MALDI-TOF MS identification of T. pallidum proteins.
Rabbits have been used for many years to propagate T. pallidum
in the absence of an effective in vitro
culture system, and T. pallidum
maintained in rabbits retains infectivity and virulence in humans (13
). We extracted T. pallidum
Nichols from the testicular tissue of infected rabbits and purified the bacteria by Percoll gradient density centrifugation. Lysates of purified bacteria were separated by IEF and NEPHGE 2DGE; NEPHGE permits the separation of highly basic polypeptides (31
). Proteins from silver-stained gels were analyzed by MALDI-TOF mass spectrometry. We identified the polypeptides present in 148 protein spots; of these, 144 corresponded to 88 different T. pallidum
proteins, and 4 corresponded to rabbit proteins (Fig. and Table ). The 56 additional spots represented charge variants, minor size variants, or degradation products of T. pallidum
proteins. The detection of only four rabbit products demonstrates the high degree of purification achievable by Percoll density gradient centrifugation (17
FIG. 1. Two-dimensional gel electrophoresis of T. pallidum proteins. T. pallidum lysates were separated by IEF at pH 5 to 7 (A) or NEPHGE at pH 3.5 to 10 (B) in the first dimension, followed by 8 to 20% SDS-PAGE in the second dimension. Gels were subsequently (more ...)
MALDI-TOF analysis of T. pallidum proteins separated by IEF and NEPHGE 2DGE and summary of reactivity of IRS and human syphilis patient sera against T. pallidum proteinsa
Multiple distinct spots in the same gel were often identified as the same protein and apparently represent charge or molecular mass variants; examples of these variants are indicated by arrows in Fig. . This phenomenon was observed at a higher frequency with IEF than with NEPHGE 2DGE, most likely due to the higher resolution and lower compression level of the gradient in the IEF gels (for example, compare spots 143 and 144 on the IEF and NEPHGE gels in Fig. ). Fifty-one of the spots identified represented apparent mass variants, based on deviation from the predicted molecular weight (MW). All of these spots corresponded to abundant proteins for which a major spot of the expected size and pI was identified; these included the cytoplasmic filament protein CfpA (9 mass variants), the 47-kDa carboxypeptidase (5 mass variants), the flagellar proteins FlaA1, FlaB1, FlaB2, and FlaB3, and the FlaA1 paralog FlaA2 (a total of 16 mass variants).
The observed Mr
and predicted MW of intact polypeptides were compared to further verify the MS identifications (Table and Fig. ). In this analysis, the MW values were adjusted to take into account either experimentally verified or predicted cleavage of the polypeptides by either signal peptidase I or signal peptidase II; for the latter, the cleavage points of 46 predicted lipoproteins as determined by Setubal et al. (34
) were utilized. No attempt was made to correct the predicted MWs for effects of lipidation or other potential modifications. Four polypeptides (the dodecameric form of TpF1, hypothetical protein TP0179, FtsH, and Tp34 [which migrates as a smear, as determined by SDS-PAGE]) were considered outliers, with ratios of observed Mr
to predicted MW of 0.68, 1.57, 0.70, and 1.53, respectively. The TpF1 dodecamer likely migrates aberrantly because of its multimeric conformation. TP0179 was found to have an Mr
much greater than that corresponding to its predicted size (101.9 kDa, compared to 66.5 kDa); the predicted gene may be truncated by a sequence error, because inclusion of the adjacent gene (TP0178) in the TP0179 reading frame results in a predicted molecular mass of ~101 kDa. Tp34 migrates as a smear, as determined by SDS-PAGE, for unknown reasons. The reason for the discrepancy in the Mr
of FtsH is not known; the spot identified may be a degradation product.
Correlation between predicted molecular weights of T. pallidum proteins and Mr values obtained in 2DGE patterns in this study. Molecular weights take into account removal of predicted signal peptides. Apparent degradation products were excluded.
Exclusion of the mass variants and the four outliers from the list of proteins identified resulted in a high degree of correlation between the observed Mr and predicted MW (Fig. ); the mean ratio ± standard deviation (SD) for observed Mr/predicted MW was 1.01 ± 0.08 (R2 = 0.9849). In contrast, the mass variants had a poor correlation (ratio = 0.75 ± 0.22; R2 = 0.39) (data not shown). Therefore, the mass variants appear to represent degradation products. These may be naturally occurring breakdown products or may arise during the purification of T. pallidum from rabbit tissue.
Although the majority of spots identified corresponded to a single protein, there were a few spots where MALDI-TOF data indicated a mixture of two protein species. Examples are spot 53 (TmpA and FliG), spot 62 (AsnA and 30S ribosomal protein S2), spots 73 and 99 (GroEL and 47-kDa carboxypeptidase degradation products), spot 92 (hypothetical protein TP0453 and elongation factor Ts), spot 97 (hypothetical protein TP0139 and lipoprotein Tpn32), and spot 100 (Pgm and hypothetical protein TP0290). Thus, these spots appear to be composed of multiple protein species with similar molecular masses and pIs that were not resolved from one another under the electrophoretic conditions utilized.
A number of the more-abundant polypeptides that we describe here have previously been identified by other methods, such as N-terminal sequencing or immunoblotting with monoclonal antibodies (18
); these proteins are CfpA, GroEL, DnaK, the 47-kDa carboxypeptidase, TmpA, TmpB, the 17-kDa and 15-kDa lipoproteins, the purine nucleoside receptor lipoprotein PnrA (TmpC) (8
), the lactoferrin-binding periplasmic lipoprotein Tp34 (TpD) (7
), and the flagellar proteins FlaA1, FlaB1, FlaB2, and FlaB3. As in previous studies, we found that the most-abundant proteins observed by silver staining were flagellins, CfpA, chaperonins, and several lipoproteins, including MglB-2, TmpA, TmpC, and the 47-kDa, 17-kDa, and 15-kDa proteins. High-level expression of lipoprotein genes is typical of Treponema
species and other spirochetes.
In addition to confirming previous protein identities, we identified 63 proteins that had not been described previously by electrophoresis or immunoblotting (Fig. and Table ). These proteins can be categorized by their predicted functions: carbohydrate metabolism (13 proteins), cell division (2 proteins), lipoproteins or structural proteins (10 proteins), flagellum-associated proteins (8 proteins), nucleotide metabolism, degradation, or salvage proteins (4 proteins), molecular chaperones (4 proteins), chemotaxis proteins (2 proteins), energy metabolism enzymes (7 proteins), ABC transporters (7 proteins), proteases (2 proteins), proteins involved in translation (9 proteins), amino acid and cofactor biosynthesis proteins (3 proteins), iron storage proteins (1 protein), cellular detoxification proteins (1 protein), and hypothetical proteins with unknown function (12 proteins). We also determined that the hypothetical protein TP0259 is the lipoprotein TpE; this T. pallidum
gene product had been described previously (19
), but its sequence was not published.
The protein expression that we observed with 2DGE was consistent with T. pallidum
mRNA level data reported previously (35
). We identified the proteins corresponding to nearly all of the highly expressed mRNAs reported by Šmajs et al. (those with cDNA/DNA signal ratios of 4.0 or higher) by 2DGE and MALDI-TOF MS (Fig. and Table ). The majority of proteins with corresponding high transcript levels identified in that study that we did not detect were ribosomal proteins. We identified only four ribosomal proteins, ribosomal proteins S2 (TP0606; spot 62), L5 (TP0201; spot 128), L10 (TP0239; spot 132), and L9 (TP0060; spot 138), in contrast to the 11 ribosomal proteins reported to be transcribed at high levels (35
Serologic reactivity of T. pallidum proteins.
The IEF and NEPHGE 2DGE patterns obtained were highly reproducible, enabling us to reliably correlate seroreactive proteins in Western blots with the corresponding silver-stained gels. We first examined the T. pallidum proteome for serological reactivity by immunoblotting with pooled sera from rabbits infected for 84 days. At this time postinfection, rabbits develop “chancre immunity,” i.e., resistance to reinfection from intradermal inoculation. In addition, the seroreactivity of human sera from patients at different stages of syphilitic infection to the 2DGE-separated proteins was determined. Our goal was to identify antigens that were consistently reactive at all stages of infection as well as those exhibiting differential reactivity at each stage of infection. A summary of the serologic reactivity against T. pallidum proteins can be found in Table . Degradation products were excluded from this analysis.
The infected-rabbit-serum (IRS) pool was reactive with a total of 33 T. pallidum
proteins in the 2DGE immunoblots (Fig. and Table ). The majority of proteins reactive in 2DGE patterns were previously described antigens (26
), including flagellar proteins and lipoproteins, including the ABC transport proteins MglB-2 and TroA (Table ). This study also confirmed IRS serologic reactivity against phosphoenolpyruvate carboxykinase (PckA; TP0122; spot 17), translation elongation factor G (FusA-2; TP0767; spot 6), and chemotaxis protein X (CheX; TP0365; spot 136), which were identified as antigens by McKevitt et al. (22
FIG. 3. Immunoreactivity of T. pallidum proteins separated by 2DGE with rabbit sera. T. pallidum lysates were separated by IEF at pH 5 to 7 (A to C) or NEPHGE at pH 3.5 to 10 (D to F) in the first dimension, followed by 8 to 20% SDS-PAGE in the second (more ...)
Thirty-two of the 106 T. pallidum
proteins found to be reactive with IRS by McKevitt et al. (22
) were identified by MALDI-TOF MS. Surprisingly, only 16 of these 32 antigens were reactive with rabbit sera in the present study. Although the other 16 antigens reported by McKevitt et al. were detected by silver staining and MS, they were not reactive with IRS in our study. However, four of those proteins were reactive with human sera (see below), including bacterioferritin TpF1 (TP1038; spot 1), oxaloacetate decarboxylase (TP0056; spot 22), the integral membrane protein (TP0453; spot 92), and peptidyl-prolyl cis
isomerase FklB (TP0862; spot 96), indicating that a sufficient amount of these proteins was present for detection of serological reactivity by immunoblotting. Possible explanations for these results are that the immunoblot reactivity in our studies was less sensitive than the reactivity obtained with the ELISA format utilized by McKevitt et al. and that the human patient sera were more reactive to some antigens than were the IRS in our analysis. Many of the most-reactive antigens identified in the McKevitt et al. study were not detected by the 2DGE immunoblotting method, including rare lipoprotein A (RlpA; TP0993), glycerophosphodiester phosphodiesterase (GlpQ; TP0257), thioredoxin (TP0100), and the hypothetical proteins TP0957, TP0625, TP0956, TP0463, TP0567, TP0326, and TP0772. Those proteins were not identified by silver staining and subsequent MALDI-TOF MS, indicating that there may not have been sufficient protein present for detection of serological reactivity against those proteins. Overexpression of those proteins in the McKevitt et al. study may have provided adequate protein levels for rabbit serological reactivity to be observed (22
). Alternately, some of these proteins may have been among the proteins that were not selected for MALDI-TOF analysis. For example, several faint spots between 22 and 38 kDa were visible by immunoblotting but were of insufficient quantities to be identified by mass spectrometry. All but two of the antigens not identified by IRS are within that size range.
A number of previously unreported antigens were detected by immunoblotting with IRS in this study, including diphosphate-fructose-6-phosphate 1-phosphotransferase (TP0108; spot 41), flavodoxin (TP0925; spot 139), the FKBP-type peptidyl-prolyl cis-trans isomerase SlyD (TP0349; spot 118), polyribonucleotide nucleotidyltransferase (Pnp; TP0886; spot 11), glyceraldehyde 3-phosphate dehydrogenase (Gap; TP0844; spot 59), and hypothetical protein TP0608 (spots 102 and 105) (Fig. ). We also found one antigen, CfpA, to be weakly reactive with sera from uninfected animals (Fig. ).
The immunoreactivity of the identified T. pallidum
proteins with human sera collected from patients diagnosed with primary, secondary, early latent, or late latent syphilis was also examined. Sera from patients at each stage were pooled as described in Materials and Methods to provide an analysis of reactivity during the course of infection. As expected, the highest level of reactivity occurred with sera from secondary- and early-latent-syphilis patients (Fig. and and Table ). All of the proteins that were strongly reactive with infected rabbit sera were also reactive with sera from syphilis patients. Many of these are lipoproteins, such as the purine nucleoside periplasmic binding protein PnrA (TmpC) (8
), the lactoferrin-binding periplasmic lipoprotein Tp34 (TpD) (7
), the 47-kDa carboxypeptidase (9
), TmpA, TmpB, TpE, and the 17-kDa and 15-kDa lipoproteins (Fig. to and Table ), and were reactive with sera from patients at all stages of syphilis. Lipoproteins thus appeared to elicit the strongest antibody response, even if they are expressed at low levels, as is the case for the 17-kDa lipoprotein (TP0435; spot 141). This protein, as well as the 47-kDa lipoprotein, has been demonstrated previously to be highly antigenic and is currently used in T. pallidum
diagnostic tests (11
). The strong immunogenicity observed with these lipoproteins appears to be dependent on the lipid moiety, as its removal diminishes the production of inflammatory cytokines and activation of immune effector cells (1
). The induction of antibody responses against the B. burgdorferi
lipoprotein outer surface protein A is highly dependent upon lipidation (10
). Therefore, the lipid portion of T. pallidum
lipoproteins is likely acting as an intrinsic adjuvant to stimulate the antibody response against these proteins.
FIG. 4. Immunoreactivity of T. pallidum proteins separated by IEF (pH 5 to 7) 2DGE with human sera. T. pallidum lysates were separated by IEF at pH 5 to 7 in the first dimension, followed by 8 to 20% SDS-PAGE in the second dimension. Gels were subsequently (more ...)
FIG. 5. Immunoreactivity of T. pallidum proteins separated by NEPHGE (pH 3.5 to 10) 2DGE with human sera. T. pallidum lysates were separated by NEPHGE at pH 3.5 to 10 in the first dimension, followed by 8 to 20% SDS-PAGE in the second dimension. Gels (more ...)
Tp34 (TpD; spot 81), also a lipoprotein, was reactive with all the serum pools, although at lower levels with human late-latent-syphilis sera and infected rabbit sera. Two lipidated periplasmic ABC transport proteins, MglB-2 and TroA, were also reactive at all stages of infection. In addition, a number of antigens that have not previously been reported were identified, including hypothetical protein TP0584 (spot 29), the V-type ATPase AtpA-1 (TP0425; spot 19), and hypothetical protein TP0608 (spots 102 and 105). These proteins were reactive with all sera tested (Fig. to ). Five antigens found to be reactive with infected human sera (but not with infected rabbit sera) included hexokinase (TP0505; spot 42), hypothetical protein TP0965 (spot 66), phosphate acetyltransferase (Pta; TP0094; spot 85), the integral membrane protein TP0453 (spot 92), and peptidyl-prolyl cis
isomerase FklB (TP0862; spot 96). These antigens were not identified as significantly reactive proteins in the Brinkman et al. study of the reactivity of recombinant T. pallidum
proteins with human syphilitic sera (3
We observed a number of acidic, high-molecular-weight spots that were strongly reactive with infected rabbit sera and sera from primary-, secondary-, and early-latent-syphilis patients (red boxes in Fig. to ). These polypeptides ranged in size from approximately 70 kDa to 120 kDa in molecular mass (Fig. and ). Due to their low abundance, we were able to detect only one of the spots by silver staining-MS (spot 7; CfpA variant). A search of the T. pallidum genome revealed 13 proteins of appropriate predicted molecular masses with predicted pIs of <6.0 that were not identified by silver staining-MS (see Table S2 in the supplemental material); these therefore represent candidate proteins for this group.
One antigen of interest that appears to be uniquely reactive in human infection is the oligomeric form of the bacterioferritin protein TpF1 (TP1038; spot 1) (Fig. and ). TpF1 functions as a dodecamer to bind iron (36
) but has been observed to migrate at several molecular masses on SDS-PAGE gel, ranging from160 kDa to >400 kDa, in the oligomeric form (12
). Multiple identical subunits form a ring structure held together by disulfide bonds, creating a very stable oligomer (32
). In its unreduced form, the basic TpF1 oligomer typically migrates at 190 kDa, but reduction by mercaptoethanol results in migration of an oligomer at 160 kDa and dissociated monomers at 19 kDa (32
). We observed the 160-kDa form by 2DGE and Western blot analysis. We did not observe serologic reactivity against the monomeric form of TpF1 (spot 134), which is consistent with previous findings. In prior studies by Borenstein et al. (2
), TpF1 was cloned and expressed as a recombinant protein from E. coli
, and serologic reactivity from syphilis patients against a 190-kDa oligomeric form of the protein was observed. However, no reactivity against the dissociated 19-kDa monomeric form of the expressed protein was observed (2
). Furthermore, immunization of rabbits with recombinant TpF1 provided partial protection against challenge with viable treponemes (2
). It may also be of interest to determine the identity of the low-abundance polypeptides in the “red box” as proteins that might be of diagnostic or immunogenic value.
We observed stronger reactivity with pooled sera from primary-syphilis patients than expected. The serum pool used for Fig. and was comprised of three samples, i.e., the two tested for Fig. S1 in the supplemental material and an additional serum sample with an RPR titer of 1:64; insufficient serum from the latter sample was available for performance of a separate immunoblot analysis. Therefore, two of the sera from the primary-syphilis-patient pool were examined for seroreactivity with NEPHGE 2DGE immunoblots (see Fig. S1 in the supplemental material). Figure S1B in the supplemental material shows the reactivity of serum from a primary syphilis patient with an RPR titer of 1:16, and Fig. S1C in the supplemental material exhibits the reactivity of serum from a primary syphilis patient with an RPR titer of 1:64. As expected, the serum sample with an RPR titer of 1:16 was reactive to fewer proteins than the serum sample with an RPR titer of 1:64. For example, reactivity to TpF1, hypothetical protein TP0965, and AhpC was not detectable in the sample with an RPR titer of 1:16 (see Fig. S1B in the supplemental material). The high RPR titers of two of these samples correlate with the unexpected strong reactivity that we observed with pooled primary-syphilis sera.
The antibody reactivities obtained with human sera and IRS in the 2DGE immunoproteome analysis correlated well in general, with some differences (Table ). Of the 87 T. pallidum polypeptides identified by 2DGE-MS, 40 were found to be reactive with sera from humans at some stage of infection, whereas only 32 were reactive with IRS; 31 of the proteins were reactive with both human sera and IRS. Nine of the human serum-reactive proteins were not detectably reactive with IRS, whereas only one of the IRS-reactive proteins was not reactive with the human sera tested. In most cases, these represented faint reactions indicative of low antibody titers. However, moderate immunoblot reactivity with some human serum pools against AtpA-1 (V-type ATPase, subunit a; TP0424), elongation factor Ts (TP0605), and FklB (peptidyl-prolyl cis-trans isomerase; TP0862) was observed (Table ), whereas a reaction with IRS was not detected. The IRS used in this study were collected at 84 days postinfection and are considered to be highly reactive. Therefore, the apparent differential reactivity observed for these three proteins may reflect differences between the antibody responses of humans and rabbits to certain T. pallidum polypeptides, but additional studies using purified proteins would be needed to verify this finding. Other possible explanations for the differences observed in rabbit and human immunoreactivity are that (i) multiple time points were tested for humans, compared to the single time point tested for rabbits, (ii) fewer subjects were used in the rabbit serum pool than in the human serum pools, resulting in a smaller array of immunoreactive proteins, and (iii) laboratory rabbits are more inbred than the human population and thus may have reduced antibody repertoire diversity.
Comparison of the antigenicity results obtained in the present study with those obtained in prior recombinant protein immunoproteome analyses (3
) indicates that the two approaches provide overlapping but somewhat disparate results. In the current study, moderate to high levels of reactivity of human serum pools against CfpA (TP0748), the three flagellar filament core proteins FlaB1, FlaB2, and FlaB3 (TP0868, TP0792, and TP0870, respectively), flagellar motor protein FliG (TP0400), the V-type ATPase subunit AtpA-1 (TP0426), hypothetical protein TP0584, and elongation factor Ts (TP0606) were observed (Fig. and and Table ; see also Fig. S1 in the supplemental material), whereas these proteins were nonreactive in both of the prior studies using expression of recombinant proteins in E. coli
). In addition, Tpp15 (TP0171), GroEL (TP0030), PckA (TP0122), hypothetical protein TP0453, and membrane fusion protein TP0965 were reactive in the current study and the prior IRS analysis (22
) but not in the prior human serum analysis (3
). The lack of reactivity of the flagellar core proteins and CfpA in the prior immunoproteome studies was particularly surprising, in that these proteins had been shown previously to be highly immunogenic and to induce antibody responses during infection (reviewed in references 27
). The lack of reactivity in the immunoproteome studies (3
) may have been due to poor expression, rapid degradation, or improper folding with loss of antibody binding activity. For some of the relatively minor spots in the 2DGE pattern, it is possible that the antigenic reactivity detected in the immunoblots was due to comigrating proteins that were not detected in the MS analysis, yielding a “false-positive” result. There were also 5 proteins for which clones were not obtained in the previous recombinant protein studies but were found to be highly reactive by 2DGE immunoblot analysis; these proteins were phosphofructokinase (Pfk; TP0108), flagellar sheath protein (FlaA-1; TP0249), membrane lipoprotein TpE (TP0259), hypothetical protein TP0608, and polyribonucleotide nucleotidyltransferase (Pnp; TP0886) (Table ).
Conversely, four proteins identified by 2DGE-MS were not found to be reactive with human sera by immunoblot analysis in our study but were reactive in the prior recombinant protein analyses (3
). These proteins were FlaA2/Tromp2 (TP0663), translation elongation factor G (FusA-2), hypothetical protein TP0789, and lipoprotein Tpn32 (TP0821). All of these are relatively minor spots in the 2D gels and may be present in too small of a quantity to yield a visible antibody reaction under the conditions used in the current study. An even greater discrepancy was observed with the IRS chemiluminescence enzyme immunoassay (EIA) recombinant protein results reported by McKevitt et al., in which 17 proteins reactive in this prior study were not detectably reactive by our 2DGE IRS immunoblot analysis. However, 11 and 13 of these proteins were not reactive with human syphilis sera in the current study (Fig. and ; see also Fig. S1 in the supplemental material) or in the Brinkman et al. analysis (3
). Therefore, many of these disparities may have resulted from a low positive-value threshold or procedural differences, resulting in detection of weakly positive or potentially false-positive results.
We assessed whether polypeptides expressed at high levels were more likely to evoke a strong antibody response than proteins expressed at low levels. To provide a rough estimate of expression and relative antigenicity, we compared the apparent amounts of protein of 22 polypeptides (see Table S3 in the supplemental material) in the stained gels to the intensities of antibody staining using ImageQuantTL, version 7.0 (General Electric), software. Immunostaining intensity did not correlate with silver staining intensity, as exemplified by the values obtained with early-latent-syphilis human sera and with IRS (see Fig. S2 in the supplemental material). Several low-abundance proteins exhibited high immunoreactivity, whereas certain abundant proteins had low immunoreactivity. Of particular interest was the very strong reactivity observed for the 15-kDa lipoprotein, TpF1, and the unidentified polypeptides highlighted in the red box (Fig. to ; see also Fig. S1 in the supplemental material). TpF1 and the 15-kDa lipoprotein account for >25% of the total immunostaining intensity obtained with early-latent-syphilis human sera. However, when the silver staining intensities of all immunoreactive proteins were quantitated, these proteins were found to represent <0.6% of the total reactive protein. In IRS stained immunoblots, the proteins in the red box and the 15-kDa lipoprotein account for <1% of the total reactive protein, while accounting for 20% of the immunoreactivity. An important caveat to note is that silver staining is not very quantitative, especially for smaller proteins in higher-percentage polyacrylamide (16
). However, staining intensity tends to be relatively reduced for highly expressed proteins rather than proteins expressed at low levels, suggesting that our quantitation might overrepresent any correlation between the quantity and immunogenicity of a polypeptide. Thus, the immunogenicity of a polypeptide does not appear to be closely related to its abundance.
In contrast, a relatively good correlation was obtained between the reactivity of individual proteins with human sera from patients at different stages or with IRS, as exemplified by the comparisons of early-latent-syphilis sera/primary-latent-syphilis sera and early-latent-syphilis sera/IRS shown in Fig. S3 in the supplemental material. This analysis further emphasized differences in the reactivities of sera from infected humans and rabbits (see Fig. S3A in the supplemental material). Most notably, TpF1 (spot 1) had ~12-fold-higher staining intensity with human early-latent-syphilis sera than with IRS, and the unidentified “red box” antigens were essentially nonreactive with human sera but were highly reactive with IRS. In addition, FlaB2 (spot 82) reacted 3.4 times more intensely with human early-latent-syphilis sera than with IRS, and IRS were ~3-fold more reactive with TpE (spot 117) and TmpA (spot 53) than early-latent-syphilis sera. Removal of these 5 “outliers” from the correlation shown in Fig. S3A in the supplemental material increased the R2 value from 0.364 to 0.897. These results indicate that the immune responses to some T. pallidum proteins may differ in humans and experimentally infected rabbits. Overall, the 22 polypeptides analyzed quantitatively had similar reactivities with human primary- and early-latent-syphilis serum samples (see Fig. S3B in the supplemental material).
In reality, it is likely that nearly all bacterial proteins induce an adaptive immune response during an infection, due to the foreign nature of these proteins and the exquisite sensitivity of the immune system. The degree of immunogenicity of T. pallidum
proteins may therefore represent a continuum. Only those proteins with the highest-level responses are potentially useful for immunodiagnostics, while those that are surface exposed are most likely to be immunoprotective. The data presented in this study confirmed the identity of previously reported antigens. Many new immunoreactive T. pallidum
proteins were also revealed by 2DGE and MS analysis, demonstrating the value of analyzing the immunoproteome by a variety of methods. These antigens may provide useful future directions for the development of vaccines and immunodiagnostics. Five antigens of particular interest are the bacterioferritin TpF1, the integral membrane protein TP0453, TP0965 (a putative membrane fusion protein), and the hypothetical proteins TP0584 and TP0608. All five of these antigens were reactive with sera from patients with primary syphilis, suggesting that these antigens might be useful in early diagnostic studies. TP0453 has been tested by an enzyme immunoassay in the serodiagnosis of syphilis, was found to be highly reactive with sera from primary-syphilis patients, and exhibited 100% specificity and sensitivity when reacted with sera from syphilis, relapsing-fever, Lyme disease, or leptospirosis patients (39
). The outer membrane location of TP0453 (18
) may also make this antigen useful for vaccine development. TP0965, TP0584, and TP0608 were reactive with sera from patients at all syphilis stages, indicating that these antigens may also be useful in the serodiagnosis of syphilis. The cellular location and protective activity of these antigens have yet to be determined. Two-dimensional gel electrophoresis coupled with MALDI-TOF MS and serological analysis is a valuable tool for the identification of new antigens and virulence factors and can be applied to a variety of microbiological systems. These tools are especially useful for organisms like T. pallidum
that cannot be cultured in vitro