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Identification and sequence analysis of Treponema pallidum tprJ, a member of a polymorphic multigene family
FEMS Microbiology Letters 169 (1998) 155^163
Identi¢cation and sequence analysis of Treponema pallidum tprJ, a member of a polymorphic multigene family Lola V. Stamm *, Shermalyn R. Greene, Heather L. Bergen, John M. Hardham 1 , Natalie Y. Barnes 2 Program in Infectious Diseases, Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, NC 27599-7400, USA Received 7 July 1998; received in revised form 29 September 1998; accepted 30 September 1998
Abstract TnphoA mutagenesis was used to identify genes encoding exported proteins in a genomic DNA library of Treponema pallidum, the syphilis agent. The nucleotide sequence of an open reading frame (tprJ) that encodes a 755-amino acid protein with a predicted molecular mass of 81.1 kDa was determined. The deduced amino acid sequence of TprJ has homology to the major surface protein of Treponema denticola, a periodontal pathogen. Southern hybridization and genomic DNA sequence analysis indicate that tprJ is a member of a polymorphic multigene family. RT-PCR data showed that tprJ is expressed in treponemes during syphilitic infection. A putative tprJ gene was sequenced from T. pertenue, the closely related yaws agent. The deduced amino acid sequence of T. pertenue TprJ is 87.3% identical to that of T. pallidum TprJ. This is the first report of significant sequence differences within homologous genes of T. pallidum and T. pertenue. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Syphilis ; Yaws ; Multigene family; Treponema pallidum; Treponema pertenue
1. Introduction Treponema pallidum subsp. pallidum is the etiologic agent of syphilis, a chronic, sexually transmitted infection that is a risk factor for the acquisition * Corresponding author. Tel.: +1 (919) 966-3882; Fax: +1 (919) 966-2089; E-mail: [email protected] 1 Present address: Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77225, USA. 2 Present address: Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
and transmission of HIV [1]. The noncultivable nature of T. pallidum and the lack of a genetic system have hindered research progress on this spirochete. A major goal of our studies is the identi¢cation and characterization of T. pallidum genes encoding exported proteins. To facilitate these studies, we used alkaline phosphatase fusion methodology to screen a T. pallidum genomic DNA library [2]. This approach resulted in the identi¢cation of treponemal genes whose products are involved in motility [3], nutrient acquisition [4,5], and cell shape [2]. Using TnphoA mutagenesis, we identi¢ed a T. pallidum gene, tprJ, that is a member of a polymorphic multigene family and encodes a protein with homology to the major
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surface protein (Msp) of Treponema denticola, a periodontal pathogen. Additionally, we cloned and sequenced tprJ from T. pallidum subsp. pertenue, the etiologic agent of yaws. Here we describe the analysis of the tprJ genes.
2. Materials and methods 2.1. Identi¢cation of clone 3E3 Recombinant plasmids from a T. pallidum Nichols strain genomic DNA library [2] were transformed into Escherichia coli strain CC118 (phoA v20) [6]. Individual transformants were infected with VTnphoA as previously described [2]. Alkaline phosphatase (PhoA) positive colonies were obtained following infection of E. coli cells harboring plasmid p3E3. Partial nucleotide sequences of p3E3 (T3) and p3E3 (T4), which contain TnphoA insertions in the treponemal DNA fragment of p3E3, were determined using a phoA speci¢c primer [7]. 2.2. DNA sequencing and analysis Plasmids p3E3, p3E3(T3), and p3E3(T4) were isolated from E. coli strains CC118 or DH5K using the Wizard Plus Minipreps DNA Puri¢cation System (Promega Corp., Madison, WI). The nucleotide sequence of the T. pallidum DNA insert present in the plasmids was determined using the Taq DyeDeoxy1 Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) at the UNC-CH Automated DNA Sequencing Facility on models 373A and 377 DNA sequencers (Applied Biosystems). Both DNA strands were sequenced to ensure accuracy. DNA and amino acid sequence analyses were performed using the AssemblyLIGN Program (International Biotechnologies, New Haven, CT), Analyze, MacMolly Tetra version 1.2.1 (Europa Scienti¢c Software Corp., Hollis, NH) and the Wisconsin Genetic Computer Group software package version 8.1 (University of Wisconsin Biotechnology Center, Madison, WI). Amino acid sequences were used to search the protein databases at the National Center for Biotechnology Information (National Library of Medicine, Washington, DC) using the Blast algorithm [8].
2.3. In vitro expression of the tprJ gene The tprJ gene was ampli¢ed from p3E3 using the Expand1 Long Template PCR System Kit (Boehringer-Mannheim, Indianapolis, IN). The puri¢ed PCR product was cloned using the pGEM-T Easy Vector System kit (Promega Corp.) and the identity of the gene was con¢rmed by nucleotide sequence analysis. The tprJ gene was expressed from the T7 RNA polymerase promoter and the synthesized protein was labeled using [35 S]methionine (1000 Ci/ mmol; Amersham Life Sciences) with the TNT Quick Coupled Transcription/Translation System (Promega Corp.). The radiolabeled protein was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described [9]. 2.4. Southern hybridization T. pallidum Nichols strain was cultivated by intratesticular infection of adult male New Zealand White rabbits with approximately 5U107 treponemes/testis as previously described [9]. Testes were aseptically removed from the killed rabbits on the day of maximal orchitis and the treponemes were extracted, washed, enumerated by dark-¢eld microscopy, and frozen at 370³C. Genomic DNA was isolated from the treponemes using the Wizard Genomic DNA Puri¢cation kit (Promega Corp.). The DNA was digested to completion with BamHI, electrophoresed on a 0.8% agarose gel, and transferred to Hybond N (Amersham Life Sciences, Arlington Heights, IL). Hybridizations were performed with a PCR product corresponding to nucleotides 264^674 of the tprJ gene. A control hybridization was performed with the single copy T. pallidum gyrB gene [10]. The probes were £uorescein labeled and the results were visualized with the ECL Direct Nucleic Acid Labeling and Detection System (Amersham Life Sciences). 2.5. Reverse transcriptase (RT)-PCR Total RNA for RT-PCR analysis was isolated from T. pallidum cells using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol. RT-PCRs were performed using primer pairs speci¢c to the variable region of
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Fig. 1. Nucleotide and deduced amino acid sequence of the T. pallidum tprJ gene. The putative start and stop codons are in bold, the putative 335 and 310 sequences are underlined, and the putative rbs is double underlined. The sequence corresponding to the PCR product used in Southern blotting is dash underlined. Arrowheads indicate the position of two in-frame TnphoA insertions (T3 and T4, respectively). The arrow indicates the putative signal peptidase I cleavage site. The nucleotide sequence of T. pallidum tprJ has been deposited in GenBank under accession number U88957.
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each gene with the RT-PCR Access System kit (Promega Corp.). Each RT-PCR was accompanied by a PCR reaction using the same oligonucleotide primer pair and T. pallidum genomic DNA as template. The resulting DNA products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. 2.6. Cloning of T. pertenue tprJ The tprJ gene was ampli¢ed from T. pertenue Gauthier strain genomic DNA (provided by A. Centurion-Lara and S. Lukehart) using the Expand1 Long Template PCR System (Boehringer-Mannheim) and oligonucleotide primers based on sequences £anking the T. pallidum tprJ gene. The gel puri¢ed PCR product was cloned into the pGEM-T Easy vector (Promega Corp.). Identical nucleotide sequences obtained from two independent clones were analyzed as described above.
Fig. 2. Southern blot analysis of BamHI digested T. pallidum chromosomal DNA with a PCR product representing V0.4 kb of the 5P end of the tprJ gene (lane A) or with the gyrB gene (lane B). HindIII digested V DNA size standards are indicated on the left in kb.
quences, are present at 86 and 60 nucleotides, respectively, upstream of the rbs.
3. Results and discussion 3.1. Identi¢cation and sequence analysis of plasmid p3E3 E. coli clones containing recombinant plasmids from a T. pallidum genomic DNA library were screened for the expression of exported proteins by TnphoA insertion mutagenesis. Clone 3E3, which yielded PhoA positive colonies, was chosen for further study based on partial nucleotide sequence obtained from plasmids p3E3(T3) and p3E3(T4) [7] (Fig. 1). The remainder of the nucleotide sequence was determined from p3E3. Analysis of the nucleotide sequence indicated the presence of a large open reading frame (ORF). The nucleotide and deduced amino acid sequences of this ORF are shown in Fig. 1. The ORF is 2268 nucleotides long and encodes a 755-amino acid protein with a predicted molecular mass of 81.1 kDa and a pI of 8.96. We designated this ORF tprJ based on the nomenclature of Hardham et al. [11]. A putative ribosome binding site (rbs) (GAGGT) is located 12 nucleotides upstream of the putative ATG start codon. Putative 335 (TGGATA) and 310 (TAGAAA) sequences, similar to those of E. coli c70 -type consensus promoter se-
3.2. Analysis of the TprJ protein Analysis of the deduced amino acid sequence of the TprJ protein indicated that it has homology (49^ 53% similarity; 27^29% identity) to the Msps of T. denticola strains OTK, ATCC 33520, and ATCC 35405 [12]. Identi¢cation of an Msp-like homolog in T. pallidum is not surprising since sequence data obtained from genomic DNA (Greene and Stamm, unpublished) and 16S rRNA [13] suggest that T. pallidum and T. denticola are more closely related than previously proposed. The T. denticola ATCC 34505 Msp has been implicated in the interaction between this spirochete and gingival epithelium. Studies have shown that Msp has porin activity, depolarizes epithelial cell membranes, binds to extracellular matrix components, and forms a regular hexagonal surface array in the T. denticola outer membrane [14]. A hydrophilicity pro¢le of TprJ shows that it contains an N-terminal hydrophobic region (data not shown). ALOM analysis indicates that this region (amino acids 4^20) is the only extended transmembrane region in the protein. The TprJ N-terminus (amino acids 1^32) contains features characteristic
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Fig. 3. Amino acid sequence alignments of the T. pallidum TprJ protein with the TprE and TprG proteins [15]. In each alignment, the vertical bars denote identity with the TprJ amino acid sequence ; the colons and dots denote similarity with the TprJ amino acid sequence (based on the Dayho¡ PAM-250 similarity matrix).
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Fig. 4. Transcriptional analysis of the T. pallidum tprJ, tprE, and tprG genes. RT-PCR analysis was performed using primer pairs speci¢c to the variable region of each gene. For each primer pair, PCRs were performed with an RT reaction minus the RT (3) or plus the RT (+) or with T. pallidum genomic DNA as a control (C).
of a signal peptide [positive (+1) charge, hydrophobic core, signal peptidase type I processing site (GSA)]. The observation that TprJ-PhoA fusion proteins synthesized in E. coli are PhoA positive is consistent with the prediction of a signal peptide at the TprJ N-terminus. Studies to determine the cellular location of the native TprJ are contingent upon the further re¢nement of T. pallidum cell fractionation protocols. 3.3. In vitro expression of the recombinant tprJ gene The tprJ gene was PCR ampli¢ed, cloned, and expressed in vitro. The [35 S]methionine labeled TprJ protein was visualized by SDS-PAGE and £uorography (data not shown). The observed molecular mass of the protein (72 kDa) is 9.1 kDa smaller than the predicted molecular mass. The observed molecular masses of Msps from T. denticola strains range from 53 to 62 kDa [12]. As expected, the molecular mass of the in vitro synthesized TprJ protein was not a¡ected by heat treatment (data not shown). However, since the native T. denticola Msps are heat modi¢able [12], further experiments are needed to assess if this is the case with the native TprJ protein. 3.4. Southern hybridization analysis Southern analysis of BamHI digested T. pallidum genomic DNA was performed with a PCR product representing V0.4 kb of the 5P end of the tprJ gene. The probe hybridized, with varying degrees of a¤nity, to at least eight T. pallidum DNA fragments indicating that the tprJ gene is present in more than a single copy and the nucleotide sequences of some of the homologs are divergent from that of tprJ
(Fig. 2, lane A). A control hybridization performed with the single copy T. pallidum gyrB gene showed reactivity with two T. pallidum DNA fragments due to a BamHI site that is present in the gene (Fig. 2, lane B). The copy number of tprJ could not be conclusively determined based on hybridization results since polymorphisms in the BamHI site (which is external to the tprJ gene) could be present in the other genes. However, analysis of data from a T. pallidum whole genome sequencing project con¢rmed that tprJ is a member of a polymorphic multigene family [11,15]. This family contains twelve genes designated tprA-tprL that are present in eight loci evenly distributed around the treponemal chromosome. Seven of the twelve tpr genes, including tprJ, encode proteins with putative N-terminal signal peptides. 3.5. Comparison of TprJ to other members of the Tpr family The predicted sizes of the Tpr proteins range from 42.8 kDa to 81.1 kDa. Comparative analysis of the deduced amino acid sequences of the Tpr proteins revealed the presence of conserved (N- and C-termini) and variable (central) regions. Based on their predicted molecular mass, percent identity, and presence of a putative signal peptide, the Tpr proteins can be divided into three subgroups: TprA, B, H, K, L; TprC, D, F, I; and TprE, G, J [11,15]. Only the proteins in the latter two subgroups contain putative N-terminal signal peptides. The TprJ protein has highest identity to TprE and TprG (69.9% and 85.5%, respectively). A multiple amino acid sequence alignment of TprJ with TprE and TprG is shown in Fig. 3. 3.6. Transcriptional analysis of the tprJ, tprE, and tprG genes RT-PCR was used to determine if mRNA for the tprJ, trpE, and tprG genes is present in total RNA from T. pallidum isolated from infected rabbit testes. The oligonucleotide primer pairs used were speci¢c to each tpr gene. No products were obtained when the reactions were performed in the absence of RT (Fig. 4 (3)). RT-PCRs for trpJ, trpE, and tprG yielded products of 333 bp, 300 bp, and 381 bp, respectively (Fig. 4 (+)). The RT-PCR products
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Fig. 5. Amino acid sequence alignments of the T. pallidum and T. pertenue TprJ proteins. Symbols are as in Fig. 3. The nucleotide sequence of T. pertenue tprJ has been deposited in GenBank under accession number AF073527.
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were identical in size to PCR products obtained with T. pallidum genomic DNA template (Fig. 4 (C)). These results con¢rm the data of Hardham et al. [11] and show that the tprJ, trpE, and tprG genes are expressed during syphilitic infection. 3.7. Analysis of T. pertenue tprJ Availability of the T. pallidum tprJ nucleotide sequence prompted us to clone and sequence T. pertenue tprJ. This spirochete is the agent of yaws, a nonvenereal treponematosis. Although the clinical di¡erences between syphilis and yaws are distinct, previous studies of T. pallidum and T. pertenue revealed only very minor di¡erences in these organisms at the molecular level [13,16]. To ensure cloning of tprJ and not other closely related tpr genes, oligonucleotide primers based on sequences £anking the T. pallidum tprJ gene were used for PCR of T. pertenue genomic DNA. Analysis of sequences immediately upstream and downstream of the putative tprJ gene indicated that they were virtually identical to the corresponding T. pallidum sequences. Analysis of the putative T. pertenue tprJ sequence indicated that this gene has 89.8% nucleotide identity to T. pallidum tprJ and is six nucleotides shorter. T. pertenue tprJ encodes a 753-amino acid protein with a predicted molecular mass of 80.7 kDa that has 71.2%, 87.3%, and 97.6% identity to T. pallidum TprE, TprJ, and TprG, respectively. As expected the amino acid sequences of the N- and C-termini of the TprJ proteins are highly conserved, whereas the central regions are more variable (Fig. 5). This is the ¢rst report of signi¢cant di¡erences within the nucleotide sequences of homologous genes of T. pallidum and T. pertenue. Computer predictions of restriction endonuclease cleavage sites located within the tprJ genes indicate that PCR products of T. pallidum and T. pertenue tprJ can be di¡erentiated based on restriction fragment length polymorphism analysis. This information could provide a molecular tool to di¡erentiate syphilis and yaws in areas of the world where these two infections co-exist. In conclusion, we have identi¢ed and analyzed T. pallidum tprJ which is a member of a polymorphic multigene family. The presence of twelve tpr genes is an intriguing observation since the small genome size
of T. pallidum (V1.1 Mb) [13,15] would seem to pose a selective pressure against multiple homologs. Polymorphic multigene families have been identi¢ed in several bacterial pathogens. The proteins encoded by these multigene families are cell surface exposed and undergo phenotypic (phase and/or antigenic) variation facilitating evasion of host defense mechanisms and modulating functional activities (attachment, invasion, etc.) [17]. Although the cellular location(s) and function(s) of the Tpr proteins are currently unknown, it is possible that some of these proteins localize to the T. pallidum cell surface. Phenotypic variation of such proteins could account for certain clinical features of syphilis and for the observed heterogeneity of treponemal subpopulations in phagocytosis, attachment, and invasion assays [18^20]. Further studies of the Tpr proteins are needed to determine their role in the pathogenesis of syphilis.
Acknowledgments We thank J. Frye, L. Shepanski, M. Price, and P. Padgett for technical assistance and A. CenturionLara and S. Lukehart for T. pertenue genomic DNA. This research was supported by National Institutes of Health Grant U19 AI31496.
References [1] Quinn, T.C., Cannon, R.O., Glasser, D., Groseclose, S.L., Brathwaite, W.S., Fauci, A.S. and Hook, E.W. III (1990) The association of syphilis with risk of human immunode¢ciency virus infection in patients attending sexually transmitted disease clinics. Arch. Intern. Med. 150, 1297^1302. [2] Hardham, J.M. and Stamm, L.V. (1994) Identi¢cation and characterization of the Treponema pallidum tpn50 gene, an ompA homolog. Infect. Immun. 62, 1015^1025. [3] Hardham, J.M., Frye, J.G. and Stamm, L.V. (1995) Identi¢cation and sequences of the Treponema pallidum £iMP, £iY, £iP, £iQ, £iR, and £hBP genes. Gene 166, 57^64. [4] Stamm, L.V., Young, N.R., Frye, J.G. and Hardham, J.M. (1996) Identi¢cation and sequences of the Treponema pallidum mglA and mglC genes. DNA Sequence 6, 293^298. [5] Hardham, J.M., Stamm, L.V., Porcella, S.F., Frye, J.G., Barnes, N.Y., Howell, J.K., Mueller, S.L., Radolf, J.D., Weinstock, G.M. and Norris, S.J. (1997) Identi¢cation and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor
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[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
protein homolog, and a glycolytic pathway enzyme homolog. Gene 197, 47^64. Manoil, C. and Beckwith, J. (1985) Tnpho:A : A transposon probe for protein export signals. Proc. Natl. Acad. Sci. USA 82, 8129^8133. Hardham, J.M. (1995) Identi¢cation and Characterization of Treponema pallidum Genes Encoding Exported and Membrane Proteins. Ph.D. Thesis, University of North Carolina at Chapel Hill, NC. Altschul, S.F., Madden, S.F., Shafer, A.A., Zhang, J., Zang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402. Stamm, L.V., Kerner, T.C. Jr., Bankaitis, V.A. and Bassford, P.J. Jr. (1983) Identi¢cation and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coli. Infect. Immun. 41, 709^721. Stamm, L.V., Greene, S.R. and Barnes, N.Y. (1997). Identi¢cation and characterization of the gyrB gene from Treponema pallidum subsp. pallidum. FEMS Microbiol. Lett. 153, 129^134. Hardham, J.M., McLeod, M.P., Howell, J.K., Sodergren, E., Weinstock, G.M. and Norris, S.J. (1998) Identi¢cation, expression, and transcriptional analysis of the twelve Treponema pallidum tpr genes. In: Abstracts of the 98th General Meeting of the American Society for Microbiology 1998, abstr. D-56, p. 222. American Society for Microbiology, Washington, DC. Fenno, J.C., Wong, G.W.K., Hannam, P.M., Muller, K.-H., Leung, W.K. and McBride, B.C. (1997) Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp. J. Bacteriol. 179, 1082^1089. Norris, S.J. and the Treponema pallidum Polypeptide Research Group (1993) Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Microbiol. Rev. 57, 750^779.
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[14] Fenno, J.C., Muller, K.-H. and McBride, B.C. (1996) Sequence, analysis, expression and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola. J. Bacteriol. 178, 2489^2497. [15] Fraser, C.M., Norris, S.J., Weinstock, G.M., White, O., Sutton, G.G., Dodson, R., Gwinn, M., Hickey, E.K., Clayton, R., Ketchum, K.A., Sodergren, E., Hardham, J.M., McLeod, M.P., Salzberg, S., Peterson, J., Khalak, H., Richardson, D., Howell, J.K., Chidambaram, M., Utterback, T., McDonald, L., Artiach, P., Bowman, C., Cotton, M.D., Fujii, C., Garland, S., Hatch, B., Horst, K., Roberts, K., Sandusky, M., Weidman, J., Smith, H.O. and Venter, J.C. (1998) Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 281, 375^388. [16] Centurion-Lara, A., Castro, C., Van Voorhis, W.C. and Lukehart, S.A. (1996) Two 16S-23S ribosomal DNA intergenic regions in di¡erent Treponema pallidum subspecies contain tRNA genes. FEMS Microbiol. Lett. 143, 235^240. [17] Deitsch, K.W., Moxon, E.R. and Wellems, T.E. (1997) Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections. Microbiol. Mol. Biol. Rev. 61, 281^293. [18] Lukehart, S.A., Sha¡er, J.M. and Baker-Zander, S.A. (1992) A subpopulation of Treponema pallidum is resistant to phagocytosis: possible mechanisms of persistence. J. Infect. Dis. 166, 1449^1453. [19] Fieldsteel, A.H., Stout, J.G. and Becker, F.A. (1979) Comparative behavior of virulent strains of Treponema pallidum and Treponema pertenue in gradient cultures of various mammalian cells. Infect. Immun. 24, 337^345. [20] Riviere, G.R., Thomas, D.D. and Cobb, C.M. (1989) In vitro model of Treponema pallidum invasiveness. Infect. Immun. 57, 2267^2271.
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Identi¢cation and sequence analysis of Treponema pallidum tprJ, a member of a polymorphic multigene family Lola V. Stamm *, Shermalyn R. Greene, Heather L. Bergen, John M. Hardham 1 , Natalie Y. Barnes 2 Program in Infectious Diseases, Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, NC 27599-7400, USA Received 7 July 1998; received in revised form 29 September 1998; accepted 30 September 1998
Abstract TnphoA mutagenesis was used to identify genes encoding exported proteins in a genomic DNA library of Treponema pallidum, the syphilis agent. The nucleotide sequence of an open reading frame (tprJ) that encodes a 755-amino acid protein with a predicted molecular mass of 81.1 kDa was determined. The deduced amino acid sequence of TprJ has homology to the major surface protein of Treponema denticola, a periodontal pathogen. Southern hybridization and genomic DNA sequence analysis indicate that tprJ is a member of a polymorphic multigene family. RT-PCR data showed that tprJ is expressed in treponemes during syphilitic infection. A putative tprJ gene was sequenced from T. pertenue, the closely related yaws agent. The deduced amino acid sequence of T. pertenue TprJ is 87.3% identical to that of T. pallidum TprJ. This is the first report of significant sequence differences within homologous genes of T. pallidum and T. pertenue. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Syphilis ; Yaws ; Multigene family; Treponema pallidum; Treponema pertenue
1. Introduction Treponema pallidum subsp. pallidum is the etiologic agent of syphilis, a chronic, sexually transmitted infection that is a risk factor for the acquisition * Corresponding author. Tel.: +1 (919) 966-3882; Fax: +1 (919) 966-2089; E-mail: [email protected] 1 Present address: Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77225, USA. 2 Present address: Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
and transmission of HIV [1]. The noncultivable nature of T. pallidum and the lack of a genetic system have hindered research progress on this spirochete. A major goal of our studies is the identi¢cation and characterization of T. pallidum genes encoding exported proteins. To facilitate these studies, we used alkaline phosphatase fusion methodology to screen a T. pallidum genomic DNA library [2]. This approach resulted in the identi¢cation of treponemal genes whose products are involved in motility [3], nutrient acquisition [4,5], and cell shape [2]. Using TnphoA mutagenesis, we identi¢ed a T. pallidum gene, tprJ, that is a member of a polymorphic multigene family and encodes a protein with homology to the major
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 4 7 7 - 7
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surface protein (Msp) of Treponema denticola, a periodontal pathogen. Additionally, we cloned and sequenced tprJ from T. pallidum subsp. pertenue, the etiologic agent of yaws. Here we describe the analysis of the tprJ genes.
2. Materials and methods 2.1. Identi¢cation of clone 3E3 Recombinant plasmids from a T. pallidum Nichols strain genomic DNA library [2] were transformed into Escherichia coli strain CC118 (phoA v20) [6]. Individual transformants were infected with VTnphoA as previously described [2]. Alkaline phosphatase (PhoA) positive colonies were obtained following infection of E. coli cells harboring plasmid p3E3. Partial nucleotide sequences of p3E3 (T3) and p3E3 (T4), which contain TnphoA insertions in the treponemal DNA fragment of p3E3, were determined using a phoA speci¢c primer [7]. 2.2. DNA sequencing and analysis Plasmids p3E3, p3E3(T3), and p3E3(T4) were isolated from E. coli strains CC118 or DH5K using the Wizard Plus Minipreps DNA Puri¢cation System (Promega Corp., Madison, WI). The nucleotide sequence of the T. pallidum DNA insert present in the plasmids was determined using the Taq DyeDeoxy1 Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) at the UNC-CH Automated DNA Sequencing Facility on models 373A and 377 DNA sequencers (Applied Biosystems). Both DNA strands were sequenced to ensure accuracy. DNA and amino acid sequence analyses were performed using the AssemblyLIGN Program (International Biotechnologies, New Haven, CT), Analyze, MacMolly Tetra version 1.2.1 (Europa Scienti¢c Software Corp., Hollis, NH) and the Wisconsin Genetic Computer Group software package version 8.1 (University of Wisconsin Biotechnology Center, Madison, WI). Amino acid sequences were used to search the protein databases at the National Center for Biotechnology Information (National Library of Medicine, Washington, DC) using the Blast algorithm [8].
2.3. In vitro expression of the tprJ gene The tprJ gene was ampli¢ed from p3E3 using the Expand1 Long Template PCR System Kit (Boehringer-Mannheim, Indianapolis, IN). The puri¢ed PCR product was cloned using the pGEM-T Easy Vector System kit (Promega Corp.) and the identity of the gene was con¢rmed by nucleotide sequence analysis. The tprJ gene was expressed from the T7 RNA polymerase promoter and the synthesized protein was labeled using [35 S]methionine (1000 Ci/ mmol; Amersham Life Sciences) with the TNT Quick Coupled Transcription/Translation System (Promega Corp.). The radiolabeled protein was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described [9]. 2.4. Southern hybridization T. pallidum Nichols strain was cultivated by intratesticular infection of adult male New Zealand White rabbits with approximately 5U107 treponemes/testis as previously described [9]. Testes were aseptically removed from the killed rabbits on the day of maximal orchitis and the treponemes were extracted, washed, enumerated by dark-¢eld microscopy, and frozen at 370³C. Genomic DNA was isolated from the treponemes using the Wizard Genomic DNA Puri¢cation kit (Promega Corp.). The DNA was digested to completion with BamHI, electrophoresed on a 0.8% agarose gel, and transferred to Hybond N (Amersham Life Sciences, Arlington Heights, IL). Hybridizations were performed with a PCR product corresponding to nucleotides 264^674 of the tprJ gene. A control hybridization was performed with the single copy T. pallidum gyrB gene [10]. The probes were £uorescein labeled and the results were visualized with the ECL Direct Nucleic Acid Labeling and Detection System (Amersham Life Sciences). 2.5. Reverse transcriptase (RT)-PCR Total RNA for RT-PCR analysis was isolated from T. pallidum cells using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol. RT-PCRs were performed using primer pairs speci¢c to the variable region of
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Fig. 1. Nucleotide and deduced amino acid sequence of the T. pallidum tprJ gene. The putative start and stop codons are in bold, the putative 335 and 310 sequences are underlined, and the putative rbs is double underlined. The sequence corresponding to the PCR product used in Southern blotting is dash underlined. Arrowheads indicate the position of two in-frame TnphoA insertions (T3 and T4, respectively). The arrow indicates the putative signal peptidase I cleavage site. The nucleotide sequence of T. pallidum tprJ has been deposited in GenBank under accession number U88957.
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each gene with the RT-PCR Access System kit (Promega Corp.). Each RT-PCR was accompanied by a PCR reaction using the same oligonucleotide primer pair and T. pallidum genomic DNA as template. The resulting DNA products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. 2.6. Cloning of T. pertenue tprJ The tprJ gene was ampli¢ed from T. pertenue Gauthier strain genomic DNA (provided by A. Centurion-Lara and S. Lukehart) using the Expand1 Long Template PCR System (Boehringer-Mannheim) and oligonucleotide primers based on sequences £anking the T. pallidum tprJ gene. The gel puri¢ed PCR product was cloned into the pGEM-T Easy vector (Promega Corp.). Identical nucleotide sequences obtained from two independent clones were analyzed as described above.
Fig. 2. Southern blot analysis of BamHI digested T. pallidum chromosomal DNA with a PCR product representing V0.4 kb of the 5P end of the tprJ gene (lane A) or with the gyrB gene (lane B). HindIII digested V DNA size standards are indicated on the left in kb.
quences, are present at 86 and 60 nucleotides, respectively, upstream of the rbs.
3. Results and discussion 3.1. Identi¢cation and sequence analysis of plasmid p3E3 E. coli clones containing recombinant plasmids from a T. pallidum genomic DNA library were screened for the expression of exported proteins by TnphoA insertion mutagenesis. Clone 3E3, which yielded PhoA positive colonies, was chosen for further study based on partial nucleotide sequence obtained from plasmids p3E3(T3) and p3E3(T4) [7] (Fig. 1). The remainder of the nucleotide sequence was determined from p3E3. Analysis of the nucleotide sequence indicated the presence of a large open reading frame (ORF). The nucleotide and deduced amino acid sequences of this ORF are shown in Fig. 1. The ORF is 2268 nucleotides long and encodes a 755-amino acid protein with a predicted molecular mass of 81.1 kDa and a pI of 8.96. We designated this ORF tprJ based on the nomenclature of Hardham et al. [11]. A putative ribosome binding site (rbs) (GAGGT) is located 12 nucleotides upstream of the putative ATG start codon. Putative 335 (TGGATA) and 310 (TAGAAA) sequences, similar to those of E. coli c70 -type consensus promoter se-
3.2. Analysis of the TprJ protein Analysis of the deduced amino acid sequence of the TprJ protein indicated that it has homology (49^ 53% similarity; 27^29% identity) to the Msps of T. denticola strains OTK, ATCC 33520, and ATCC 35405 [12]. Identi¢cation of an Msp-like homolog in T. pallidum is not surprising since sequence data obtained from genomic DNA (Greene and Stamm, unpublished) and 16S rRNA [13] suggest that T. pallidum and T. denticola are more closely related than previously proposed. The T. denticola ATCC 34505 Msp has been implicated in the interaction between this spirochete and gingival epithelium. Studies have shown that Msp has porin activity, depolarizes epithelial cell membranes, binds to extracellular matrix components, and forms a regular hexagonal surface array in the T. denticola outer membrane [14]. A hydrophilicity pro¢le of TprJ shows that it contains an N-terminal hydrophobic region (data not shown). ALOM analysis indicates that this region (amino acids 4^20) is the only extended transmembrane region in the protein. The TprJ N-terminus (amino acids 1^32) contains features characteristic
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Fig. 3. Amino acid sequence alignments of the T. pallidum TprJ protein with the TprE and TprG proteins [15]. In each alignment, the vertical bars denote identity with the TprJ amino acid sequence ; the colons and dots denote similarity with the TprJ amino acid sequence (based on the Dayho¡ PAM-250 similarity matrix).
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Fig. 4. Transcriptional analysis of the T. pallidum tprJ, tprE, and tprG genes. RT-PCR analysis was performed using primer pairs speci¢c to the variable region of each gene. For each primer pair, PCRs were performed with an RT reaction minus the RT (3) or plus the RT (+) or with T. pallidum genomic DNA as a control (C).
of a signal peptide [positive (+1) charge, hydrophobic core, signal peptidase type I processing site (GSA)]. The observation that TprJ-PhoA fusion proteins synthesized in E. coli are PhoA positive is consistent with the prediction of a signal peptide at the TprJ N-terminus. Studies to determine the cellular location of the native TprJ are contingent upon the further re¢nement of T. pallidum cell fractionation protocols. 3.3. In vitro expression of the recombinant tprJ gene The tprJ gene was PCR ampli¢ed, cloned, and expressed in vitro. The [35 S]methionine labeled TprJ protein was visualized by SDS-PAGE and £uorography (data not shown). The observed molecular mass of the protein (72 kDa) is 9.1 kDa smaller than the predicted molecular mass. The observed molecular masses of Msps from T. denticola strains range from 53 to 62 kDa [12]. As expected, the molecular mass of the in vitro synthesized TprJ protein was not a¡ected by heat treatment (data not shown). However, since the native T. denticola Msps are heat modi¢able [12], further experiments are needed to assess if this is the case with the native TprJ protein. 3.4. Southern hybridization analysis Southern analysis of BamHI digested T. pallidum genomic DNA was performed with a PCR product representing V0.4 kb of the 5P end of the tprJ gene. The probe hybridized, with varying degrees of a¤nity, to at least eight T. pallidum DNA fragments indicating that the tprJ gene is present in more than a single copy and the nucleotide sequences of some of the homologs are divergent from that of tprJ
(Fig. 2, lane A). A control hybridization performed with the single copy T. pallidum gyrB gene showed reactivity with two T. pallidum DNA fragments due to a BamHI site that is present in the gene (Fig. 2, lane B). The copy number of tprJ could not be conclusively determined based on hybridization results since polymorphisms in the BamHI site (which is external to the tprJ gene) could be present in the other genes. However, analysis of data from a T. pallidum whole genome sequencing project con¢rmed that tprJ is a member of a polymorphic multigene family [11,15]. This family contains twelve genes designated tprA-tprL that are present in eight loci evenly distributed around the treponemal chromosome. Seven of the twelve tpr genes, including tprJ, encode proteins with putative N-terminal signal peptides. 3.5. Comparison of TprJ to other members of the Tpr family The predicted sizes of the Tpr proteins range from 42.8 kDa to 81.1 kDa. Comparative analysis of the deduced amino acid sequences of the Tpr proteins revealed the presence of conserved (N- and C-termini) and variable (central) regions. Based on their predicted molecular mass, percent identity, and presence of a putative signal peptide, the Tpr proteins can be divided into three subgroups: TprA, B, H, K, L; TprC, D, F, I; and TprE, G, J [11,15]. Only the proteins in the latter two subgroups contain putative N-terminal signal peptides. The TprJ protein has highest identity to TprE and TprG (69.9% and 85.5%, respectively). A multiple amino acid sequence alignment of TprJ with TprE and TprG is shown in Fig. 3. 3.6. Transcriptional analysis of the tprJ, tprE, and tprG genes RT-PCR was used to determine if mRNA for the tprJ, trpE, and tprG genes is present in total RNA from T. pallidum isolated from infected rabbit testes. The oligonucleotide primer pairs used were speci¢c to each tpr gene. No products were obtained when the reactions were performed in the absence of RT (Fig. 4 (3)). RT-PCRs for trpJ, trpE, and tprG yielded products of 333 bp, 300 bp, and 381 bp, respectively (Fig. 4 (+)). The RT-PCR products
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Fig. 5. Amino acid sequence alignments of the T. pallidum and T. pertenue TprJ proteins. Symbols are as in Fig. 3. The nucleotide sequence of T. pertenue tprJ has been deposited in GenBank under accession number AF073527.
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were identical in size to PCR products obtained with T. pallidum genomic DNA template (Fig. 4 (C)). These results con¢rm the data of Hardham et al. [11] and show that the tprJ, trpE, and tprG genes are expressed during syphilitic infection. 3.7. Analysis of T. pertenue tprJ Availability of the T. pallidum tprJ nucleotide sequence prompted us to clone and sequence T. pertenue tprJ. This spirochete is the agent of yaws, a nonvenereal treponematosis. Although the clinical di¡erences between syphilis and yaws are distinct, previous studies of T. pallidum and T. pertenue revealed only very minor di¡erences in these organisms at the molecular level [13,16]. To ensure cloning of tprJ and not other closely related tpr genes, oligonucleotide primers based on sequences £anking the T. pallidum tprJ gene were used for PCR of T. pertenue genomic DNA. Analysis of sequences immediately upstream and downstream of the putative tprJ gene indicated that they were virtually identical to the corresponding T. pallidum sequences. Analysis of the putative T. pertenue tprJ sequence indicated that this gene has 89.8% nucleotide identity to T. pallidum tprJ and is six nucleotides shorter. T. pertenue tprJ encodes a 753-amino acid protein with a predicted molecular mass of 80.7 kDa that has 71.2%, 87.3%, and 97.6% identity to T. pallidum TprE, TprJ, and TprG, respectively. As expected the amino acid sequences of the N- and C-termini of the TprJ proteins are highly conserved, whereas the central regions are more variable (Fig. 5). This is the ¢rst report of signi¢cant di¡erences within the nucleotide sequences of homologous genes of T. pallidum and T. pertenue. Computer predictions of restriction endonuclease cleavage sites located within the tprJ genes indicate that PCR products of T. pallidum and T. pertenue tprJ can be di¡erentiated based on restriction fragment length polymorphism analysis. This information could provide a molecular tool to di¡erentiate syphilis and yaws in areas of the world where these two infections co-exist. In conclusion, we have identi¢ed and analyzed T. pallidum tprJ which is a member of a polymorphic multigene family. The presence of twelve tpr genes is an intriguing observation since the small genome size
of T. pallidum (V1.1 Mb) [13,15] would seem to pose a selective pressure against multiple homologs. Polymorphic multigene families have been identi¢ed in several bacterial pathogens. The proteins encoded by these multigene families are cell surface exposed and undergo phenotypic (phase and/or antigenic) variation facilitating evasion of host defense mechanisms and modulating functional activities (attachment, invasion, etc.) [17]. Although the cellular location(s) and function(s) of the Tpr proteins are currently unknown, it is possible that some of these proteins localize to the T. pallidum cell surface. Phenotypic variation of such proteins could account for certain clinical features of syphilis and for the observed heterogeneity of treponemal subpopulations in phagocytosis, attachment, and invasion assays [18^20]. Further studies of the Tpr proteins are needed to determine their role in the pathogenesis of syphilis.
Acknowledgments We thank J. Frye, L. Shepanski, M. Price, and P. Padgett for technical assistance and A. CenturionLara and S. Lukehart for T. pertenue genomic DNA. This research was supported by National Institutes of Health Grant U19 AI31496.
References [1] Quinn, T.C., Cannon, R.O., Glasser, D., Groseclose, S.L., Brathwaite, W.S., Fauci, A.S. and Hook, E.W. III (1990) The association of syphilis with risk of human immunode¢ciency virus infection in patients attending sexually transmitted disease clinics. Arch. Intern. Med. 150, 1297^1302. [2] Hardham, J.M. and Stamm, L.V. (1994) Identi¢cation and characterization of the Treponema pallidum tpn50 gene, an ompA homolog. Infect. Immun. 62, 1015^1025. [3] Hardham, J.M., Frye, J.G. and Stamm, L.V. (1995) Identi¢cation and sequences of the Treponema pallidum £iMP, £iY, £iP, £iQ, £iR, and £hBP genes. Gene 166, 57^64. [4] Stamm, L.V., Young, N.R., Frye, J.G. and Hardham, J.M. (1996) Identi¢cation and sequences of the Treponema pallidum mglA and mglC genes. DNA Sequence 6, 293^298. [5] Hardham, J.M., Stamm, L.V., Porcella, S.F., Frye, J.G., Barnes, N.Y., Howell, J.K., Mueller, S.L., Radolf, J.D., Weinstock, G.M. and Norris, S.J. (1997) Identi¢cation and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor
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[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
protein homolog, and a glycolytic pathway enzyme homolog. Gene 197, 47^64. Manoil, C. and Beckwith, J. (1985) Tnpho:A : A transposon probe for protein export signals. Proc. Natl. Acad. Sci. USA 82, 8129^8133. Hardham, J.M. (1995) Identi¢cation and Characterization of Treponema pallidum Genes Encoding Exported and Membrane Proteins. Ph.D. Thesis, University of North Carolina at Chapel Hill, NC. Altschul, S.F., Madden, S.F., Shafer, A.A., Zhang, J., Zang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST : a new generation of protein database search programs. Nucleic Acids Res. 25, 3389^3402. Stamm, L.V., Kerner, T.C. Jr., Bankaitis, V.A. and Bassford, P.J. Jr. (1983) Identi¢cation and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coli. Infect. Immun. 41, 709^721. Stamm, L.V., Greene, S.R. and Barnes, N.Y. (1997). Identi¢cation and characterization of the gyrB gene from Treponema pallidum subsp. pallidum. FEMS Microbiol. Lett. 153, 129^134. Hardham, J.M., McLeod, M.P., Howell, J.K., Sodergren, E., Weinstock, G.M. and Norris, S.J. (1998) Identi¢cation, expression, and transcriptional analysis of the twelve Treponema pallidum tpr genes. In: Abstracts of the 98th General Meeting of the American Society for Microbiology 1998, abstr. D-56, p. 222. American Society for Microbiology, Washington, DC. Fenno, J.C., Wong, G.W.K., Hannam, P.M., Muller, K.-H., Leung, W.K. and McBride, B.C. (1997) Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp. J. Bacteriol. 179, 1082^1089. Norris, S.J. and the Treponema pallidum Polypeptide Research Group (1993) Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Microbiol. Rev. 57, 750^779.
163
[14] Fenno, J.C., Muller, K.-H. and McBride, B.C. (1996) Sequence, analysis, expression and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola. J. Bacteriol. 178, 2489^2497. [15] Fraser, C.M., Norris, S.J., Weinstock, G.M., White, O., Sutton, G.G., Dodson, R., Gwinn, M., Hickey, E.K., Clayton, R., Ketchum, K.A., Sodergren, E., Hardham, J.M., McLeod, M.P., Salzberg, S., Peterson, J., Khalak, H., Richardson, D., Howell, J.K., Chidambaram, M., Utterback, T., McDonald, L., Artiach, P., Bowman, C., Cotton, M.D., Fujii, C., Garland, S., Hatch, B., Horst, K., Roberts, K., Sandusky, M., Weidman, J., Smith, H.O. and Venter, J.C. (1998) Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 281, 375^388. [16] Centurion-Lara, A., Castro, C., Van Voorhis, W.C. and Lukehart, S.A. (1996) Two 16S-23S ribosomal DNA intergenic regions in di¡erent Treponema pallidum subspecies contain tRNA genes. FEMS Microbiol. Lett. 143, 235^240. [17] Deitsch, K.W., Moxon, E.R. and Wellems, T.E. (1997) Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections. Microbiol. Mol. Biol. Rev. 61, 281^293. [18] Lukehart, S.A., Sha¡er, J.M. and Baker-Zander, S.A. (1992) A subpopulation of Treponema pallidum is resistant to phagocytosis: possible mechanisms of persistence. J. Infect. Dis. 166, 1449^1453. [19] Fieldsteel, A.H., Stout, J.G. and Becker, F.A. (1979) Comparative behavior of virulent strains of Treponema pallidum and Treponema pertenue in gradient cultures of various mammalian cells. Infect. Immun. 24, 337^345. [20] Riviere, G.R., Thomas, D.D. and Cobb, C.M. (1989) In vitro model of Treponema pallidum invasiveness. Infect. Immun. 57, 2267^2271.
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