant T. pallidum multiplication and lesionformation occurs only in exposed areas of the skin and inthe testes (Hollander and Turner, 1954). Infection and lesiondevelopment are optimal at cool room temperatures of 16to 20°C. There is also a lower temperature limit in vivo, asdemonstrated in a dramatic experiment by Hollander andTurner (Hollander and Turner, 1954). Rabbits inoculatedintradermally on the ear did not develop lesions. If, however,a unilateral cervical sympathectomy was performed(causing vasodilation of the ipsolateral face and ear),lesions developed in the warm, vasodilated ear (meantemperature = 29°C to 34°C) but not the cold ear (22°C to27°C). From these studies, the authors concluded that theoptimal tissue temperature range for T. pallidum infectionis between 30 and 35°C.

Very similar results have been obtained in vitro, wherethe optimal range is 33 to 35°C. Survival is extendedprogressively as temperatures are reduced from 40° to20°C (Weber, 1960), and motility and virulence can bepreserved for at least a week at 4°C (Turner and Hollander,1957; J. N. Miller, unpublished). However, the organismsare also metabolically inactive at the lower temperatures.Fieldsteel et al. (Fieldsteel et al., 1982) showed that optimalmultiplication of T. pallidum occurred at temperatures of33 to 35°C. Similarly, Baseman and Hayes (Baseman andHayes, 1974) found that incorporation of [3H]-labeled aminoacids into protein was maximal at 32 to 36°C. T. pallidumrapidly loses its motility and anabolic potential attemperatures > 38°C, indicating the presence of yet anotherphysiologic limit. Suspensions can be rendered nonviableby incubation at 45°C for 30 minutes.

The upper temperature limit may be due in part to thelack of a heat shock response in T. pallidum (Norris, 1991;

Stamm et al., 1991). In most organisms, exposure to hightemperatures results in increased expression of proteinsof the heat shock regulon, including GroEL and DnaK(Neidhardt and van Bogelin, 1987). These chaperonins areinvolved in protein folding and apparently help to counteractthe detrimental effects of high temperatures. No increasein the expression of these proteins has been observed inT. pallidum incubated at elevated temperatures. Thisunusual deficiency may represent an evolutionary loss ofadaptibility.

Metabolism

Much of the experimental evidence regarding the catabolicactivities and energy production of T. pallidum is based onstudies conducted during the 1970’s in the laboratories ofC. D. Cox and J. B. Baseman (reviewed by (Cox, 1983)).Glucose apparently serves as the principal carbon andenergy source of T. pallidum, although pyruvate can alsobe metabolized. Of 22 carbohydrates tested, only glucoseand pyruvate were degraded to CO2 (Nichols andBaseman, 1975; Schiller and Cox, 1977). D-glucose, itsdisaccharide maltose, and mannose are capable ofsupporting multiplication of T. pallidum in the Fieldsteel etal. system, whereas a number of other hexoses, pentoses,

Figure 7. Assessment of carbohydrate utilization by in vitro culture. T.pallidum subsp. pallidum Nichols was incubated in the Sf1Ep cell culturesystem in the presence of 2.5 mg/ml of the carbohydrates shown, and thefold increase in numbers of T. pallidum per culture was assessed on days7, 10, and 12 of culture. Substantial multiplication of T. pallidum occurredonly in the presence of D-glucose, maltose, and mannose. The datarepresent the combined results of 6 experiments (S. J. Norris and N. J.Farley, unpublished data).

52 Norris et al.

and pyruvate are ineffective in this regard (Figure 7; S. J.Norris and N. J. Farley, unpublished). The spirochete isincapable of metabolizing fatty acids via ß-oxidation(Schiller and Cox, 1977), further restricting the substratesavailable for energy production.

T. pallidum has an active glycolytic pathway, but genesencoding the enzymes involved in the Krebs tricarboxylicacid cycle are completely absent from the genome (Fraseret al., 1998). Schiller and Cox (Schiller and Cox, 1977)showed that cell-free extracts contain the enzymaticactivities associated with the Embden-Meyerhoff-Parnasand the hexose monophosphate shunt pathways. Althoughtwo of the eight enzyme activities of the Krebs cycle weredetected in T. pallidum extracts by Schiller et al. (Schillerand Cox, 1977), genes encoding these well-conservedenzymes could not be identified in the T. pallidum genomesequence (Fraser et al., 1998). Therefore it is likely thatthe activities detected resulted from contamination withrabbit testicular tissue. The final products of glucosecatabolism are acetate, lactate, and CO2, with the relativequantities of acetate and lactate produced being dependentupon oxygen concentration (Nichols and Baseman, 1975;Barbieri and Cox, 1979)

Oxygen Utilization and ToxicityLysko et al. (Lysko and Cox, 1978) reported the presenceof an active terminal electron transport system in T.pallidum, with oxygen serving as the final electron acceptor.Flavoproteins and cytochromes b, c, and o were detectedspectrophotometrically in these studies. Again, theseactivities were apparently due to contaminating rabbitconstituents, inasmuch as genes encoding highlyconserved electron transport proteins could not be identifiedin the T. pallidum genome. Enzymes responsible forsynthesis of quinones and other electron transportcompounds are also absent. If a terminal electron transportsystem exists in T. pallidum, it must consist of gene productswith no apparent homology to known electron transportconstituents.

The only gene product in the T. pallidum genome thatclearly utilizes O2 as a substrate is NADH oxidase (Fraseret al., 1998), which converts NADH and O2 to NAD+ andwater. NADH oxidase activity is apparently associated withthe inner membrane in crude lysates of T. pallidum (Norrisand Group, 1993). The homologous enzyme was shownrecently to have a protective effect against oxygen toxicityin B. hyodysenteriae (Stanton et al., 1999).

Overall, T. pallidum appears to be most similar to lacticacid bacteria in terms of its relationship with oxygen. Lacticacid bacteria, which include Lactobacillus, Streptococcus,and some Bacillus species, are facultatively or obligatelyanaerobic bacteria. Like T. pallidum, they lack a functionaltricarboxylic cycle, cytochromes and other heme proteins,and other evidence of an electron transport chain resultingin oxidative phosphorylation. In lactic acid bacteria, ATPproduction is primarily dependent upon the glycolyticpathway and is therefore largely independent of thepresence of O2. NADH oxidase (Nox) appears to play tworoles in these organisms: 1) oxidation of excess NADH;and 2) removal of molecular O2 and the potential forreactive oxygen intermediate (ROI) production. NADH isproduced by the glycolytic pathway, and an NADH/NAD+

imbalance would result if the excess NADH were notdissipated. Nox fulfills this role in the absence of an electrontransport system. O2 by itself is relatively non-reactive andnon-toxic, and only becomes toxic when converted toreactive oxygen intermediates (ROI’s) such as H2O2,superoxide anion, and hydroxyl radical. Because lactic acidbacteria lack catalase and (in some cases) superoxidedismutase, it is necessary to limit ROI exposure by othermeans. One of the best-studied systems is that ofAmphibacillus xylanus, which utilizes Nox coupled with alkylhydroperoxide reductase (AhpC) (Niimura et al., 1993;Niimura et al., 1995; Niimura and Massey, 1996). In thiscase, Nox actually converts O2 to H2O2, which is thenscavenged through a coupled reaction with AhpC. A similarsystem exists in S. typhimurium, and A. xylanus Nox andS. typhimurium AhpC components can complement oneanother to catalyze the 4-electron reduction of oxygen towater (Niimura et al., 1995). In Streptococcus mutans, thesystem is more complex, with the coexistence of two NADHoxidase isoforms, Nox-1 and Nox-2, along with AhpC. Nox-1 produces H2O2 and is coupled with AhpC to convert thefinal product to water (Poole et al., 2000). Nox-2 catalyzesthe conversion of NADH and O2 into H2O without an H2O2intermediate. In S. mutans, the combination of Nox-1 andAhpC appears to play an important role in reducing oxygentoxicity, whereas Nox-2 is primarily involved in maintainingan appropriate NADH/NAD+ red-ox state (Higuchi et al.,1999). The putative NADH oxidase of T. pallidum (TP0921)appears to be “Nox-2 like”, in that it is highly homologousto S. mutans Nox-2 (59% identity/75% similarity) and onlyweakly similar to Nox-1 (24%/41%). The T. pallidumgenome also encodes a highly conserved AhpC ortholog(TP0509), but potential Nox-1 candidates are more similarto thioredoxin reductase. Further functional studies will berequired to define the roles of the Nox and AhpC orthologsof T. pallidum in oxygen scavenging or utilization pathways.

Anabolic ActivityT. pallidum is fully capable of DNA, RNA, and proteinsynthesis as measured by incorporation of radiolabeledprecursors (Baseman and Hayes, 1974; Baseman et al.,1976; Baseman and Hayes, 1977; Nichols and Baseman,1978; Sandok et al., 1978; Baseman et al., 1979; Norris etal., 1980). Amino acids, adenine, and uracil are readilyincorporated; nucleosides are not incorporated asefficiently, probably because of reduced uptake.

(Barbieri et al., 1981) further demonstrated that carbonfrom glucose was incorporated into lipids, nucleic acids,and proteins. In lipids, this incorporation was restricted tophospholipids and glycolipids and was not found in freefatty acids and cardiolipin. These results indicate that fattyacids are not synthesized de novo, consistent with theobservation that fatty acids are acquired from exogenoussources and incorporated into lipids in an unmodified form(Nichols and Baseman, 1975; Schiller and Cox, 1977). Thedistribution of labeled carbon atoms was limited to theribose and deoxyribose portions in nucleic acids and wasprimarily associated with the amino acid aspartate inproteins. These findings are consistent with the absenceof recognizable pathways for the synthesis of nucleic acidbases and most amino acids, based on the genomesequence (Fraser et al., 1998). However, some studies


show that T. pallidum is capable of at least limitedmultiplication in medium lacking amino acids and vitamins(see Nutritional Requirements below). In addition,Gherardini et al. (Gherardini et al., 1990) showed that T.pallidum DNA contained genes capable of complementingE. coli proC mutants defective in proline biosynthesis,indicating that T. pallidum is capable of synthesizing at leastsome amino acids. The gene complementation approachshould be extremely valuable as a surrogate geneticsystem for further defining the metabolic capabilities of T.pallidum based on the genome sequence.

Nutritional RequirementsAssessment of the nutritional requirements of T. pallidumhas been complicated by the inability to obtain multiplicationin pure culture. In addition, most T. pallidum survival mediaare extremely complex and include undefined components(such as serum or tissue extract). Although the mediumdeveloped by Nelson (Nelson, 1948) is relatively simple,survival is poor unless the infected tissue is extracted forprolonged periods (~1 hour) permitting the diffusion oftissue components into the medium. The availability of theFieldsteel et al. system (Fieldsteel et al., 1981) permitsthe analysis of factors affecting multiplication, although thepresence of tissue culture cells complicates interpretation.

As indicated above, there appears to be an absoluterequirement for D-glucose as a carbon and energy source(Nichols and Baseman, 1975; Schiller and Cox, 1977). Asshown in Figure 7, D-glucose, the glucose disaccharidemaltose, and mannose were the only sugar compoundsthat were capable of supporting the in vitro multiplicationof T. pallidum (S. J. Norris and N. J. Farley, unpublisheddata). To further delineate the growth requirements, theamino acid and vitamin components were omitted fromthe culture medium (BRMM) with the expectation thattreponemal multiplication would not be supported. Instead,the spirochetes in this deficient medium (BRMM-D)multiplied as well as, and in some cases better than,cultures in the complete medium. Treponemal survival andmultiplication were observed in BRMM-D even if dialyzedserum was used in place of whole fetal bovine serum.Therefore T. pallidum was capable of surviving andmultiplying in a medium containing glucose, salts,dithiothreitol and dialyzed serum. It is of course possible(or likely) that the tissue culture cells are secreting nutrientsrequired by the treponemes. Surprisingly, the Sf1Ep rabbitepithelial cells used in the cultures also survived well underthese conditions, probably a reflection of the slow growthrate and low metabolic activity of these cells.

It has long been recognized that serum is an essentialmedium component for T. pallidum survival. Removal ofserum (and tissue extract, which also contains serumcomponents) results in loss of motility within minutes. Riceand Nelson (Rice and Nelson, 1951) identified a crystallinecompound derived from serum ultrafiltrate that replacedthe serum requirement; the compound was identified as anon-reducing hydrocarbon with a formula of C15H26O10,but was not characterized further. Only certain commerciallots of fetal bovine serum are effective in supporting thegrowth of T. pallidum. Many lots appear to contain toxiccomponents, which may relate to the method ofpreparation. Serum lots may also vary in the amounts of

beneficial components. D. L. Cox and coworkers(unpublished data) examined 70 lots of serum fromHyClone Laboratories, which routinely analyzes each lotof serum for more than 200 biochemical components. Byanalyzing the amount of treponemal multiplication relativeto serum composition, the concentrations of sevencompounds were found to correlate with improvedtreponemal growth: prostaglandin F1, erythrose,arachidonic acid, cytosine, malic acid, methionine, andestrone. Compounds that correlated with decreasedtreponemal multiplication were hemoglobin, 17-ß-estradiol,methionine sulfoxide, and free fatty acids (D. L. Cox,unpublished data). Further studies in which thesecompounds are added in varied amounts are needed toverify that they have stimulatory or inhibitory activities.

Norris and Edmondson (1986) found that serumcomponent(s) required for in vitro multiplication of T.pallidum were not soluble small molecular weightcompounds, but were instead associated with the proteinfraction. Dialyzed serum or a serum protein fractionprecipitated with polyethylene glycol supported the growthof T. pallidum in the Fieldsteel et al. culture system, whereasserum ultrafiltrate (containing small molecular weightcompounds) did not support either growth or retention ofmotility. It is likely that the required component(s) includelipids carried by albumin and other serum proteins. Asindicated previously, T. pallidum apparently is incapable ofsynthesizing long chain fatty acids, and incorporates themin lipids and lipoproteins in an unmodified form (Nicholsand Baseman, 1975; Schiller and Cox, 1977). All easilycultivable parasitic spirochetes, including Leptospira,Borrelia, and nonpathogenic Treponema species, requirefatty acids complexed with albumin or other serum proteins(Smibert, 1973; Ellinghausen, 1976; Smibert, 1976; VanHorn and Smibert, 1983). Studies with T. denticola and T.vincentii by Van Horn and Smibert (1983) indicated thatproteins present in the α-globulin fraction of serum mayserve as the principal source of lipids. Fatty acids may bereleased by lipases produced by the treponemes and thenbound by albumin, which serves a detoxifying function.Alderete and Baseman (Alderete and Baseman, 1989)showed that human plasma lipoproteins are bound by T.pallidum suspensions, and that this binding is enhancedby the presence of dextran sulfate. It is possible that T.pallidum may acquire lipids by direct binding to high-densityor low-density lipoproteins.

As described above, T. pallidum has many metabolicsimilarities to lactic acid bacteria. Although iron is requiredby most organisms, lactic acid bacteria are unusual in thatthey lack iron acquisition mechanisms, have few if anyproteins that require iron as a cofactor, and possesses Mn-requiring enzymes that fulfill functions usually provided byheme-containing proteins (Archibald, 1986). Posey andGherardini (2000) demonstrated recently that B. burgdorferigrew normally in the presence of iron chelators, and thatthe genome of B. burgdorferi has few detectable sequencespredicted to encode iron metalloproteins. In addition, B.burgdorferi does not accumulate detectable levels of iron,but accumulates manganese, calcium, and magnesium andrequires these divalent cations for growth (Posey andGherardini, 2000). T. pallidum also lacks genes encodingiron metalloproteins such as cytochromes, superoxide

54 Norris et al.

dismutase, catalase, and succinate dehydrogenase. Inaddition, it possesses an ABC transport operon that is tightlyregulated by Mn concentrations (Posey et al., 1999). It ispossible that T. pallidum, like lactic acid bacteria and B.burgdorferi, has developed an iron-independent lifestyle.However, Alderete et al. (Alderete et al., 1988) found thatlactoferrin and transferrin bound to the surface of T.pallidum, and the spirochete was particularly efficient atacquiring radiolabeled Fe++ from transferrin. This bindingactivity could potentially be involved in the acquisition ofiron during in vivo infection.

Interactions with Mammalian Cells

The association of T. pallidum with mammalian cells isimportant in terms of understanding both the physiology ofthe bacterium and the complex host-parasite interactionsthat occur during infection. The key aspects of thisinteraction have been reviewed previously (Fitzgerald,1983; Baseman et al., 1986) and are considered below.

AdherenceT. pallidum readily and tenaciously attaches to the surfaceof mammalian cells in culture (Fitzgerald et al., 1975;Fitzgerald et al., 1977; Fitzgerald et al., 1977; Hayes etal., 1977; Fitzgerald et al., 1982; Oakes et al., 1982; Quistet al., 1983; Wong et al., 1983; Baseman et al., 1986;Thomas et al., 1989). Literally hundreds of treponemescan associate with each tissue culture cell within minutes.Although adherence has been said to occur through a “tip-like organelle” (Hayes et al., 1977; Baseman et al., 1986),binding of T. pallidum to tissue culture cells can occur atany location on the spirochete (Konishi et al., 1986). Thepredominant occurrence of treponemes attached by theirend is due to the motility of the organism, which tends to

draw the attachment point to the end opposite of thedirection of translational motion; when the treponemereverses its direction of rotation, the attachment point shiftsalong the length of the organism to the other end. Thesame phenomenon is observed with spirochetes attachedto antibody-coated polystyrene beads (Charon et al., 1984).Therefore it is likely that the receptors for attachment areevenly distributed over the surface of the bacterium, ratherthan localized on the ends. In transmission electronmicroscopy studies, Konishi et al. (Konishi et al., 1986)noted the presence of an electron dense layer at thesuspected site of attachment which may represent a fusionpoint between the treponemal OM and the host cell’splasma membrane.

Adherence seems to be a common property amongpathogenic spirochetes, including B. burgdorferi.Attachment requires structural integrity and viability of boththe spirochete and the mammalian cell. Although somevariation in the extent of surface attachment has beenobserved, T. pallidum will adhere to virtually any nucleatedcell type, including fibroblasts, epithelial cells, endothelialcells, muscle fibers, and neurons; it does not adhere toerythrocytes.

It has been postulated that fibronectin plays a majorrole in this adherence (Hardy and Levin, 1983; Fitzgeraldet al., 1984; Fitzgerald and Repesh, 1985; Steiner and Sell,1985; Thomas et al., 1985; Thomas et al., 1985; Thomaset al., 1985; Baseman et al., 1986; Baughn, 1986; Baughn,1987). Fibronectin is a host extracellular matrix proteininvolved in cell-cell adherence in tissue. This dimericglycoprotein has several functional domains, includingthose that bind to heparin, gelatin, and cell surfacereceptors. In a model developed by Baseman andcoworkers (Baseman et al., 1986), the cell-binding domainsof fibronectin binds to both the treponeme and mammaliancell surface, essentially crosslinking the two cells. Severallines of evidence support this hypothesis. 1) T. pallidumadheres to fibronectin-coated surfaces (Hardy and Levin,1983; Fitzgerald et al., 1984). 2) Anti-fibronectin antibodyinhibits attachment; in particular, monoclonal antibodiesagainst the cell-binding domain decrease adherence(Thomas et al., 1985). 3) heptapeptides containing thespecific cell-binding domain sequence Arg-Gly-Asp-Serblocked adherence of T. pallidum to fibronectin, whereaspeptides in which one of these amino acids is changedare not inhibitory (Thomas et al., 1985).

The fact that attachment can be blocked to some extentby addition of anti-T. pallidum antiserum (Fitzgerald et al.,1984; Thomas et al., 1985) indicates that specific receptorson the surface of T. pallidum may be involved in cell binding.There is disagreement in the literature regarding thenumber and affinity of fibronectin receptors on T. pallidum(Steiner and Sell, 1985; Thomas et al., 1985; Baughn, 1986;Baughn, 1987; Cox et al., 1992), but most estimatesindicate the presence of ~3,000 to 5,000 receptors per celland an affinity (Ka) of ~2 x 107 M-1 (Baughn, 1987).

Using a surface-specific radioimmunoassay, Cox etal. (Cox et al., 1992) provided evidence that the amount offibronectin bound to the OM of intact T. pallidum is muchless than that indicated by previous studies. Significantbinding of either 125I-labeled fibronectin or antibodiesdirected against fibronectin was not detected with this

Figure 8. In vitro system for assessing the susceptibility of T. pallidum subsp.pallidum (Norris and Edmondson, 1988), as demonstrated with thecephalosporin ceftriaxone. The Nichols strain of T. pallidum was incubatedin the Fieldsteel et al. culture system in presence of varied concentrationsof ceftriaxone. After 7 days of incubation, the number of T. pallidum perculture was determined and compared with the original inoculum. Eachcurve represents a separate experiment, and each data point is the meanof three separate cultures. The minimal inhibitory concentration (MIC) isdefined as the lowest concentration that completely inhibits multiplication(culture yield < inoculum); in this instance, the average MIC for ceftriaxonewas 0.0007 micrograms/ml. This system provides a means of determiningboth the bacteriostatic and bactericidal activities of compounds against T.pallidum.


assay, although fibronectin readily associated withorganisms treated with detergent to remove the OM. Thusestimates of fibronectin binding is highly dependent on thecondition of the treponemes. The picture is complicatedfurther by the variable presence of host ground substance(Ziegler et al., 1976; Fitzgerald et al., 1979; Strugnell etal., 1984; Fitzgerald et al., 1985; van der Sluis et al., 1985;Strugnell et al., 1986; van der Sluis et al., 1987) and otherhost proteins (Lugtenberg and Van Alphen, 1983) on thesurface of freshly extracted T. pallidum, which could alsoincrease or decrease fibronectin binding.

Baseman and Hayes (Nelson and Mayer, 1949)identified three T. pallidum proteins as putative celladhesion receptors (adhesins). Detergent solubilized,radiolabeled T. pallidum proteins were incubated withformalin-fixed human epithelial cells; the cell monolayerwas washed and then assessed for the presence of boundtreponemal proteins. Three polypeptides, called P1 (Mr =89,500), P2 (37,000), and P3 (32,000) were found to beassociated with the cells. These same proteins are capableof binding fibronectin (Peterson et al., 1983), and arethought to share a common ‘domain’ involved in fibronectinbinding and cell adhesion (Thomas et al., 1985).Unfortunately, there is little evidence that these proteinsare surface-associated; in fact, P1 appears to be sameprotein as CfpA, the 83 kDa subunit of the cytoplasmicfilaments, an internal structure (Norris and Group, 1993;You et al., 1996). The genes encoding P1 and P2 werecloned into E. coli (Peterson et al., 1987), but furthercharacterization of these proteins has not been reported.

Host Cell Association and T. pallidum Survival andMultiplicationAs mentioned previously, the presence of mammalian cellspromotes the survival and multiplication of T. pallidum invitro (Steinhardt, 1913; Kast and Kolmer, 1943; Perry, 1948;Fitzgerald et al., 1975; Fieldsteel et al., 1977; Fitzgerald etal., 1977; Sandok et al., 1978; Fieldsteel et al., 1981;Fitzgerald, 1983). Sf1Ep, a slow-growing cottontail rabbitepithelial cell culture, is particularly effective in this regard(Fieldsteel et al., 1977). Mammalian cells may produce atissue-like microenvironment by providing nutrients,protective activities, or an anchoring site. Close associationis required; separation of the treponemes from themammalian cells by a permeable filter prevents theirgrowth. In addition, treponemal survival and multiplicationappears to depend on host cell protein synthesis, because2.5 µg/ml cycloheximide (which inhibits eukaryotic but notprokaryotic protein synthesis) decreases T. pallidummultiplication (D. L. Cox, unpublished). Further analysis isrequired to examine the nature of host cell dependenceand to determine whether medium factor(s) can beidentified which can supplant this requirement and hencepermit growth of T. pallidum in a host cell-free environment.

Tissue Invasion and DisseminationT. pallidum’s motility coupled with its ability to bind to hostcells provides a powerful mechanism for local and systemicdissemination. Most of the dissemination of treponemesfrom the initial nidus of infection is thought to behematogenous. Infectious T. pallidum adheres to isolatedcapillaries (Quist et al., 1983) and endothelial cells (Thomas

et al., 1989). T. pallidum also readily penetrates throughthe intercellular junctions of endothelial cell monolayers,as shown in a model system described by Thomas et al.(Thomas et al., 1988). Human or rabbit aortic endothelialcells cultured on polycarbonate filters form continuous tightjunctions, as demonstrated by transendothelial electricalresistance measurements (Thomas et al., 1988). Viable T.pallidum rapidly penetrate through the intercellularjunctions, so that treponemes placed on the top side ofthe monolayer can be found on the other side within 2hours. Heat-killed T. pallidum or the nonpathogenictreponeme T. phagedenis do not cross the endothelialbarrier in significant numbers. Riviere et al. (Riviere et al.,1989) took this model one step further by measuring themigration of T. pallidum through the mouse abdominal wall.Segments of the abdominal wall (consisting of the serosa,connective tissue and muscle layers) were placed in aculture chamber, forming a watertight barrier between thetwo sides. After 24 hours, up to 6 x 107 of the 5 x108

organisms placed in the chamber had migrated across theabdominal wall, whereas few, if any, T. phagedenis or heat-killed T. pallidum had crossed the barrier. Migrationoccurred only from the epithelial side to the connectivetissue side, not vice versa. The highly invasive nature ofthis pathogen is impressively demonstrated by this system.

This invasive activity may also be important in theestablishment of latent infection. Latency represents asteady state between active infection and eradication ofthe pathogen by the immune system. Because of itsinvasiveness, T. pallidum may penetrate to areas that areinaccessible to the humoral and cellular arms of the immuneresponse and hence avoid elimination (Medici, 1972). Sellet al. (Sell et al., 1985) examined the mechanisms ofimmunity to syphilis by reinfecting immune rabbitsintradermally with large numbers of viable T. pallidum. Mostof the organisms were eliminated with little inflammatoryresponse. Several days following infection, T. pallidumcould still be found in certain ‘protective niches’ (such asnerves, arrector pili muscles, hair follicles, and densecollagen bundles), thereby evading the immune response.During natural infection, small numbers of T. pallidum couldpersist in similar protective niches, leading to long-termlatent infection with the possible development of latemanifestations.

Apparently viable treponemes can be found within hostcells, either during active infection in vivo or in tissuecultures (Sykes and Miller, 1971; Lauderdale and Goldman,1972; Sykes et al., 1974). Intracellular localization couldpotentially be involved in latent infection.

CytotoxicityOne possible mechanism in the pathogenesis of syphilisis the creation of toxic products by T. pallidum. Fitzgeraldand coworkers (Fitzgerald et al., 1982; Oakes et al., 1982;Quist et al., 1983) have described cell disruption and othercytopathic effects in cultures of fibroblasts, epithelial cells,isolated capillaries, muscle cells, and neurons followingincubation with extremely high concentrations of T. pallidum(>9 x 107/ml). Lower concentrations of T. pallidum or cell-free supernatants have little, if any, toxic effect onmammalian cells.

Cytopathic effects have also been observed during in

56 Norris et al.

vitro culture of T. pallidum (D. L. Cox, unpublished).Enhanced treponemal multiplication and viability wasobtained with the Cox et al. modification (Cox et al., 1990),resulting in increased numbers of T. pallidum per host cell.Under these conditions, Sf1Ep and RAB-9 cells exhibitedcytopathic effects after 10 to 12 days of culture, and theseverity increased during the next 3 to 5 days. Titrationstudies determined that the effects occurred within 48 hoursafter there were >100 treponemes per cell; uninfectedcultures did not show these effects. The cellular swelling,detachment, and disintegration observed were similar tothe effects previously reported by Fitzgerald and coworkers(Fitzgerald et al., 1982; Oakes et al., 1982; Quist et al.,1983). The pH and glucose concentration of the mediumwere not abnormally low (pH 6.7-7.0; glucose 0.7 to 1.0mg/ml). The mitochondria of heavily infected cells exhibitedgreater fluorescence in the presence of the fluorescent dyerhodamine 123 than did lightly infected or non-infectedcells, indicating a higher membrane potential and metabolicactivity (D. L. Cox, unpublished). This phenomenoncommonly occurs in cells that have been damaged andmay correspond to the activation of cellular repairmechanisms (Goldstein and Korczack, 1981; Johnson etal., 1981; DeBiosio et al., 1987).

It remains to be determined whether this type ofcytotoxicity is active in vivo. Also, it is unclear if the toxicityobserved is specific in nature (i.e. involving certain toxinsor virulence factors) or is nonspecific. High concentrationsof any bacterium may bring about changes in cultureconditions leading to mammalian cell death; suchnonspecific cytotoxic effects are commonly seen inbacterially contaminated tissue cultures. Most tissuenecrosis that occurs during syphilitic infection seems to bedue to vessel occlusion or rupture in heavily infected tissue(such as chancres), or to tissue displacement caused byspace-filling inflammatory reactions (as in gummas).

Weinstock and coworkers (Weinstock et al., 1998;Norris and Weinstock, 2000; Weinstock et al., 2000;Weinstock et al., 2000) identified five T. pallidum genesencoding potential cytotoxins, based on amino acidsimilarities. These have been termed tlyC (TP0649), hlyIII(TP1037), hlyA (TP0026), hlyB (TP0027), and hlyC(TP0936). The identification of the homologous proteins inother organisms as cytolysins is tenuous, in that in mostcases the purified protein has not been demonstrated tohave hemolytic or cytolytic activity. Therefore it is necessaryto express and characterize the activities of the T. pallidumproteins. As is often the case with heterologous bacterialproteins, expression these T. pallidum proteins in E. colihas been problematic, requiring the examination of severaldifferent vectors (Weinstock et al., 2000). The reactivity ofthe recombinant proteins with sera from syphilis patientswas varied, with TlyC exhibiting the most consistentreactivity (Weinstock et al., 2000). The cytolytic activity ofthese products is under investigation.

Mucoid MaterialIt has long been noted that a mucoid material accumulatesin T. pallidum lesions, particularly during experimentalinfection of rabbits (Turner and Hollander, 1957;Christiansen, 1963; Ziegler et al., 1976; Fitzgerald andJohnson, 1979a; Fitzgerald et al., 1979; Strugnell et al.,

1984; Fitzgerald et al., 1985; van der Sluis et al., 1985;Strugnell et al., 1986; van der Sluis et al., 1987). Coupledwith the knowledge that freshly extracted treponemes areresistant to antibody binding and killing, it has beenpostulated that this material is a capsule or slime layerproduced by the bacterium, and that this layer protects T.pallidum from the immune response. Studies by Strugnelland coworkers (Strugnell et al., 1984; Strugnell et al., 1986)demonstrated that most of this material is actuallyproteoglycans synthesized by the host. Using slices ofinfected testicular tissue incubated in vitro, they found thatincorporation of [35S]-sulfate into the mucoid material wasblocked by the eukaryotic inhibitor cycloheximide but notby prokaryotic inhibitor erythromycin (Strugnell et al., 1986).[3 H]-labeled N-acetyl glucosamine is incorporated into highmolecular-weight material by T. pallidum, but this materialhas not been characterized (Strugnell et al., 1984). Thegenome of T. pallidum lacks the complex operonsassociated with capsular synthesis found in otherorganisms (Fraser et al., 1998). The host tissue apparentlyproduces excessive amounts of extracellular matrixcomponents in response to T. pallidum infection, particularlywhen the infected animals are treated with corticosteroids(Turner and Hollander, 1954).

Fitzgerald and coworkers (Fitzgerald and Johnson,1979b; Fitzgerald and Gannon, 1983; Fitzgerald andRepesh, 1987) have found that hyaluronidase activity isassociated with T. pallidum extracted from rabbit tissue,although it is unclear whether the activity is of bacterial orhost origin. It was hypothesized that hyaluronidasepromotes the dissemination of T. pallidum during the courseof infection (Fitzgerald and Repesh, 1987). Gene(s)responsible for this activity have not been identified.

Antimicrobial Susceptibility

One of the most clinically important properties of anyinfectious agent is its susceptibility to antimicrobial agents.In vivo therapy is still the gold standard in assessing theefficacy of antimicrobials against T. pallidum (Lukehart etal., 1984; Lukehart and Baker-Zander, 1987; Lukehart etal., 1990), but in vitro assays provide a means of screeningcompounds and defining effective concentrations. In thepast, loss of motility or virulence of T. pallidum or inhibitionof growth of nonpathogenic Treponema species have beenused to evaluate in vitro susceptibility (Rein, 1976).

Two additional methods have been developed. Thefirst utilizes the incorporation of [35S]-methionine to assessthe inhibitory effects of agents on macromolecular synthesisby T. pallidum (Stapleton et al., 1985; Stamm et al., 1988).This approach is particularly useful for assaying the activityof protein synthesis inhibitors and was used to demonstratethe resistance of a venereal syphilis isolate to erythromycin(Stamm et al., 1988). This isolate, Street Strain 14, hassince been shown to contain a single point mutation inboth copies of 23S rRNA, which may account for thisstrain’s resistance to macrolides (Stamm and Bergen,2000).

The other procedure (Norris and Edmondson, 1988)involves the addition of varied concentrations of anantimicrobial compound to T. pallidum cultured by theFieldsteel et al. procedure (Fieldsteel et al., 1981). After 7


days of incubation, the number of T. pallidum per culture isdetermined. This technique is a very sensitive measure ofgrowth inhibition, and can reproducibly measure activitywithin a two-fold dilution (Figure 8). The minimal inhibitoryconcentration (MIC) is calculated as the concentration thatcompletely inhibits T. pallidum multiplication (culture yieldat 7 days < inoculum). The minimal bactericidalconcentration (MBC) is estimated by inoculation of the T.pallidum cultures into rabbits after the 7-day incubationperiod; the lowest concentration that destroys infectivity isconsidered to be the MBC. This system has been used toassess the in vitro activity of cephalosporins, quinolones,and other compounds (Table 4) (Norris and Edmondson,1988; S.J. Norris and N.J. Farley, unpublished data). Thecephalosporins have low MICs in this assay, in agreementwith the high efficacy of ß-lactam antibiotics against T.pallidum. In contrast, the quinolone compounds testedgenerally have a low activity against T. pallidum, as is alsoindicated by in vivo studies in rabbits (S. J. Norris,unpublished). This insensitivity is consistent with thepredicted similarity of T. pallidum GyrA and GyrB to thoseof relatively quinolone-resistant organisms (Huang, 1996;Stamm et al., 1997; Fraser et al., 1998).

Summary

T. pallidum is an unusual organism in a number of respects.In addition to its spirochetal morphology, its outermembrane contains no lipopolysaccharide and relativelyfew intramembranous proteins; as such, it may evade theimmune response by presenting an antigenically inertsurface. It is also one of the few important humanpathogens that has not been cultured continuously in vitro.This hindrance is most likely related to a combination offactors including extreme nutritional requirements due todeficiencies in biosynthetic capabilities, the narrowequilibrium between oxygen dependence and toxicity, and

an undetermined dependence on mammalian cells thatmay involve one or both of these factors. In keeping withits invasive nature, the genome of T. pallidum yielded arelatively short list of potential virulence factors. However,further characterization of the tpr gene family, putativehemolysins, and other virulence factor candidates mayreveal the importance of these proteins in T. palliduminfection. The genome sequence provides raw data thatwill greatly enhance the examination of each of these areas.For example, gene products with signal peptidase I leadersequences would be expected to include outer membraneproteins, the apparent relationship between T. pallidum andlactic acid bacteria identifies Nox/AhpF, AhpC, andneelredoxin as being potential key factors in oxygenmetabolism, and ‘mining’ of the genome may yield as yetunidentified adhesins and other factors important indissemination. The real benefit of the genome sequencelies not in sequence gazing, but in facilitating the applicationof functional genomic approaches to understanding thebiology of T. pallidum.

References

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Table 4. Minimal inhibitory concentrations (MICs) and minimal bactericidalconcentrations (MBCs) of cephalosporins and quinolones against T. pallidumsubsp. pallidum (Nichols) in a tissue culture system (Norris and Edmondson,1988); S. J. Norris and N. J. Farley, unpublished).

Agent MIC (µg/ml) MBC (µg/ml)

CephalosporinsCeftriaxone 0.0007 0.002Cefetamet 0.04 NDCefteram 0.004 NDCeftazidime 0.007 ND

QuinolonesClinafloxacin 1 2Ciprofloxacin 5 4Difloxacin 10 NDNorfloxacin 6 NDPefloxacin 8 NDTemafloxacin 8 ND

Other agentsPenicillin G 0.0005 0.0025Tetracycline 0.2 0.5Erythromycin 0.005 0.005Spectinomycina 0.5 0.5

a Lack of efficacy in vivo due to poor tissue penetration (Rice et al., 1988).

58 Norris et al.

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Blanco, D.R., Walker, E.M., Haake, D.A., Champion, C.I., Miller, J.N. andLovett, M.A. 1990. Complement activation limits the rate of in vitrotreponemicidal activity and correlates with antibody-mediated aggregationof Treponema pallidum rare outer membrane protein. J. Immunol. 144:1914-1921.

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