Expression and sequence analysis of a Treponema pallidum gene, tpn38(b), encoding an exported protein with homology to T. pallidum and Borrelia burgdorferi proteins

ELSEVIER

FEMS Microbiology

Letters 135 (1996) 57-63

Expression and sequence analysis of a Treponema pallidum gene, tpn38(b), encoding an exported protein with homology to T. pallidum and Borrelia burgdo$eri proteins Lola V. Stamm a,*, John M. Hardham b,‘, Jonathan G. Frye a32 ;’ Program

in Infectious Discuses,

Department

of Epidemiology, CB#7400,

School of Public He&h.

Chapel

h Drpurtment of A4icrobiolog.vand Immunology. School

of Medicine.

Received 8 September

University of North Carolina,

242 Rosenuu Hull.

Hill, NC 27599-7400. USA University

1995; accepted

ofNorth Carolinu,

16 October

Chapel Hill, NC 275YY, USA

1995

Abstract An Escherichia coli clone containing recombinant plasmid Cl9 was identified from a Treponema pallidurn genomic library by in situ immunoassay. E. coli maxicells containing pC19 synthesized a treponemal protein doublet of 39.2 and 38.2 kDa, designated TpN38(b). Pulse-chase and protein processing studies showed that TpN38(b) is synthesized with a cleavable amino-terminal signal peptide. A 2.0-kb fragment of pC19 containing the rpn38(b) gene was subcloned and sequenced. The rpn38(b) gene is 1029 nucleotides long and encodes a protein of 343 amino acids with a calculated molecular mass of 37.9 kDa. The deduced amino acid sequence of TpN38(b) has homology with the T. pullidum TpN35 lipoprotein and the Borrelia burgdotferi BmpA, BmpB, BmpC, and BmpD proteins. DNA

h’ey-vords:

Treponrma

pullidum;

Syphilis;

Borrelia

burgdotferi;

TpN35; BmpA; BmpB; BmpC

1. Introduction Treponema pallidum subsp. pallidum is the etiological agent of syphilis, a sexually transmitted disease that continues to be a significant public health problem [1,2]. Efforts to study the structure, physiology, and host-parasite interaction of T. pallidum

* Corresponding author. Tel.: + 1 (919) 966 3882; Fax: + 1 (919) 966 2089; E-mail: [email protected]. ’ Present address: Department of Pathology and Laboratory Medicine. University of Texas Medical School at Houston, Houston, TX 77225, USA. ’ Present address: Department of Microbiology, University of Georgia. Athens, GA 30605, USA. Federation SSDI 0378-

of European

Microbiological

IO97(95)00429-7

Societies

have been hindered by the non-cultivable nature of this spirochete and the fragility of the treponemal outer membrane [1,2]. Additionally, the lack of any identified genetic exchange mechanisms precludes the use of standard genetic manipulations in the native organism. The application of recombinant DNA technology has facilitated the cloning and expression of several T. pallidum genes in Escherichia coli [l]. However, in most cases it has been difficult to assign specific cellular functions to proteins synthesized by the cloned genes [l-3]. The goal of our research is to clone and characterize T. pallidum genes encoding proteins that are exported beyond the cytoplasmic membrane to the treponemal petiplasm or outer membrane. A subset of these proteins are

likely to be involved in functions critical to the survival and dissemination of T. pallidurn. In addition, some of these proteins may be targets of the host treponemicidal immune response. Here, we report the expression and sequence analysis of a T. pallidurn gene, tpn38(b), that encodes an exported protein with amino acid sequence homology to the T. pallidurn TpN35 lipoprotein [4,5] and to members of a newly identified family of B. burgdotjki proteins [6,71.

2. Materials and methods

2. I. Maxicell analysis Specific labelling of plasmid-encoded proteins with Tran 35S-label was performed in E. coli strain SE5000 maxicells as previously described [8]. Immunoprecipitations of the solubilized maxicell extracts were performed with normal rabbit serum (NRS), normal human serum (NHS), experimental rabbit syphilitic serum (ERSS), and human syphilitic serum (HSS) [S]. Samples of the solubilized, radiolabelled maxicell extracts and the precipitates were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography

A 2.0-kb Sal1 DNA fragment containing the tpn38(b) gene was subcloned from pC 19 into pEBH2 I [3], creating pC19-El. The nucleotide sequence of the entire fragment was determined by the dideoxynucleotide chain termination method with the Sequenase version 2.0 kit (United States Biochemicals, Cleveland, OH). Reaction products were labelled with [“‘S]dATP and separated by electrophoresis using 8% polyacrylamide gels. The nucleotide sequence was obtained using specifically designed oligonucleotide primers or M 13 - 20 and reverse primers. Both DNA strands were sequenced a minimum of three times to ensure accuracy. The nucleotide sequence was confirmed by the UNC-CH Automated DNA Sequencing Facility on a model 373A DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) using the Taq DyeDeoxyTM Terminator Cycle Sequencing kit (Applied Biosystems). Plasmid DNA for automated sequencing was prepared from E. coli DHSQ (MCR).

3. Results and discussion

3.1. Expression qf tpn38(b)

k31. 2.2. Processing

2.3. DNA sequencing

studies

For pulse-chase experiments, E. coli maxicells containing pC 19 or pLVS3 (synthesizing TpN39(b)) were pulse-labelled for 1 min with Tran ““S-label as previously described [9]. The chase was initiated by the addition of one-tenth volume of unlabelled 4% methionine. Samples were removed at various time points, precipitated with 10% trichloroacetic acid, washed with acetone, and solubilized [9]. Samples of the solubilized precipitates were analysed by SDSPAGE and fluorography [8]. For experiments involving inhibition of processing, E. coli maxicells containing pC 19 or pCH3 (synthesizing TpN35) were treated with ethanol, sodium azide, or globomycin as previously described [4] prior to labelling with Tran 35S-label. Samples of the solubilized extracts were analysed by SDS-PAGE and fluorography.

E. coli clones synthesizing treponemal protein antigens were identified from a T. pallidurn genomic DNA library by screening colony blots with ERSS [lo]. Plasmid-encoded proteins were labelled in E. coli strain SE5000 maxicells with Tran 35S-label and immunoprecipitations of the solubilized maxicell extracts were performed with NRS and ERSS [S]. A recombinant plasmid, designated pC 19, that contains a 10.6-kb T. pallidurn DNA insert was chosen for further investigation. E. coli maxicells containing pC19 synthesized a treponemal protein doublet of 39.2 and 38.2 kDa (Fig. 1, lane A) that was precipitated with ERSS, but not NRS (Fig. 1, lanes C and B, respectively). We have designated this protein TpN38(b) in accordance with the proposed nomenclature for T. pallidurn proteins [l]. HSS from patients in the secondary and latent stages of syphilis [8] precipitated TpN38(b), indicating that this protein is synthesized during the course of syphilitic infec-

L. V. Stamm et al. / FEMS Micmhiolog~

Letters 135

f IYY6J57-63

a

s9

c

b

d

A

45-

B

+

Fig. 3. Processing of recombinant TpN38tb) (A) or TpN39t b) (B) synthesized in E. co/i maxicells exposed to protein export or processing inhibitors. Lanes: A, untreated control: B. IOr?r ethanol; C, 5 mmol sodium azide; D, 100 ~g per ml globomycin.

31Fig. I, SDS-PAGE analysis of plasmid encoded proteins synthesized in E. c,oli SE5000 maxicells containing pC 19, and immunoprecipitation of the TpN38(b) protein. Lanes: A, solubilized extract of Tran ‘SS-labelled E. co/i SE5000 maxicells containing pC19: B and C. precipitates obtained with NRS and ERSS, respectively. Molecular mass standards are indicated in kDa. The arrowhead indicates the position of the recombinant TpN38tb) protein doublet of 39.2 and 38.2 kDa. The 28-kDa doublet in lane A is the vector encoded p-lactamase.

tion and evokes an IgG antibody response (data not shown). Pooled NHS did not precipitate TpN38(b). 3.2. Processing studies

of TpN38Cb)

The appearance of TpN38(b) as a doublet following SDS-PAGE suggested that this protein may be exported. To determine if TpN38(b) is processed by cleavage of a signal peptide during translocation across the cytoplasmic membrane, pulse-chase studies were performed with E. coli SE5000 maxicells containing pC 19 [9]. A parallel pulse-chase experi-

0

1

2.5 5

Fig. 2. Pulse-chase analysis of recombinant TpN38(b) (A) and TpN39(b) containing pC19 or pLVS3 were pulse-labelled with Tran’5S-label for processed, and analysed by SDS-PAGE and fluorography.

ment was performed with E. coli maxicells containing pLVS3. This plasmid encodes TpN39(b), a 39kDa basic membrane protein synthesized with a signal peptide that is cleaved by signal peptidase I [9]. The results of these experiments clearly demonstrated the slow processing of TpN38(b) from the 39.2-kDa precursor form to the 38.2-kDa mature form (Fig. 2A). Interestingly, the processing of TpN38(b) was still incomplete after 60 min. In contrast, TpN39(b) showed rapid processing from a precursor to a mature form that was virtually complete at 10 min (Fig. 2B). Protein export or processing can be inhibited by exposure of cells to agents such as ethanol, which acts as a membrane perturbant and inhibits translocation [ 1 I]; sodium azide, which inhibits export of SecA-dependent proteins [ 121; or globomycin, which inhibits lipoprotein-specific signal peptidase II [ 131. To determine whether the export or processing of TpN38(b) is affected by these agents, E. coli maxicells containing pC19 were treated with the compounds prior to labelling with Tran 35S-labcl [41. As

10

20 30

(B) synthesized

60

in E. co/i maxicells.

E. co/i SE5OCtO maxicells

I min. Samples were taken at the indicated chase times (min).

60

L. V. Stamm et al. / FEMS Microbiology

Letters 135 I I9961 57-63

GGTCTTTTCAGGCTGTGGGTGCCGGTAGGGTGCACCCTACGCGAGAGAGGGATTTTTGGG

60

~TTBl'C!AACGGTGCGGTGl-GTGTTCTCAG'TGCGCTCATTGCAG'TGTTl'AC'N.G MNGAVCVLSALIAVFTC

120

CTTTTCGTGTAGGCCTGCGGTGCAAGATGAGCGCGCGCGGTGCGTATXCCGTTTTTGTCCC FSCRPAVQDERAVRIAVFVP

180

AGGTTTTCGTCACGACAGTCCl-G'l-GTATGCAA'XTTfXGTGACGG!IGTTGAGCGTGCAGT GFRHDSPVYAMLCDGVERAV

240

TACGCAGGAACGCGCGACAGGGCGCAGCATZGGCTTGATATCATCGAAGCGGGGCCGAA TQERATGRS IGLDIIEAGPN

300

CCAGGCGCTCTGGCGCGAAAAGTTGGCGCATCTTGCTGCAGAACAGCGCTATCGTTTGAT QALWREKLAHLAAEQRYRLI

360

TGTGTCTTCCAACCCTGCACTCCCGCACGTCCTTGAGCCTATTTTGCGTCAATTTCCCCT VSSNPALPHVLEPILRQFPL

420

GCAGCGGTTTTn;GTTCTAGA~CCTACGCGCCGCAGGAACCTTTCG QRFLVLDAYAPQEHSLITFR

480

CTATAACCAGTGGGAGCAAGCCTACCTTGCAGGACACCTTTCCGCGTTAGTGAGlKXGAG YNQWEQAYLAGHLSALVSAS

540

'IGCTATGCGCTTTXAAATGCAGAT AMRFANADKKIGLIAGQSYP

600

AAAAAAATCGGTCTTATTGCGGGGCAGTCGTATCC

GGTGATGACCCAGACTATATTCCTGCCTTTCTCGCAGGTGCCCGTGCAGTAGATCCTGC VMTQTII PAFLAGARAVDPA

660

CTTTGAAGTCGATGTGCGCGTGGTGGGGAACTGGTATGACGCTGCAAAAAG'TGCAGACCT FEVDVRVVGNWYDAAKSADL

720

CGCACGGATTCTCTTT'CACGAAGGGGTGGATGTTATGAl-GCCAAl-T'TGCGGCGGTGCGZUi ARILFHEGVDVMMPICGGAN

780

TCAGGGAGTACTlKXGGCCGCGCGGGAGCTCGGTTTTTATG'M"l'CGTGGTTXACGAT~ QGVLAAARELGFYVSWFDDN

840

CGGCTATGCGAGGGCACCGGGCTACGTAGTTGGCAGTTCCGTTATGGAACAGGAGCGTCT GYARAPGYVVGSSVMEQERL

900

TGCGTATGAGCAGACGCTGCGCn;CATTCGCGCGGTGAACl-GCCATCT'XAGGAGCC'IWZAC AYEQTLRCIRGELPSAGAWT

960

ATTGGGGG'TGAAAGACGGGTACGTACGTTTCATTGAAGAGGATCCCTTGTACCl-GCAX=.C LGVKDGYVRFIEEDPLYLQT

1020

GGTACCCGAACCGATTCGTG?Y3CGGCAGGCAGTCTGCGTTGCTCAGGCGTATTCAAAGCGGTGA VPEPIRVRQSALLRRIQSGE

1080

GCTTACGTl-GCCGG'TGCGIATAGCl-GAGCGCGGCGTTAGGGCATCTGCGCGCGGT LTLPVR

1140

Fig, 4. Nucleotide and deduced amino acid sequences of the T. pallidurn tpn38Cbl gene. The putative Shine-Dalgarno ribosome binding site is underlined. The putative ATG start codon and the TGA stop codon are bolded. The putative signal peptide is double underlined. The rpn38(b) sequence is deposited in the GenBank database at NCBI under accession No. U I286 1.

Fig. 5. (A) Alignment of the amino acid sequences of TpN38(b) and TpN35. (B) Multiple sequence alignment of the C-terminal regions of TpN38(b), BmpA, BmpB, and BmpC. Bars represent amino acid sequence identity with TpN38(b). Colons and dots represent amino acid sequence similarity with TpN38(b) based on the default scoring matrix (Dayhoff table) for the Wisconsin Genetics Computer Group Sequence Analysis Software Package (version 8.1).

L.V.

Stamm

et al. / FEMS

Microbiology

Letters

135 f 1996)

61

57-63

A

TpN38 (b) TpN3 5 TeN38

(b)

TpN3 5 TpN38

(b)

TpN35 TpN38

(b)

TpN3 5 TpN38

(b)

TpN3 5 TpN38

(b)

TpN35 TpN38

(b)

TpN3 5 TpN38

(b)

TpN3 5

. . . . .MNGAVCVLSALIAVFTCFSCRPAVQDERAVRIAVFVPGFRHDSPV :.: .. )::I:: : .: 1 II.:.:. :. : 11.1: Il.

45

VREKWV'RAFAAVFCAMLLIGCSKSDRPQMGNAGGAEGGDFVVGMi'I'DSGD

50

YAMLC.... ..DGVERAvTQERATGRSIGLDIIEAGPNQALWREKLAHLA :I:.( . ::.I..: : .:..:I : ..I. :I ..TASTDAEYVPSLSAFA ~~DK&NQQ~EGISRFAQENNAKCK~~....

89

AEQRYRLIVSSNPALPHVLEPILRQFPLQRFLVLDAYAPQEHSLITFRYN I...: . . .Il l:IIl:II ..: ..::. . . . I:).:.. DENM.GLWACGSFLVEAVIETSARFPKQKFLVIDAWQDRDNVVSAVFG

139

94

:. 143 189

QWEQAYLAGHLSALVSASAMRFANADKKIGLIAGQSYPVMTQTIIPAFLA 1.1. .I ..:.. :l:l.l . . ..I . : ::I I l..:I.I QNEGSFLVG..... VAAALKRKEAGKSAVGFIVGMELGMM.PLFEAGFEA

I 187 239

GARAVDPAFNDVRWGNWYDAAKSADLARILFHEGVDVMMPICGGANQG I.:llll.::l I I...: I:.l:..lI l:..II:l::.:.ll...I GVKAVDPDIQVWEVANTFSDPQKGQALAAKLYDSGVNvIFQVAGGTGNG

237 279

VLAAARE......... .LGFYVSWFDDNGYARAPGYWGSSVMEQERLAY :I. .: l..l...:I:.I I..: I : . II: VIKEARDRRLNGQDVWVIGVDRDQYMDGVYDGSKSWLTSMVKRADVAAE

l

EQTLRCIRGELPSAGAWTLGVKDGYVRFIEEDPLYLQTVPEPIRVRQSAL . . . . I .: I::.. :I:.1 I:ll:l. .II.II RISKMAYtXSFPGGQSIMFGLEDKAVGIPEENPNLSSAVMEKIR....SF

.:

LRRIQSGELTLPVR...... :I I l:.:III EEKIVSKEIWPVRSARMMN

287 329 333

343 353

B TpN38(b) BmpA BmpB BmpC TpN38tb)

KIGLIAGQSYPVMTQTIIPAFLAGARAVDPAFENWYDAAKSAD Ill:::1 . .:: :.: :: Ill: .:..:.:... :I.: l . : KIGFLGGIEGEIV.DAFRYGYEAGAKYANKDIKISTQYIGSFADLEAGRS :: :.: :: . 11: .:..:I: ..: I.. : III:I:I . KIGFIGGMKGNIV.DAFRYGYESGAKYANKDIEIISEYSNSFSDVDIGRT IIl::.I. . :.: : :I II .:I : : : . . . ..I .I: KIGFLTGPMSEHLKDFKF.GFKAG1NANPKLRLVSKKAPSLFDKEKGK.A

BmpB BmpC

201 . 206 . 215

LARILFHEG.VDVMMPICGGANQGvLUARELG..FWSWFDDNGYARAP

I::

I BmpA

216 .

I

I.:..l:III

I:

263

I::.

II

~ATR&%.LDIIHHAAG&IGAI~AKELGSGHYIIGVDE~AYLAP I. :: .I :I\: _I I. ll:.Il::ll I::. :Il ~ASKMYSKG.IDVIHFAAGLAGIGVIBAAKNLG~YYVIGAEQDQSYLAP I :.I: I:l::II.I II..II:IIl :II &.~~&KEDKVGVIFPIAGI&G~YDAAKELGPKY~IG~$&YIAP

250 II 255 II 265

BmpB

GYVVGSSVMEQERLAYEQTLRCIRGELPSAG.AWT..LGVKDGYVRFIEE 310 : \:.I...: :I I . ::.: .:I :. .l:I:l I I: : DNVITS'MXDVGRALNIFTSNHLKTNTFEGG.KLIN.YGLKEGWGFVRN 298 .:. :.: : I I :I:.: ._.I :.. :I::11 : : :. KNFITSVIKNIGDALYLITGEYIKNNNVW 304 EG.GKWQMGLRDGVIGLPNA

BmpC

QNVITSIIKDIGKVIYSISSEYINNRVFKGGIIIi.RGLKEGVIEIVKD

TpN38(b) BmpA

. TpN38(b) BmpA

I:.I

:.:

BmpC

I.

.

:I..

:

.:I

I:l:I

.DPLYLQTVPEPIRVRQSALLRRIQSGELTLPvR ..:. .:... .. I .: I.l:.:I .PKMISFELEKEI.. ..DNLSSKIINKEIIVP..

. .I BmpB

:::

I.

::

. ...:

. . .

:I..:

.&;EYIKVLER.KIVNKEIIVPCNQEEYEIFIK I.1 . 1.1.1 .I .:I Ill:.:1 PDVLNNRLVDEvI.....DLENKIISGEIIVP..

343 325

::

336 340

:

::.:

313

a control, E. coli maxicells containing pCH3, which encodes the T. pallidurn lipoprotein TpN35 (formerly designated TmpC) [4,5] were treated in an identical manner. The TpN38(b) and the TpN35 doublets (Fig. 3A, B, lanes A, respectively) were observed in solubilized extracts of the untreated control cells. Only the precursor forms of TpN38(b) and TpN35 (Fig. 3A, B, lanes B and C, respectively) were observed in extracts of the ethanol- and sodium azide-treated maxicells. The TpN38(b) doublet was observed in extracts of the globomycin-treated maxicells (Fig. 3A, lane D). In contrast, only the precursor form of TpN35 was present in extracts of the globomycintreated maxicells (Fig. 3B, lane D>. The latter results are in agreement with those of Hubbard et al. [4], who demonstrated inhibition of processing of TpN3.5 by globomycin [ 131. The inability of globomycin to affect the processing of TpN38(b) indicates that the signal peptide of this protein is not cleaved by signal peptidase II. These results are consistent with our observation that recombinant expressed TpN38(b) does not label with [3H]palmitate (data not shown).

short length of the signal peptide correlates well with the observation that the precursor and mature forms of TpN38(b) are separated by I .O kDa (Fig. 2). The signal peptide contains only two of the three regions (positively charged N-terminus, hydrophobic core, and signal peptidase cleavage site) that are present in most signal peptides [14]. The lack of a positively charged N-terminal region in the signal peptide of TpN38(b) may account for the slow and incomplete processing of this protein in E. coli maxicells. Although the positively charged N-terminal region of a signal peptide is not required for protein export, it has been shown to play an important role in efficient protein translocation across the cytoplasmic membrane [ 141. A Kyte-Doolittle hydropathy plot of the deduced amino acid sequence of TpN38(b) failed to reveal regions indicative of an integral membrane protein. Additionally, an Alom analysis of the TpN38(b) amino acid sequence predicted the Nterminal signal peptide as the only membrane-spanning domain (data not shown). 3.4. Protein homologies

3.3, Sequence

determination

and analysis

The nucleotide sequence of the entire pC 19-El insert was determined. The nucleotide and deduced amino acid sequences of the tpn38(b) gene are shown in Fig. 4. The tpn38Cb) gene is 1029 nucleotides long and encodes a protein of 343 amino acids with a calculated molecular mass of 37.9 kDa. This is in close agreement with the molecular mass of the TpN38(b) protein as judged by SDS-PAGE. An apparent Shine-Dalgarno ribosome binding site (GGAGGT) precedes the putative ATG initiation codon by four nucleotides. Regions with sequence similarity to (T’”-type promoter elements were not identified upstream of the tpn38fb) gene. The deduced amino acid sequence of TpN38(b) has a putative amino (N)-terminal signal peptide of 13 amino acids with a putative signal peptidase I cleavage site (Leu-Ile-Ala). A putative signal peptidase II cleavage site (Val-Phe-Thr-Cys) is present immediately following the signal peptidase I cleavage site. Since the processing of TpN38(b) is not affected by globomycin and the protein does not label with [3H]palmitate, the signal peptidase II cleavage site does not appear to be utilized. The

The deduced amino acid sequence of the tpn38Cb) gene was used to conduct a BLAST search of the National Center for Biotechnology Information amino acid sequence databases [ 151. Statistically significant matches were found to T. pallidum TpN35 [4,5] and to B. burgdo+ri BmpA (formerly designated P39) [7], BmpB (formerly designated Orf2) [7], and BmpC [6]. Simpson et al. [7] reported that the deduced amino acid sequences of BmpA and BmpB have homology with TpN35. Additionally, they suggested that BmpA and BmpB are lipoproteins based on the presence of putative signal peptidase II cleavage sites in the N-terminal regions of the proteins. Aron et al. [6] subsequently identified BmpC and reported that this protein has homology to TpN35, BmpA, and BmpB. Overall, TpN38(b) has 48% similarity and 27% identity to TpN35; 45% similarity and 19% identity to BmpA; 47% similarity and 21% identity to BmpB; and 48% similarity and 23% identity to BmpC. An amino acid sequence alignment of the TpN38(b) and TpN35 proteins is shown in Fig. 5A. While homology is observed throughout the proteins, the greatest degree of homology is present in the carboxy (C)-terminal regions. A multiple sequence

L.V. Stamm et al. / FEMS Microbiology

alignment of the C-terminal regions of TpN38(b), BmpA, BmpB, and BmpC is shown in Fig. 5B. Ramamoorthy et al. [ 161 have suggested that a family of related proteins (BmpA, BmpB, and BmpC) is present in B. burgdotferi. The recently identified BmpD protein also has amino acid sequence homology with BmpA, BmpB, and BmpC (R. Ramamoorthy, personal communication). TpN38(b) has 5 1% similarity and 22% identity to BmpD. Our results and those of Simpson et al. [7] show that members of the B. burgdo$eri BmpA family are also present in T. pallidurn. Since the genome size of T. pallidum appears to approach the minimum genomic coding potential for prokaryotes [l], the retention of the tpn38Cb) and tpn35 genes suggests that the encoded protein homologs may perform important functions. The cellular location of native TpN38(b) and the homologous proteins is currently unknown. Schouls et al. [S] and Hubbard et al. [4] showed that TpN35 is membrane-associated based on the results of fractionation studies. Simpson et al. [7] postulated that BmpA may be associated with the cytoplasmic or outer membrane of B. burgdogeri. Due to the presence of a signal peptide in the precursor form of TpN38(b) and the absence of predicted membrane spanning domains in the mature protein, we propose that TpN38(b) is exported beyond the cytoplasmic membrane into the T. pallidurn periplasm. Further studies are required to determine the cellular location of TpN38(b) in T. pallidurn and the potential role of this protein in syphilis pathogenesis.

Acknowledgements We thank E. Parrish and L. Shepanski for technical assistance. This research was supported by National Institutes of Health grants 1 UOI A131496, 3 T32 AI07001 and A124976.

References [I] Norris. S.J. and the

Treponema palLidurn Polypeptide Research Group (1993) Polypeptides of Treponema pallidurn: progress toward understanding their structural, functional, and immunological roles. Microbial. Rev. 57, 750-779.

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