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Published online before print
June 12, 2003, 10.1101/gr.288103 Genome Res. 13:1665-1674, 2003 ©2003 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/03 $5.00
Letter Systematic Cloning of Treponema pallidum Open Reading Frames for Protein Expression and Antigen Discovery1 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA 2 Structural and Computational Biology and Molecular Biophysics Program, Baylor College of Medicine, Houston, Texas 77030, USA 3 Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas 77030, USA 4 Department of Microbiology and Molecular Genetics, University of Texas—Houston Medical School, Houston, Texas 77030, USA 5 Department of Pathology and Laboratory Medicine, University of Texas—Houston Medical School, Houston, Texas 77030, USA
A topoisomerase-based method was used to clone PCR products encoding 991 of the 1041 open reading frames identified in the genome sequence of the bacterium that causes syphilis, Treponema pallidum subsp. pallidum. Cloning the open reading frames into the univector plasmid system permitted the rapid conversion of the original clone set to other functional vectors containing a variety of promoters or tag sequences. A computational prediction of signal sequences identified 248 T. pallidum proteins that are potentially secreted from the cell. These clones were systematically converted into vectors designed to express the encoded proteins as glutathione-S-transferase fusion proteins. To test the potential of the clone set for novel antigen discovery, 85 of these fusion proteins were expressed from Escherichia coli, partially purified, and tested for antigenicity by using sera from rabbits infected with T. pallidum. Twelve of the 85 proteins bound significant levels of antibody. Of these 12 proteins, seven had previously been identified as T. pallidum antigens, and the remaining five represent novel antigens. These results demonstrate the potential of the T. pallidum clone set for antigen discovery and, more generally, for advancing the biology of this enigmatic spirochete.
The genome sequences of many infectious microorganisms have now been determined. The availability of genome sequence information will enable new approaches to be developed to determine the function of gene products and their possible role in pathogenesis. A proteomics approach is being developed to identify proteins important for the Treponema pallidum host-pathogen interaction. As a first step, an efficient high-throughput strategy has been used to clone 96% of the predicted T. pallidum open reading frames (ORFs) into a recombination-based vector system.
T. pallidum is the causative agent of syphilis. Syphilis is a
multistage infection characterized by localized, disseminated, and chronic
manifestations interspersed between periods of latent infection. The molecular
mechanisms of T. pallidum pathogenesis are poorly understood
(Norris et al. 2001
The complete genome sequence of T. pallidum was determined in 1998
(Fraser et al. 1998 A number of factors make the T. pallidum genome an excellent model system for the development of functional genomic techniques. These factors include the small size of the genome, the correspondingly small number of ORFs, and the intractability of this organism to standard genetic approaches. Important questions regarding T. pallidum biology that a functional genomics approach can address include the identification of antigenic proteins that may aid in diagnostics and vaccine development, and identification of proteins important for attachment and invasion of human tissues.
Construction of the T. pallidum Univector Clone Set The purpose of this study was to develop a complete set of T. pallidum genes cloned into a variety of plasmids useful for protein expression and purification in Escherichia coli, as well as other functional vectors useful for methods such as phage display and two-hybrid system protein interaction studies. This goal was accomplished by using a recombination-based approach developed by Liu et al. (1998  ) to facilitate the
cloning of a PCR product into a plasmid, followed by the rapid conversion of
the plasmid to a number of different expression systems without the necessity
of cloning the PCR product multiple independent times. The method, termed the
univector plasmid fusion system (UPS), involves the insertion of a gene into
the univector plasmid (in this case, pUNI-D) and subsequent fusion of the gene
to a variety of promoters and tag sequences. The system is based upon plasmid
fusion using the Cre-loxP site–specific recombination system of
bacteriophage P1 (Fig. 1;
Sternberg et al. 1981). The
pUNI plasmid is used for the initial cloning of PCR products, whereas the
pHOST plasmid contains the appropriate promoter or tag sequences for creating
fusion proteins. The recombinant protein expression construct is made by
fusion of the pUNI and pHOST plasmids via Cre-loxP
site–specific recombination (Fig.
1; Liu et al.
1998).
Insertion of T. pallidum ORFs in a univector was accomplished by
using a cloning method based on the mechanism of action of the vaccinia virus
DNA topoisomerase I (Shuman
1994
The annotated T. pallidum genome sequence deposited in GenBank
includes the sequence position of the start and stop codons of each ORF
(Fraser et al. 1998 In the initial PCR reactions, 959 of the 1031 ORFs were successfully amplified by using the primers designed with 25 bp of complementary nucleotides. Inspection of primer pairs for the ORFs that failed to amplify indicated that below average Tm values for the primers might have been responsible for the lack of amplification of 61 of these ORFs. The region of complementarity in these primers was extended to 30 bp, and subsequent amplification yielded PCR product for 38 of the 61 genes. In addition to extending the length of primers, increasing the annealing/extension parameter of the PCR cycling program aided in acquiring PCR products for an additional 26 ORFs. To date, 1023 of the 1031 predicted genes have been successfully amplified by PCR. Of the 1023 PCR products, 991 have been inserted into the pUNI-D vector by using the topoisomerase cloning method. Briefly, PCR products were inserted into pUNI-D (Fig. 1), transformed into E. coli, and selected on Luria Bertani (LB) agar plates containing kanamycin. Transformants were screened by using a PCR technique that identifies ORFs of the correct size cloned into pUNI-D in the correct orientation.
The T. pallidum ORFs that have not been cloned successfully are
listed in a table available at
www.genome.org
as Supplemental Materials. The average size of the ORFs that were not cloned
is 1695 bp, whereas the average size ORF for the entire genome is 1013 bp.
Therefore, a partial explanation for the failure to clone these ORFs may be
related to the size of the PCR product. However, this is not a complete
explanation in that several of the uncloned ORFs are average-sized or smaller.
Another possibility is that these T. pallidum gene products are toxic
when expressed in E. coli. Although the pUNI vector is designed to
minimize transcription of the cloned insert in E. coli, it is
possible that low levels of transcription of some genes occur, and these could
have toxic effects (Liu et al.
1998
Characterization of the T. pallidum Univector Clone Set
Of the 27 mutations identified by sequencing, nine are predicted to result in frameshift or nonsense mutations and, therefore, to encode nonfunctional protein products. Five of these nine mutations were single or multiple nucleotide deletions that occurred inside the region complementary to the 5' PCR primers used to amplify ORFs TP0252, TP0415, TP459, TP0602, and TP0943. This resulted in a frameshift mutation in each of these ORFs. In addition, a frameshift mutation occurred in TP0536 due to a single nucleotide insertion within the region complementary to the 5' primer used for amplification of this ORF. Two additional frameshift mutations occurred due to single base pair deletions within the coding sequences of TP0174 and TP0324. Finally, a single nucleotide substitution resulted in the generation of a STOP codon within the coding sequence of TP0117. The high frequency of mutations detected inside PCR primer regions (six of nine) indicates that quality control of PCR primer production is an important step that may improve the systematic cloning of any genome. Of the 18 remaining mutations, six were single nucleotide substitutions that resulted in amino acid substitutions within ORFs TP0013, TP0074, TP0350, TP0471, TP0711, and TP0782. An additional single nucleotide substitution was silent, whereas another occurred in the CACC sequence that is used for cloning but is not within the coding sequence of an ORF. The remaining 10 mutations were deletions at the 3' end of cloned genes. Three single nucleotide deletions were identified in clones TP0212, TP0692, and TP0792 that resulted in silent mutations because the next nucleotide maintains the stop codon. The seven other deletions involved multiple (two to six) nucleotides; however, all of these deletions impact only the last amino acid and/or the stop codon in the translated protein of ORFs TP0011, TP0074, TP0247, TP0260, TP0309, TP0389, and TP1031.
Conversion of T. pallidum Clone Set to
glutathione-S-transferase–Fusion Protein Expression Vector
Currently, there is no vaccine available for the prevention of syphilis
(Weinstock et al. 1998
Over the past 25 years, identification and antigenic characterization of
T. pallidum polypeptides has dominated T. pallidum research
(Strugnell et al. 1990 To identify novel antigens, binding interactions between recombinant T. pallidum proteins and IgG antibodies present in rabbits infected with T. pallidum were examined. Expression of 85 GST-fusion proteins (a sample set from the proteins with predicted signal sequences) was carried out in E. coli cells grown in a 96-well format. The GST-fusion proteins present in E. coli cell lysates were incubated in ELISA wells coated with immobilized glutathione (Pierce), and washes were performed to reduce nonspecific binding interactions between E. coli proteins and the ELISA wells. The immobilized GST-fusion proteins were then exposed to serum collected from three rabbits both before inoculation (normal rabbit serum [NRS]) and 2 months after intratesticular injection of T. pallidum (infected rabbit serum [IRS]). Detection of interactions between rabbit IgG antibody and recombinant fusion proteins was accomplished by chemiluminescence with donkey antirabbit IgG antibody conjugated to horseradish peroxidase (HRP). The IgG binding data obtained by ELISA (Fig. 2A) indicates that rabbit serum collected prior to T. pallidum infection contains little or no antibody that specifically binds to T. pallidum proteins. Further, the data indicate that significant binding interactions occur between IgG antibodies present in rabbit serum collected 2 months after T. pallidum injection and several immobilized T. pallidum proteins (Fig. 2B). There is a striking difference between the binding interactions detected between the two serum types. For example, the mean level of binding of antibody infected rabbits to proteins in E. coli control lysates that were grown without T. pallidum clones (sample numbers 1 and 2) was 10 times greater than that obtained using sera from uninfected rabbits (Fig. 2A). The difference between the two samples may be a result of nonspecific binding of E. coli proteins to ELISA wells that in turn capture E. coli–specific IgG antibodies. Alternatively, it is possible that intratesticular injections of virulent T. palldium act as an adjuvant and generate an immune response against E. coli proteins that increases IgG antibody concentrations. Finally, an increase in overall IgG antibody concentration may occur as a result of T. pallidum infection, which results in increased nonspecific binding of rabbit IgG antibodies to ELISA wells. In an attempt to eliminate IgG antibodies that recognize E. coli proteins and thereby decrease the background binding levels in the assay, the rabbit serum samples were preincubated with soluble E. coli proteins (Methods). This subtraction technique, along with the fusion protein purification step, allowed the capture and detection of T. pallidum–specific IgG antibodies from serum collected from rabbits 2 months after infection.
A total of 12 GST-fusion protein samples with the highest mean
chemiluminescence proteins were identified as statistically relevant T.
pallidum antigenic proteins based on a t test for independent
samples with P scores of P < 0.001
(Fig. 2B;
Table 2). Seven of the 12
antigens have been previously identified by various methods. These proteins
include TP0100 (thioredoxin-like protein;
Radolf et al. 1995
In contrast to the above clones, the GST-fusion of lipoprotein 15 kD
(Tpp15), a previously identified major treponemal antigen, was not bound
significantly by IgG antibody in the IRS
(Radolf et al. 1988
Five previously unreported antigens (TP088, TP0133, TP0183, TP0307, and TP0469) were found to bind significant amounts of IgG in the sera from infected rabbits. Interestingly, none of these proteins have been assigned a biological function. Protein TP0088, a conserved hypothetical protein, shares identity to only one known protein, the Borrelia burgdorferi conserved hypothetical protein, BB0662. Residues 55–170 are 24% identical to BB0662, but neither TP0088 nor BB0662 has a predicted biological function. Protein TP0133, a hypothetical lipoprotein, shares significant identity to four other T. pallidum hypothetical lipoproteins, including TP0134, TP0136, and TP0462. Proteins TP0133 and TP0134 appear to be closely related because residues 8–140 of protein TP0133 are 49% identical to residues 6–376 of protein TP0134. Protein TP0133 also appears related to proteins TP0462, sharing 38% identity between residues 12–410 of protein TP133 and residues 1–392 of protein TP0462. Protein TP0462 was also among the 85 proteins assayed in this experiment, and despite the sequence homology with TP0133, protein TP0462 did not bind significant amounts of antibody from the sera from infected rabbits. However, the sequence of clone TP0462 has not been analyzed; therefore, it is possible that a mutation in this construct may reduce protein expression. Lastly, residues 6–151 of protein TP0133 are 51% identical to residues 3–146 of protein TP0136.
The binding interactions detected between immobilized protein TP0183 and
rabbit IgG generated the third highest chemiluminescent signal after that of
proteins TP0453 and TP0319 (Fig.
2B). It is difficult to make quantitative conclusions about
binding interactions because this screen does not normalize for the quantity
of immobilized fusion protein for each gene examined, but the observed
robustness of the chemiluminescent signal generated by proteins TP0183 and
TP0435, the latter of which is known to elicit a strong immune response in
rabbits (Lukehart et al.
1986 Although the most significant sequence homology of TP0307 is with a B. burgdorferi hypothetical protein of unknown function, other sequence alignment data for protein TP0307 indicates a putative function as a kinase, as well as a possible subcellular location on the outer membrane. Residues 33–191 of protein TP0307 are 30% identical to residues 441–593 of the Streptomyces toyocaensis eukaryotic-type serine/threonine kinase. In addition, residues 5–201 of protein TP0307 are 22% identical to residues 12–214 of a Leptospira interrogans putative outer membrane protein. The fact that TP0307 reacts with the sera and has homology with an outer membrane protein makes this an interesting candidate for immune protection studies. Of all the previously unreported antigens identified here, protein TP0469 appears to be the most conserved. Of the 989 sequence alignments identified in database searches, 186 possess a low enough E value to qualify as significant. Multiple alignments to proteins such as Methanothermobacter thermautotrophicus O-linked N-acetylglucosamine transferase indicate this as a putative function of protein TP0469. In addition, protein TP0469 has sequence identity to several proteins containing tetratricopeptide repeats. Lastly, residues 233–472 of protein TP0469 are 21% identical to residues 719–949 of B. burgdorferi surface-located membrane protein 1 (lmp1). Further investigations into these putative antigenic proteins are necessary to determine their relative antigenicity and potential protective activity. However, the fact that several well-characterized T. pallidum antigens were identified by the same method lends support to the findings and indicates that these antigens should be treated as potential vaccine candidates. Antigenic characterization experiments were accomplished by exposing T. pallidum proteins fused to GST to serum from rabbits 56 d after T. pallidum infection. Current protein quantitation techniques are not sensitive enough to accurately quantify the amount of fusion protein immobilized to the glutathione-coated plate. However, it is important to determine whether there is a relationship between the amounts of protein immobilized and the amounts of rabbit IgG antibody captured by the GST-fusion protein. For example, if the proteins that resulted in the strongest binding signal with antibody were also present at much higher levels than other proteins tested, it would indicate that the signal being detected is simply an increase in nonspecific binding due to increased amount of protein. Similarly, if a protein is not expressed, it cannot bind antibody and will yield a false-negative result in the ELISA experiment. The relative amount of T. pallidum fusion proteins immobilized in the ELISA wells was estimated by using an anti-GST antibody to detect the GST tag that is common to all of the fusions. The methods used to immobilize T. pallidum–GST fusion proteins on glutathione-coated wells for quantitation were identical to those used in the antigenic characterization experiments. However, instead of exposing immobilized protein to rabbit serum, an anti-GST antibody was used. Nonspecific antibody binding interactions were washed away before the HRP substrate was added to each well in order to detect chemiluminescence generated from captured HRP. The chemiluminescent signal was compared with the signal generated from captured IgG antibody present in serum from rabbits 56 d after T. pallidum infection (Fig. 3A). A scatter plot (Fig. 3B) demonstrates that there is no correlation between the amount of GST-fusion protein immobilized and the amount of IgG antibody captured by the immobilized GST-fusion proteins. Many of the E. coli extracts tested contained relatively low quantities of the T. pallidum-GST fusion proteins, based on low anti-GST reactivities (Fig. 3). This low reactivity could be due to poor transcription and or translation of the fusion product, or to proteolytic degradation of the product. Those fusion proteins that are present at low levels could potentially yield false-negative results. Thus one of the goals in future studies will be to achieve more uniform levels of the fusion products. However, it is notable that two of the gene products, TP0326 and TP0257, were highly reactive with sera from T. pallidum–infected rabbits and demonstrated relatively low reactivity with anti-GST (Fig. 3B). Furthermore, several proteins are expressed at high levels but do not exhibit marked reactivity with anti–T. pallidum antibodies (Fig. 3B). Taken together, these results indicate that high reactivity with anti-T. pallidum antibodies is a reliable indicator of high antigenicity in this system, whereas low reactivity could be due to either low antigenicity or low fusion protein levels. This is a common limitation in most recombinant antigen screening techniques (e.g., colony immunoblot assays), and this report represents the first attempt to approach this problem quantitatively. The above experiments indicate that the T. pallidum clone set will be a powerful reagent for functional genomics studies of T. pallidum biology and pathogenesis. The availability of the clone set permits the systematic evaluation of antigenicity of T. pallidum proteins. In addition, the ELISA experiments described above can be used to detect other types of protein–protein or protein ligand interactions. The clone set can also be used to examine binding interactions in vivo by using two-hybrid approaches to generate large-scale protein–protein interaction maps. Such tools are important for an organism that cannot be cultured in vitro and is therefore recalcitrant to most molecular genetic approaches for the study of gene function and pathogenesis.
Bacterial Strains and Plasmids Chemically competent E. coli PIR1 (Liu et al. 1998 ) were
provided by Invitrogen, Inc., and were used to propagate the univector plasmid
DNA containing the cloned T. pallidum ORFs. E. coli
BL21* DE3 (Invitrogen, Inc.) was used to propagate univector-host
fusion plasmid DNA after Cre-loxP recombinase reactions and to
express all of the GST-fusion proteins. The vector pUNI-D/V5-His-TOPO
(referred to as pUNI-D in Results and Discussion) was provided by Invitrogen,
Inc., and was used for the insertion of all T. pallidum PCR products.
The vector pMM110 was created by inserting a loxP site into the
XhoI-NotI site of pGex-4t-2 (Amersham Biosciences) and was
used as the host vector for expression of all GST-tagged T. pallidum
proteins.
Media
PCR Reactions
Topoisomerase Cloning
PCR Screen for Recombinant Clones
Cre-loxP Recombinase Reactions
DNA Sequence Data Collection
Rabbit Serum Preparation
Absorption of Anti–E. coli Protein Antibodies
ELISA Protocol
We thank Invitrogen, Inc., for providing the pUNI-D vector adapted with topoisomerase and Research Genetics for providing the PCR primer set. In addition, we thank Steve Elledge for helpful advice. M.M. is supported in part through the Keck Center for Computational and Structural Biology, NLM Training Grant T15 LM07093. This work was supported by NIH grants AI45842 to T.P. and AI49552 to S.N. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.288103.
6 Corresponding author. Article published online before print in June 2003. [Supplemental material is available online at www.genome.org.]
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http://www.cbs.dtu.dk/services/SignalP/; SignalP program.
Received March 19, 2002;
accepted in revised format April 22, 2003.
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ABSTRACT
RESULTS AND DISCUSSION
1100 kb and is therefore one of the
smallest known prokaryotic genomes (Fraser
et al. 1998
