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Molecular and immunochemical analysis of Treponema pallidum
FEMS Microbiology Reviews 32 (1986) 139-148 Published by Elsevier
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FER 00008
Molecular and immunochemical analysis of Treponemapallidum (Syphilis; spirochaete; outer membrane; axial filament)
C.W. Penn, M.J. Bailey and A. Cockayne Department of Microbiology, Universityof Birmingham, Birmingham BI 5 2TT, U.K. (Received 27 February, 1985) (Revision received 28 May, 1985) (Accepted 5 June 1985)
1. SUMMARY Molecular analysis of polypeptides and antigens of Treponema pallidum has been used increasingly during the past 5 years in investigation of the immunology, pathogenicity and molecular biology of this organism. Failure to culture the organism has severely limited our knowledge of its constituent polypeptides and antigens, but many profiles of these unknown constituents, revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques have been published. In order to compare meaningfully the results obtained by different groups, we have identified a standard pattern of prominent 'landmark' polypeptides in such gel profiles and where possible have assigned functional identities to them. A preliminary nomenclature for the prominent polypeptides of T. pallidum is proposed. These are: P1, 80 kDa; P2, 60 kDa; P3, 47 kDa, an outer membrane-associated polypeptide; P4, 40 kDa; P5, 37 kDa, the major polypeptide of the axial filament; P6, 34 kDa; and P7, 31.5 kDa. 2. INTRODUCTION Despite an enormous wealth of clinical data, detailing human antibody responses in syphilis to
both non-treponemal [1] anti treponemal [2] antigens, extending over most of this century, we have only recently begun to analyse the antigens and other components of T. pallidum at the molecular level. The source of T. pallidum antigen for diagnostic testing has been the whole treponeme, consisting of a mosaic of antigenic determinants of unknown individual importance in the stimulation of antibody responses in disease. The first reported use of modem techniques of biochemical and immunochemical analysis of the antigens of T. pallidum was published in 1979 [3], and since then at least 40 papers have contributed to the substantial knowledge of proteins and antigenic determinants which has now accumulated. This rapid expansion of data has, however, led to considerable difficulties of interpretation and comparison of the work of different groups, for two main reasons. The most important is that despite some recenl, progress in attempts to culture the organism in vitro [4,5], the quantities of organisms needed for meaningful analysis can still only be obtained routinely by extraction from infected rabbit testes, with attendant variation in both the condition of the extracted treponemes and the level of contamination with rabbit-derived material. This has led to great variation in the composition of treponeme preparations, and the definite identification of treponemal as distinct
0168-6445/86/$03.50 © 1986 Federation of European MicrobiologicalSocieties
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from host-derived proteins has seldom been possible, since material has not been available in sufficient quantity for the purification and characterisation of individual components. Secondly,' in the widely used technology of S D S - P A G E , the criterion of identification of polypeptides by the determination of relative molecular mass (Mr) has been difficult to use for comparison of results between laboratories, because of the use of molecular mass standards of different identity and origin, and with different 'known' values assigned to the same marker proteins. In some cases, the standards used have not been specified. If a number of prominent polypeptides had been purified and characterised by different workers, this would not have caused much difficulty, but due to the unknown identity of the majority of components, the problems of comparison are exacerbated. In this review, we attempt to describe common features of various published S D S - P A G E profiles of T. pallidum, and to assign identities to major polypeptides which we have characterised and locate them in the common profile proposed. Important antigenic components detected by immunoblotting are also described. We have confined our attention to publications which deal with the molecular analysis of polypeptides and antigens, all of which have appeared since 1979 and which in general have utilised S D S - P A G E a n d / o r immunoblotting. We have included some details of comparative work using the cultivable Reiter treponeme, Treponema phagedenis (biotype Reiterii), since this has featured prominently as a substitute source of antigens available in quantity and is known to cross-react with components of T.
pallidum.
3. DISCUSSION
3.1. S DS- PA GE analysis of treponemal polypeptides Analysis by S D S - P A G E of a very large number of independently obtained samples of treponemes in our laboratory over a period of years has shown that, despite a variable degree of contamination with non-treponemal, microscopically visible particulate material, and accompanying variation in polypeptide profiles, a recognisa-
-92-5K -66.2
47K-45 37K -31
-21-5
a
b
c
d
e
f
g
h
Fig. 1. S D S - P A G E profiles of T. pallidum (Nichols) polypeptides. Tracks (a)-(f) are from published papers ([23,10,13,9,15,11], respectively). Track (g) was obtained by us and shows polypeptides P1-P7. Positions of M r markers for track (g) are shown at (h). Track (g) was obtained in a discontinuous gel with 11% acrylamide in the separating gel [431, and stained with Coomassie brilliant blue.
ble and qualitatively constant pattern of prominent polypeptides can be observed. By careful selection of batches essentially free of particulate contamination (often obtained from testes showing no sign of haemorrhage or breakdown of tissue), and washing of treponemes to remove loosely bound soluble host proteins, a reproducible pattern of prominent bands emerged. This selection of 'clean' batches appeared preferable to the routine use of gradient purification of treponemes, with its attendant risks of loss of material and damage to treponemes due to the extra manipulations involved. We may assume that batches showing the smallest number of prominent bands (present in every preparation) are the least contaminated with host-derived proteins, and that bands constantly present are likely to be treponemal in origin. Nevertheless, we have observed variations in the pat-
141 terns of putative treponemal bands, which vary in relative intensity from batch to batch. This may result from phenotypic variation between batches, depending on growth conditions; for example, heterogeneity among the rabbits used and variation in the duration of the infection at time of harvesting. The polypeptide profile obtained from a typical batch of clean treponemes, with published profiles from 4 other independent groups, is shown in Fig. 1. The similarities in all these profiles validate the adoption of a numbering system for the prominent polypeptides PI-P7 as shown, with M r of 80, 60, 47, 40, 37, 34 and 31.5 kDa, respectively. The M r values given for our own profile are based on the use of Bio-Rad (Watford, U.K.) molecular mass markers, using the values given by the manufacturer, and a Beckman DU8 spectrophotometer with gel scanning attachment and 'slab gel' programme for objective determination of Mr. A major problem in the correlation of results from different groups is the use of different (or unspecified) molecular mass standard preparations and different values for the same marker proteins. We used lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), bovine serum albumin (66.2 kDa) and phosphorylase B (92.5 kDa). In addition, variation can arise from differences in the methods of measurement and interpolation of migration distances of unknowns and their relation to those of the standards. Thus differences of at least ±1000 kDa can be expected between values for the same polypeptide determined in different laboratories. However, profile characteristics can be observed, e.g., distinctive spacing between prominent 'landmark' polypeptides, and these can be used in definitive comparisons. Notable features of all the profiles are the near-equal spacing between Pl, P2 and P3 (slightly less between Pl and P2), and the formation Of 2 similarly spaced doublets by P4 and P5, and by P6 and P7. This pattern has enabled us to identify the bands corresponding to P1-P7 in the other published profiles, as shown by the lines drawn between them across the diagram (Fig. 1). We have aligned the profiles so that P5 is level throughout. We have confined our numbering system to the polypepfides Pl-P7 because these are the only ones
that can be identified with some certainty in comparisons of work from different laboratories. There are undoubtedly other important components, for example in the lower M r range of 12-20, which may be prominent antigens revealed by immunoblotting if not by protein staining. Similar definitive identification of these will require widespread use of high acrylamide concentration or gradient gels. Certain bands can be firmly or tentatively identified. We have shown that P5 is the major polypeptide of the axial filament [6], and that P3 is the most prominent polypeptide in a Triton X-100soluble fraction [7] which appears to contain components of the outer membrane [7,8]. Thus, P3
N
B
Fig. 2. SDS-PAGE profiles of Nichols (N) and Birmingham (B) strains of T. pallidmn. A reproducibledifferencebetween the strainsis seen at approximately90 kDa (arrow).
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may be an outer membrane protein. Polypeptides approximating to the M r assigned to P3 (47000) feature prominently in the literature [9-25] as immunodominant antigens and as the target antigen of several reported examples of monoclonal antibodies. Genes encoding polypeptides approximating to this M r value have also been cloned [16,18,19]. We have noted that in some of our profiles of whole treponeme preparations (unpublished), as well as in most of those shown in Fig. 1, that a trio of polypeptides is often present in the P3 region, and it is often difficult to be certain which of these is the one described by certain authors. However, of this trio, Triton X-100 selectively extracts P3 from whole treponemes in our laboratory, which is thus a definitive property of this polypeptide. The great majority of molecular studies on T. pallidum has utilised the virulent Nichols strain, originally isolated in 1913. We have compared the profiles of Nichols and a strain isolated in Birmingham in 1979 [26], and the two appear identical in respect of polypeptides P1-P7 (Fig. 2). The only reproducible difference seen, in comparisons of several batches, was in the M r of one or two polypeptides of approx. 90 kDa. The relative faintness of polypeptides P6 and P7 on this gel is an example of the variability noted above. The overall similarity in profile of these two strains isolated at an interval of 66 years indicates that studies on the Nichols strain continue to be largely valid in relation to modern isolates.
3.2. Outer membrane-associated polypeptide P3 A number of reports describe a prominent a n d / o r immunodominant polypeptide(s) at or near the P3 band of 47 kDa. Hanff and colleagues demonstrated prominent antigens of 45 kDa [9] or 49 kDa [11] (relative to ovalbumin markers at 43 kDa) which reacted with human primary sera and may have been identical with P3. In contrast, some primary human sera did not contain antibody to antigens in this M r range [15], although antibody to polypeptides of 43.5, 46 and 49 kDa appeared in secondary sera. A possibly significant difference between the latter work and that of others was that radioimmunoprecipitation was used instead of Western blotting, and P3 may thus not be easily
precipitable by antibody. Comparisons of results obtained by these two different methods should therefore be made cautiously [14]. Reactions of polypeptides, similar in M r to P3, with sera from infected rabbits, have also been described: such polypeptides of 48 kDa [10] and 46 kDa [12] were almost certainly identical with P3, which reacted strongly with sera from rabbits 10-14 days post-infection in our laboratory (M.J. Bailey, unpublished observation). An antigenic 45kDa polypeptide (relative to Bio-Rad markers with the manufacturer's values) was prominent in immune complexes isolated from sera of rabbits infected with 7". pallidum [25] and may have been P3, since it appeared slightly above the 45-kDa marker band, although we cannot identify it with any certainty. Rabbit sera were also used in comparisons of T. pallidum and Treponema pertenue [13,14]. An antigenic 48-kDa polypeptide relative to identical M r markers and values to those used by us was probably P3 [13], as was a prominent 45-kDa polypeptide against which a monoclonal antibody had been obtained [14]. An antigenic polypeptide, approximating in M r to P3, has been particularly effective in eliciting monoclonal antibodies in mice. Immunization with live treponemes led to isolation of hybridomas reactive with a 46-kDa polypeptide of T. pallidum, and in one instance also with a cloned 44-kDa polypeptide which may thus have been a fragment of the 46 kDa; M r markers used or their values were not given in this study [16]. A cloned antigen of 44 kDa relative to ovalbumin at 46 kDa [19] was strongly expressed and apparently secreted in Escherichia coli, and it was suggested this might also be a fragment of the immunodominant 47-48-kDa polypeptide reported elsewhere [10]. Nevertheless the fragment was unreactive with monoclonal antibodies against a 47-kDa polypeptide, and confirmation of its relationship to P3 awaits further information. Whole treponemes in Freund's complete adjuvant (FCA) elicited a hybridoma [14] reactive with a 45-kDa polypeptide (relative to ovalbumin at 44 kDa) which was almost certainly identical to P3. Norgard and colleagues showed that a combination of whole treponemes in FCA and live treponemes elicited numerous hybridomas reactive with a 47-kDa
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polypeptide relative to ovalbumin at 45 kDa [20,21,27], again almost certainly identical to P3. This conclusion is reinforced by our own observations (M.J. Bailey, unpublished) that such immunisation schedules preferentially elicit hybridomas reactive with P3. The biological significance of immunodominant P3 a n d / o r closely adjacent polypeptides is clear. The association of P3 with the treponemal surface [7] suggests that it may be important in interactions of the organism with host defence mechanisms. Norgard and colleagues demonstrated biological activity of monoclonal antibodies to a 47kDa polypeptide in immobilisation tests and in neutralisation of infectivity, and surface iodination indicated its surface exposure [28]. Some evidence for surface exposure of this antigen has also been obtained from antibody binding and immunogold-labelling experiments [21]. We should however be cautious in accepting these data uncritically. Firstly, experiments indicating the binding of antibody to the treponemal surface do not appear to have been performed on intact living treponemes. Secondly, our own work indicates that the intact treponemal surface is antigenically inert [29] and that no treponemal proteins appear fully exposed to surface iodination [7]. Thirdly, it has been known for many years that an 'ageing' process is necessary for the full antigenic reactivity of treponemes to develop. The T. pallidum immobilisation test is ~nusual among bactericidal reactions in its requirement for overnight incubation of treponemes with antibody and complement, suggesting that some ageing of treponemes or other surface alteration takes place before killing can occur. Furthermore, recent and more critically derived data on surface iodination [23] do not show a 47-kDa polypeptide to be surface-exposed. In much of this work, methodology developed for examination of the surface architecture of typical Gram-negative bacteria has been used. It appears that extrapolation of knowledge of the latter to T. pallidum and the assumption that T. pallidum has a fundamentally similar surface structure may be misleading.
3.3. Major axial filament polypeptide P5 Purification of axial filaments of T. pallidum
and their analysis by SDS-PAGE clearly identifies P5 as the major constituent polypeptide of the axial filament [6]. A number of workers have attempted to identify the axial filament polypeptides [10-12], by exploiting the known antigenic cross-reactivity with axial filaments of the Reiter treponeme [30,31], in the expectation that a common axial filament polypeptide (i.e., identical in M r between the 2 species) might be found. We have now shown that the latter M r values differ ([6]; see below). Strongly antigenic polypeptides in the region of P5 have, however, frequently been described. Antibody to 35- and 35.5-kDa polypeptides (relative to ovalbumin at 43 kDa) and thus possibly identical with P5, appeared in human primary and secondary syphilitic sera [9]. Studies with rabbit antibody [10] which included an examination of cross-reaction with the Reiter treponeme, revealed antigenic T. pallidum polypeptides of 37, 35 and 33 kDa relative to ovalburain at 43 kDa. Location of this 37-kDa polypeptide approximately mid-way between carbonic anhydrase and ovalbumin markers, indicates that it is indeed P5. Antigens from 7". pallidum and the Reiter treponeme were also compared using normal and syphilitic human sera [11], and an antigenic polypeptide of T. pallidum of 35.5 kDa (relative to ovalbumin at 43 kDa) could be P5, were it not also present with an identical Mr in the Reiter treponeme. These authors suggested 33- and 30-kDa polypeptides as axial filament components, values too low for the major polypeptide. Further studies of the development of the humoral response in rabbits [12] showed antibody to a 36-kDa polypeptide to be prominent in early sera, and our own data (M.J. Bailey, C.W. Penn and A. Cockayne, Proc. 102nd Mtg. Soc. Gen. Microbiol., Birmingham, 1985) suggest very strongly that this polypeptide was P5. Antibody responses to 30.5-, 33-, 35- and 37-kDa polypeptides were demonstrated using early human syphilitic sera [15], and the 37-kDa polypeptide appeared identical to P5. Sera raised against either T. pallidum or T. pertenue were compared in their reactions with antigens of T. pallidum [13,14]. Some antigens of T. pallidum were reactive with both sera, including a trio of 37, 35 and 33 kDa [13] relative to M r standards and values identical to those we used.
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Thus, the 37-kDa polypeptide was certainly P5. Clearly then, P5 is an important antigen, and antibodies against it are prominent in early infection.
3.4. Cross-reactions with other treponemal species The Reiter treponeme is the most-used 'model' for studies of treponemal immunochemistry. It has been hoped that such work would yield knowledge of the antigens of T. pallidum, primarily because the Reiter treponeme was known to cross-react antigenically with T. pallidum, and the organism has indeed been used as a source of both diagnostic antigen for the Reiter protein complement fixation test and of 'sorbent' for enhancing the specificity of immunofluorescence tests for T. pallidurn antibody. The Reiter treponeme has been very thoroughly investigated by Strandberg-Petersen and co-workers [32-39] who have extensively analysed the antigens of the Reiter treponeme by 2-dimensional immunoelectrophoresis using both homologous antiserum and rabbit and human syphilis antibody. While this technique does not necessarily separate antigens as individual polypeptides, and is thus not strictly analogous to the immunoblotting technique used in most of the work described so far, it has the advantage that native antigenic species can be analysed and compared. These authors have identified a number of immunoprecipitates formed by antibodies common to T. pallidum and the Reiter treponeme [32,33], and from these common immunoprecipitates they have characterized axial filaments [34], a heat-stable antigen, possibly a polysaccharide [35], protein antigens with subunits of 66 [36], 70 [37] and 48 kDa [38], and an RNA antigen [391. The protein of 48 kDa [38] relative to ovalbumin at 43 kDa may be analogous to P3. We have also identified the immunoprecipitate of the Reiter treponeme formed by axial filaments [40], confirming the observations of StrandbergPedersen et al. [34], and we have shown an identical immunoprecipitate to be formed by T. pallidum axial filaments [6]. We have discussed above the literature concerning axial filament polypeptides, whether or not shared with the Reiter treponeme, and we have evidence from studies with monoclonal antibodies and other data (M.J. Bailey and
A. Cockayne, unpublished observations) that P1 and P3, in addition to P5, may have antigenically or functionally related equivalent polypeptides in the Reiter treponeme which differ significantly in M r between the 2 species. Cross-species comparisons should therefore be interpreted with caution in the context of the immunochemistry of T. pallidum. We believe that the relevance of Reiter treponeme antigens as potential diagnostic reagents and otherwise, is in theory limited in comparison to antigens of T. pallidum which may have unique, possibly dominant epitopes of greater importance in the human antibody response. Comparisons of T. pallidum with a number of other pathogenic spirochaetes have also been made, using Western blotting of T. pallidum antigens with antisera against Treponema paraluiscuniculi, Treponema hyodysenteriae, Borrelia herrnsii and Leptospira interrogans [22]. T. pallidum antigens of 80, 69, 48, 37 and 30 kDa (relative to Bio-Rad markers with the manufacturer's values) were frequently detected by heterologous sera. It was assumed that where polypeptides of T. pallidum were reactive with heterologous antisera, identical polypeptides would be found in the heterologous species, although, as we have seen in the case of axial filaments, such reactions may result simply from shared epitopes on polypeptides differing in
Mr. 3.5. Studies of the treponemal surface Surface properties are fundamental in determining the interaction between bacteria and the host's immune and non-specific defences, and several groups have attempted to identify important surface components by surface labelling or receptor-ligand binding experiments. Baseman and colleagues [3,14,41-45] have made extensive use of these procedures, but comparison of their work with that of others is often difficult, due to heavy exposure of autoradiographs of gel profiles, which obscures the pattern of prominent polypeptides we have described. Also, use of lower concentrations of acrylamide in gels than those used by most groups, and often of M r markers of undefined origin, makes comparison difficult. A polypeptide profile somewhat similar to our standard pattern was initially published [3] but no M r information
145 was given. Following surface iodination of density-gradient-purified, washed treponemes polypeptides of 89.5, 29.5, 25.5 and 20 kDa, and to a lesser extent 59 and 42.5 kDa (markers undefined), were labelled and assumed to be outer membrane components. We cannot identify these with certainty relative to P1-P7, although release of polypeptides 2, 5 and 6 from the treponeme by octyl glucoside suggests that 6 could possibly be the Triton-extractable P3. Radioimmunoprecipitation identified a number of other proteins, making a total of 11 surface-exposed, 'outer-membrane' proteins. Our own experience with surface iodination of intact, 'viable' treponemes [7] and a comparison of lactoperoxidase- and iodogen-mediated iodination [23], in both of which minimal iodination was achieved under mild conditions, suggests that some damage to the treponemal surface or penetration of label may have led to labelling of internal polypeptides in the above work. Receptor binding studies using formalinised HEp-2 cells and SDS-Triton-solubilised treponemes, showed that the proteins designated 1, 2 and 3 [411 bound strongly to cells [42]. In radioimmunoprecipitation experiments these proteins were prominent antigens. Mr markers and their values were not specified, and we cannot identify these proteins. Subsequently, these authors analysed the antibody response in rabbits to polypeptides 1-6 by radioimmunoprecipitation and demonstrated early responses to 2, 3 and 6; 29.5, 25.5 and 42.5 kDa, respectively. If these values had been higher, the polypeptides of 29.5 and 42.5 kDa could have been P5 and P3, respectively. Further studies on binding of plasma proteins by the surface of T. pallidum showed that fibronectin was particularly avidly bound, and proteins 1, 2 and 3 interacted strongly and specifically with fibronectin on affinity chromatography [45]. While the M r of these proteins were given as 89, 29 and 25 (relative to ovalbumin at 43) and all marker proteins were specified, uncertainty remains as to their identity. In a recent paper from this group [14], similar M r markers were used, but the values assigned to them were different. The protein 6 was clearly located above the ovalbumin marker at 44 kDa and could not have had the value of 42.5 kDa claimed previously. Similarly,
protein 2 was about mid-way between the xchymotrypsinogen marker at 27 kDa and ovalbumin at 44 kDa, and could not have had the value of 29.5 kDa previously claimed. It therefore appears possible that protein 6 could be identical to P3, and protein 2 to P5. It remains unclear, however, whether identical polypeptides were being studied throughout the series of papers published by this group. Surface iodination has been thoroughly investigated by Norris and Sell [23], who showed that, particularly with lactoperoxidase-catalysed iodination, penetration of iodine radicals through surface layers appeared to occur, so that the results should be interpreted with caution. Our own studies [7] showed that, using intact treponemes, viable until addition of iodination reagents, little label could be incorporated into any treponemal proteins in comparison with presumed host-derived albumin. However, iodination of washed treponemes [23] under mild conditions preferentially labelled a 39-kDa protein (relative to ovalbumin at 43 kDa) which appears to have been P4. This accords with our own observation of partial extraction of P4 by Triton correlating with removal of the outer membrane [7]. A most useful development is the use of 2-dimensional gel electrophoresis of proteins [23]. Combined with surface labelling or other identification procedures, this promises to assist in unequivocal identification of treponemai proteins. We were, however, unable fully to correlate 2-dimensional gel patterns with 1-dimensional protein profiles, the latter as shown by these authors correlating well with our standard pattern (Fig. 1).
3.5. Treponemal antigen gene cloning Several groups have now reported the successful cloning in E. coli of genes from T. pallidum encoding antigens reactive with human or animal syphilitic sera [16,18,19,24,46,47]. Polypeptides of 46, 43, 38, 24, 23, 20 and 18.5 kDa were revealed by reaction with human secondary syphilitic serum [18], and were said to correspond with antigenic polypeptides of T. pallidum described subsequently by Hanff and colleagues [9,11,12]. Thus, the cloned 48-kDa polypeptide might have been identical with P3. We have already discussed the
146 cloned polypeptides of 46 [16] and 44 k D a [19], which might be fragments of P3. If all these cloned antigens are P3-related it appears that this antigen is particularly easy to clone, possibly as a result of multiple gene copies or promoters which function strongly in E. coil The nature of most other cloned antigens is less easy to assess. Cloned polypeptides of 39, 35 and 25 k D a relative to Bio-Rad standards [47] c a n n o t be clearly identified relative to a total treponemal protein profile since none is shown, and it appears unlikely that any of these is P5 since they did not react strongly with primary h u m a n syphilitic sera. It should of course be remembered that cloned antigens will not necessarily be identical in M r to their counterparts in T. pallidum, due for example to the expression of incomplete genes or differences in post-translational processing between species, and indeed, antigenically related but physically disparate polypeptides might occur in different recombinant clones derived from the same T. pallidum genes [47]. The best-characterised cloned antigen at present is a protease-resistant ' 4 D ' antigen of 190 k D a [24] which was strongly antigenic. Boiling in SDS broke down this antigen to 19-kDa fragments, which were not antigenically reactive with h u m a n or rabbit syphilitic sera, but to which antib o d y could be induced by immunisation with the cloned, purified antigen. Immunisation with the intact 4 D antigen also induced treponemal immobilising antibody, but the serum was not reactive with commercially available T. pallidum preparations in immunofluorescence tests.
4. C O N C L U S I O N It now appears possible to impart some order to the diverse and confusing range of observations that have been m a d e on the antigenic and other polypeptides of T. pallidum. M a n y problems remain in the interpretation of data from various laboratories, and because we remain ignorant of the functional identity of most of the polypeptides of interest, it would be desirable that in future reports these be located as far as possible within the patterns we have recognised in S D S - P A G E
profiles. By comparing and correlating the work of different groups, faster progress can be made towards a full understanding of the structure and antigenicity of this pathogen.
ACKNOWLEDGEMENTS We thank Dr. J. Stephen for a critical reading of the manuscript and helpful comments. The work was supported by the Wellcome Trust and by the British Technology G r o u p .
REFERENCES [1] Nicholas, L. (1979) Non-treponemal tests for syphilis, in lmmunoserology in the Diagnosis of Infectious Diseases, (Friedman, H., Ed.). University Park Press, Baltimore. [21 Robertson, R.G. and Walsh, W.T. (1979) Treponemal tests in syphilis, in Immunoserology in the Diagnosis of Infectious Diseases, (Friedman, H., Ed.) University Park Press, Baltimore. [31 Alderete, J.F. and Baseman, J.B. (1979) Surface-associated host proteins on virulent Treponemapallidum. Infect. Immun. 26, 1048-1056. [4] Fieldsteel, A.H., Cox, D.L. and Moeckli, R.A. (1981) Cultivation of Treponemapallidum in tissue culture. Infect. Immun. 32, 908-915. [5] Norris, S.J. (1982) In vitro cultivation of Treponemapallidum: independent confirmation. Infect. Immun. 36, 437-439. [6] Penn, C.W., Bailey, M.J. and Cockayne, A. (1985) The axial filament antigen of Treponemapallidum. Immunology, 54, 635-641. [7] Penn, C.W., Cockayne, A. and Bailey, (1986) The outer membrane of Treponemapallidum: biological significance and biochemical properties. J. Gen. Microbiol. (in press). [81 Penn, C.W. and Lichfield, J.D. (1982) The outer membrane of Treponema pallidum: solubilization to release axial filaments. FEMS Microbiol. Left. 14, 61-64. [9] Hanff, P.A., Fehniger, T.E., Miller, J.N. and Lovett, M.A. (1982) Humoral immune response in human syphilis to polypeptides of Treponema pallidurn. J. Immunol. 129, 1287-1291. [101 Lukehart, S.A., Baker-Zander, S.A. and Gubish, E.R. (1982) Identification of Treponema pallidum antigens: comparison with a non-pathogenic treponeme. J. Immunol. 129, 833-838. [111 Hanff, P.A., Miller, J.N. and Loven, M.A. (1983) Molecular characterization of common treponemal antigens. Infect. Immun. 40, 825-828. [12] Hanff, P.A., Bishop, N.H., Miller, J.N. and Lovett, M.A. (1983) Humoral immune response in experimental syphilis
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to polypcptides of Treponemapallidur,t J. Immunol. 131, 1973-1977. Baker-Zander, S.A. and Lukehart, S.A. (1983) Molecular basis of immunological cross-reactivity between Treponema pallidum and Treponema pertenue. Infect. Immun. 42, 634-638. Thoruburg, R.W. and Baseman, J.B. (1983) Comparison of major protein antigens and protein profiles of Treponema pallidum and Treponema pertenue. Infect. Immun. 42, 623-627. Moskophidis, M. and Muller, F. (1984) Molecular analysis of immunoglobulins M and G immune response to protein antigens of Treponemapallidum in human syphilis. Infect. Immun. 43, 127-132. Van Embden, J.D., Van der Donk, H.J., Van Eijk, R.V., Van der Heide, H.G., De Jong, J.A., Van Olderen, M.F., Osterhaus, A.D. and Scouls, L.M. (1983) Molecular cloning and expression of Treponema pallidum DNA in Escherichia coil K12. Infect. Immun. 42, 187-196. Van Eijk, R.V.M. and Van Embden, J.D. (1982) Molecular characterization of Treponemapallidum proteins responsible for the human immune response to syphilis. A. van Leeuwenhock J. Microbiol. Serol. 48, 486-487. Walfield, A.M., Hanff, P.A. and Lovett, M.A. (1982) Expression of' Treponemapallidum antigens in Escherichia coll. Science 216, 522-523. Norgard, M. and Miller, J.N. (1983) Cloning and expression of Treponema pallidum (Nichols) antigen genes in Escherichia coll. Infect. Immun. 42, 435-445. Norgard, M.V., Sclland, C.K., Kettman, J.R. and Miller, J.N. (1984) Sensitivity and specificity of monoclonal antibodies directed against antigenic determinants of Treponemapallidum Nichols in the diagnosis of syphilis. J. Clin. Microbiol. 20, 711-717. Marchitto, K.S., Jones, S.A., SchelI, R.F., Holmans, P.L. and Norgard, M.V. (1984) Monoclonal antibody analysis of specific antigenic similarities among pathogenic Treponema pallidum subspecies. Infect. Immun. 45, 660-666. Baker-Zander, S.A. and Lukehart, S.A. (1984) Antigenic cross-reactivity between Treponema pallidum and other pathogenic members of the family Spirochaetaceae. Infect. Immun. 46, 116-121. Norris, S.J. and Sell, S. (1984) Antigenic complexicity of Treponema pallidum: antigenicity and surface localization of major polypeptides. J. Immunol. 133, 2686-2692. Fehniger, T.E., Walfield, A.M., Cunningham, T.M., Radolf, J.D.. Miller, J.N. and Lovett, M.A. (1984) Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen. Infect. Immun. 46, 598-607. Baughn, R.E. and Musher, D.M. (1983) Isolation and preliminary characterization of circulating immune complexes from rabbits with experimental syphilis. Infect. Immun. 42, 579-584. Penn, C.W. and Clay, J.C. (1982) Comparison of the morphology, antigenicity and pathology of Nichols and
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
Birmingham isolates of Treponemapallidunt Br. J. Vener. Dis. 58, 286-291. Robertson, S.M., Kettman, J.R., Miller, J.N. and Norgard, M.V. (1982) Murine monoclonal antibodies specific for virulent Treponemapallidum (Nichols). Infect. Immun. 36, 1076-1085. Jones, S.A., Marchitto, K.S., Miller, J.N. and Norgard, M.V. (1984) Monocional antibody with haemagglutination, immobilization, and neutralization activities defines an immunodominant, 47,000 mol. wt., surface-exposed immunogen of Treponema pallidum (Nichols). J. Exp. Meal. 160, 1404-1420. Penn, C.W. (1981) Avoidance of host defences by Treponema pallidum in situ and on extraction from infected rabbit testes. J. Gen. Microbiol. 126, 69-75. Hardy, P.H., Fredericks, W.R. and Nell, E.E. (1975) Isolation and antigenic characteristics of axial filaments from the Reiter treponeme. Infect. Immun. 11,380-386. Nell, E.E. and Hardy, P.H. (1978) Counterimmunoeiectrophoresis of Reiter treponeme axial filaments as a diagnostic test for syphilis. J. Clin. Microbiol. 8, 148-152. Pedersen, N.S., Axelsen, N.H., Jorgensen, B.B. and Petersen, C.S. (1980) Antibodies in secondary syphilis against five of forty Reiter treponeme antigens. Scand. J. Immunol. 11,629-623. Pedersen, N.S., Axelsen, N.H. and Petersen, C.S. (1981) Antigenic analysis of Treponemapallidum: cross-reactions between individual antigens of T. pallidum and T. relier. Scand. J. Immunol. 13, 143-150. Petersen, C.S., Pedersen, N.S. and Axelsen, N.H. (1981) A simple method for the isolation of flagella from Treponema Reiter. Acta Path. Microbiol. Scand. C89, 379-385. Pedersen, N.S., Petersen, C.S., Axelsen, N.H., BirchAndersen, A. and Hovind-Hougen, K. (1981) Isolation of a heat-stable antigen from Treponema Reiter, using an immunoadsorbent with antibodies from syphilitic patients. Scand. J. Immunol. 14, 137-144. Petersen, C.S., Pedersen, N.S. and Axelsen, N.H. (1982) Purification of a Reiter treponemal protein that is immunologically related to an antigen in Treponema pallidum. Infect. Immun. 35, 974-978. Petersen, C.S., Pedersen, N.S. and Axelsen, N.H. (1982) Purification of TR-b, a Reiter treponeme protein antigen precipitating with antibodies in human syphilitic sera. Infect. Immun. 38, 35-40. Petersen, C.S., Pedersen, N.S. and Axelsen,'N.H. (1982) Isolation and characterization of TR-c, an antigen of the Reiter treponeme precipitating with antibodies in syphilis. Scand. J. Immunol. 15, 459-465. Pedersen, N.S., Petersen, C.S. and Axelscn, N.H. (1982) Ribonucleic acid antigen from the Reiter treponeme used in an EL1SA for antibodies in syphilis. Scand. J. Immunol. 16, 431-436. Penn, C.W. and Rhodes, J. (1982) Surface-associated antigens of Treponemapallidum concealed by an inert outer layer. Immunology 46, 9-16. Alderete, J.F. and Baseman, J.B. (1980) Surface characteri-
148 zation of virulent Treponemapallidum. Infect. Immun. 30, 814-823. [42] Baseman, J.B. and Hayes, E.C. (1980) Molecular characterization of receptor binding proteins and immunogens of virulent Treponemapallidum. J. Exp. Med. 151,573-586. [431 Alderete, J.F. and Baseman, J.B. (1981) Analysis of serum IgG against Treponemapallidum protein antigens in experimentally infected rabbits. Br. J. Vener. Dis. 57, 302- 308. [44] Morrison-Plummer, J., Alderete, J.F. and Baseman, J.B. (1983) Enzyme-linked immunosorbent assay for the detection of serum antibody to outer membrane proteins of Treponemapallidu~ Br. J. Vener. Dis. 59, 75-79.
[45] Peterson, K.M., Baseman, J.B. and Alderete, J.F. (1983) Treponema pallidum receptor binding proteins interact with fibronectin. J. Exp. Meal. 157, 1958-1970. [46] Stamm, L.V., Folds, J.D. and Bassford, P.J. (1982) Expression of Treponemapallidum antigens in Escherichia coil KI2. Infect. lmmun. 36, 1238-1241. [47] Stamm, L.V., Kemer, T.C., Bankaitis, V.A. and Bassford, P.J. (1983) Identification and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coil Infect. Immun. 41,709-721. [48] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
139
FER 00008
Molecular and immunochemical analysis of Treponemapallidum (Syphilis; spirochaete; outer membrane; axial filament)
C.W. Penn, M.J. Bailey and A. Cockayne Department of Microbiology, Universityof Birmingham, Birmingham BI 5 2TT, U.K. (Received 27 February, 1985) (Revision received 28 May, 1985) (Accepted 5 June 1985)
1. SUMMARY Molecular analysis of polypeptides and antigens of Treponema pallidum has been used increasingly during the past 5 years in investigation of the immunology, pathogenicity and molecular biology of this organism. Failure to culture the organism has severely limited our knowledge of its constituent polypeptides and antigens, but many profiles of these unknown constituents, revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques have been published. In order to compare meaningfully the results obtained by different groups, we have identified a standard pattern of prominent 'landmark' polypeptides in such gel profiles and where possible have assigned functional identities to them. A preliminary nomenclature for the prominent polypeptides of T. pallidum is proposed. These are: P1, 80 kDa; P2, 60 kDa; P3, 47 kDa, an outer membrane-associated polypeptide; P4, 40 kDa; P5, 37 kDa, the major polypeptide of the axial filament; P6, 34 kDa; and P7, 31.5 kDa. 2. INTRODUCTION Despite an enormous wealth of clinical data, detailing human antibody responses in syphilis to
both non-treponemal [1] anti treponemal [2] antigens, extending over most of this century, we have only recently begun to analyse the antigens and other components of T. pallidum at the molecular level. The source of T. pallidum antigen for diagnostic testing has been the whole treponeme, consisting of a mosaic of antigenic determinants of unknown individual importance in the stimulation of antibody responses in disease. The first reported use of modem techniques of biochemical and immunochemical analysis of the antigens of T. pallidum was published in 1979 [3], and since then at least 40 papers have contributed to the substantial knowledge of proteins and antigenic determinants which has now accumulated. This rapid expansion of data has, however, led to considerable difficulties of interpretation and comparison of the work of different groups, for two main reasons. The most important is that despite some recenl, progress in attempts to culture the organism in vitro [4,5], the quantities of organisms needed for meaningful analysis can still only be obtained routinely by extraction from infected rabbit testes, with attendant variation in both the condition of the extracted treponemes and the level of contamination with rabbit-derived material. This has led to great variation in the composition of treponeme preparations, and the definite identification of treponemal as distinct
0168-6445/86/$03.50 © 1986 Federation of European MicrobiologicalSocieties
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from host-derived proteins has seldom been possible, since material has not been available in sufficient quantity for the purification and characterisation of individual components. Secondly,' in the widely used technology of S D S - P A G E , the criterion of identification of polypeptides by the determination of relative molecular mass (Mr) has been difficult to use for comparison of results between laboratories, because of the use of molecular mass standards of different identity and origin, and with different 'known' values assigned to the same marker proteins. In some cases, the standards used have not been specified. If a number of prominent polypeptides had been purified and characterised by different workers, this would not have caused much difficulty, but due to the unknown identity of the majority of components, the problems of comparison are exacerbated. In this review, we attempt to describe common features of various published S D S - P A G E profiles of T. pallidum, and to assign identities to major polypeptides which we have characterised and locate them in the common profile proposed. Important antigenic components detected by immunoblotting are also described. We have confined our attention to publications which deal with the molecular analysis of polypeptides and antigens, all of which have appeared since 1979 and which in general have utilised S D S - P A G E a n d / o r immunoblotting. We have included some details of comparative work using the cultivable Reiter treponeme, Treponema phagedenis (biotype Reiterii), since this has featured prominently as a substitute source of antigens available in quantity and is known to cross-react with components of T.
pallidum.
3. DISCUSSION
3.1. S DS- PA GE analysis of treponemal polypeptides Analysis by S D S - P A G E of a very large number of independently obtained samples of treponemes in our laboratory over a period of years has shown that, despite a variable degree of contamination with non-treponemal, microscopically visible particulate material, and accompanying variation in polypeptide profiles, a recognisa-
-92-5K -66.2
47K-45 37K -31
-21-5
a
b
c
d
e
f
g
h
Fig. 1. S D S - P A G E profiles of T. pallidum (Nichols) polypeptides. Tracks (a)-(f) are from published papers ([23,10,13,9,15,11], respectively). Track (g) was obtained by us and shows polypeptides P1-P7. Positions of M r markers for track (g) are shown at (h). Track (g) was obtained in a discontinuous gel with 11% acrylamide in the separating gel [431, and stained with Coomassie brilliant blue.
ble and qualitatively constant pattern of prominent polypeptides can be observed. By careful selection of batches essentially free of particulate contamination (often obtained from testes showing no sign of haemorrhage or breakdown of tissue), and washing of treponemes to remove loosely bound soluble host proteins, a reproducible pattern of prominent bands emerged. This selection of 'clean' batches appeared preferable to the routine use of gradient purification of treponemes, with its attendant risks of loss of material and damage to treponemes due to the extra manipulations involved. We may assume that batches showing the smallest number of prominent bands (present in every preparation) are the least contaminated with host-derived proteins, and that bands constantly present are likely to be treponemal in origin. Nevertheless, we have observed variations in the pat-
141 terns of putative treponemal bands, which vary in relative intensity from batch to batch. This may result from phenotypic variation between batches, depending on growth conditions; for example, heterogeneity among the rabbits used and variation in the duration of the infection at time of harvesting. The polypeptide profile obtained from a typical batch of clean treponemes, with published profiles from 4 other independent groups, is shown in Fig. 1. The similarities in all these profiles validate the adoption of a numbering system for the prominent polypeptides PI-P7 as shown, with M r of 80, 60, 47, 40, 37, 34 and 31.5 kDa, respectively. The M r values given for our own profile are based on the use of Bio-Rad (Watford, U.K.) molecular mass markers, using the values given by the manufacturer, and a Beckman DU8 spectrophotometer with gel scanning attachment and 'slab gel' programme for objective determination of Mr. A major problem in the correlation of results from different groups is the use of different (or unspecified) molecular mass standard preparations and different values for the same marker proteins. We used lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), bovine serum albumin (66.2 kDa) and phosphorylase B (92.5 kDa). In addition, variation can arise from differences in the methods of measurement and interpolation of migration distances of unknowns and their relation to those of the standards. Thus differences of at least ±1000 kDa can be expected between values for the same polypeptide determined in different laboratories. However, profile characteristics can be observed, e.g., distinctive spacing between prominent 'landmark' polypeptides, and these can be used in definitive comparisons. Notable features of all the profiles are the near-equal spacing between Pl, P2 and P3 (slightly less between Pl and P2), and the formation Of 2 similarly spaced doublets by P4 and P5, and by P6 and P7. This pattern has enabled us to identify the bands corresponding to P1-P7 in the other published profiles, as shown by the lines drawn between them across the diagram (Fig. 1). We have aligned the profiles so that P5 is level throughout. We have confined our numbering system to the polypepfides Pl-P7 because these are the only ones
that can be identified with some certainty in comparisons of work from different laboratories. There are undoubtedly other important components, for example in the lower M r range of 12-20, which may be prominent antigens revealed by immunoblotting if not by protein staining. Similar definitive identification of these will require widespread use of high acrylamide concentration or gradient gels. Certain bands can be firmly or tentatively identified. We have shown that P5 is the major polypeptide of the axial filament [6], and that P3 is the most prominent polypeptide in a Triton X-100soluble fraction [7] which appears to contain components of the outer membrane [7,8]. Thus, P3
N
B
Fig. 2. SDS-PAGE profiles of Nichols (N) and Birmingham (B) strains of T. pallidmn. A reproducibledifferencebetween the strainsis seen at approximately90 kDa (arrow).
142
may be an outer membrane protein. Polypeptides approximating to the M r assigned to P3 (47000) feature prominently in the literature [9-25] as immunodominant antigens and as the target antigen of several reported examples of monoclonal antibodies. Genes encoding polypeptides approximating to this M r value have also been cloned [16,18,19]. We have noted that in some of our profiles of whole treponeme preparations (unpublished), as well as in most of those shown in Fig. 1, that a trio of polypeptides is often present in the P3 region, and it is often difficult to be certain which of these is the one described by certain authors. However, of this trio, Triton X-100 selectively extracts P3 from whole treponemes in our laboratory, which is thus a definitive property of this polypeptide. The great majority of molecular studies on T. pallidum has utilised the virulent Nichols strain, originally isolated in 1913. We have compared the profiles of Nichols and a strain isolated in Birmingham in 1979 [26], and the two appear identical in respect of polypeptides P1-P7 (Fig. 2). The only reproducible difference seen, in comparisons of several batches, was in the M r of one or two polypeptides of approx. 90 kDa. The relative faintness of polypeptides P6 and P7 on this gel is an example of the variability noted above. The overall similarity in profile of these two strains isolated at an interval of 66 years indicates that studies on the Nichols strain continue to be largely valid in relation to modern isolates.
3.2. Outer membrane-associated polypeptide P3 A number of reports describe a prominent a n d / o r immunodominant polypeptide(s) at or near the P3 band of 47 kDa. Hanff and colleagues demonstrated prominent antigens of 45 kDa [9] or 49 kDa [11] (relative to ovalbumin markers at 43 kDa) which reacted with human primary sera and may have been identical with P3. In contrast, some primary human sera did not contain antibody to antigens in this M r range [15], although antibody to polypeptides of 43.5, 46 and 49 kDa appeared in secondary sera. A possibly significant difference between the latter work and that of others was that radioimmunoprecipitation was used instead of Western blotting, and P3 may thus not be easily
precipitable by antibody. Comparisons of results obtained by these two different methods should therefore be made cautiously [14]. Reactions of polypeptides, similar in M r to P3, with sera from infected rabbits, have also been described: such polypeptides of 48 kDa [10] and 46 kDa [12] were almost certainly identical with P3, which reacted strongly with sera from rabbits 10-14 days post-infection in our laboratory (M.J. Bailey, unpublished observation). An antigenic 45kDa polypeptide (relative to Bio-Rad markers with the manufacturer's values) was prominent in immune complexes isolated from sera of rabbits infected with 7". pallidum [25] and may have been P3, since it appeared slightly above the 45-kDa marker band, although we cannot identify it with any certainty. Rabbit sera were also used in comparisons of T. pallidum and Treponema pertenue [13,14]. An antigenic 48-kDa polypeptide relative to identical M r markers and values to those used by us was probably P3 [13], as was a prominent 45-kDa polypeptide against which a monoclonal antibody had been obtained [14]. An antigenic polypeptide, approximating in M r to P3, has been particularly effective in eliciting monoclonal antibodies in mice. Immunization with live treponemes led to isolation of hybridomas reactive with a 46-kDa polypeptide of T. pallidum, and in one instance also with a cloned 44-kDa polypeptide which may thus have been a fragment of the 46 kDa; M r markers used or their values were not given in this study [16]. A cloned antigen of 44 kDa relative to ovalbumin at 46 kDa [19] was strongly expressed and apparently secreted in Escherichia coli, and it was suggested this might also be a fragment of the immunodominant 47-48-kDa polypeptide reported elsewhere [10]. Nevertheless the fragment was unreactive with monoclonal antibodies against a 47-kDa polypeptide, and confirmation of its relationship to P3 awaits further information. Whole treponemes in Freund's complete adjuvant (FCA) elicited a hybridoma [14] reactive with a 45-kDa polypeptide (relative to ovalbumin at 44 kDa) which was almost certainly identical to P3. Norgard and colleagues showed that a combination of whole treponemes in FCA and live treponemes elicited numerous hybridomas reactive with a 47-kDa
143
polypeptide relative to ovalbumin at 45 kDa [20,21,27], again almost certainly identical to P3. This conclusion is reinforced by our own observations (M.J. Bailey, unpublished) that such immunisation schedules preferentially elicit hybridomas reactive with P3. The biological significance of immunodominant P3 a n d / o r closely adjacent polypeptides is clear. The association of P3 with the treponemal surface [7] suggests that it may be important in interactions of the organism with host defence mechanisms. Norgard and colleagues demonstrated biological activity of monoclonal antibodies to a 47kDa polypeptide in immobilisation tests and in neutralisation of infectivity, and surface iodination indicated its surface exposure [28]. Some evidence for surface exposure of this antigen has also been obtained from antibody binding and immunogold-labelling experiments [21]. We should however be cautious in accepting these data uncritically. Firstly, experiments indicating the binding of antibody to the treponemal surface do not appear to have been performed on intact living treponemes. Secondly, our own work indicates that the intact treponemal surface is antigenically inert [29] and that no treponemal proteins appear fully exposed to surface iodination [7]. Thirdly, it has been known for many years that an 'ageing' process is necessary for the full antigenic reactivity of treponemes to develop. The T. pallidum immobilisation test is ~nusual among bactericidal reactions in its requirement for overnight incubation of treponemes with antibody and complement, suggesting that some ageing of treponemes or other surface alteration takes place before killing can occur. Furthermore, recent and more critically derived data on surface iodination [23] do not show a 47-kDa polypeptide to be surface-exposed. In much of this work, methodology developed for examination of the surface architecture of typical Gram-negative bacteria has been used. It appears that extrapolation of knowledge of the latter to T. pallidum and the assumption that T. pallidum has a fundamentally similar surface structure may be misleading.
3.3. Major axial filament polypeptide P5 Purification of axial filaments of T. pallidum
and their analysis by SDS-PAGE clearly identifies P5 as the major constituent polypeptide of the axial filament [6]. A number of workers have attempted to identify the axial filament polypeptides [10-12], by exploiting the known antigenic cross-reactivity with axial filaments of the Reiter treponeme [30,31], in the expectation that a common axial filament polypeptide (i.e., identical in M r between the 2 species) might be found. We have now shown that the latter M r values differ ([6]; see below). Strongly antigenic polypeptides in the region of P5 have, however, frequently been described. Antibody to 35- and 35.5-kDa polypeptides (relative to ovalbumin at 43 kDa) and thus possibly identical with P5, appeared in human primary and secondary syphilitic sera [9]. Studies with rabbit antibody [10] which included an examination of cross-reaction with the Reiter treponeme, revealed antigenic T. pallidum polypeptides of 37, 35 and 33 kDa relative to ovalburain at 43 kDa. Location of this 37-kDa polypeptide approximately mid-way between carbonic anhydrase and ovalbumin markers, indicates that it is indeed P5. Antigens from 7". pallidum and the Reiter treponeme were also compared using normal and syphilitic human sera [11], and an antigenic polypeptide of T. pallidum of 35.5 kDa (relative to ovalbumin at 43 kDa) could be P5, were it not also present with an identical Mr in the Reiter treponeme. These authors suggested 33- and 30-kDa polypeptides as axial filament components, values too low for the major polypeptide. Further studies of the development of the humoral response in rabbits [12] showed antibody to a 36-kDa polypeptide to be prominent in early sera, and our own data (M.J. Bailey, C.W. Penn and A. Cockayne, Proc. 102nd Mtg. Soc. Gen. Microbiol., Birmingham, 1985) suggest very strongly that this polypeptide was P5. Antibody responses to 30.5-, 33-, 35- and 37-kDa polypeptides were demonstrated using early human syphilitic sera [15], and the 37-kDa polypeptide appeared identical to P5. Sera raised against either T. pallidum or T. pertenue were compared in their reactions with antigens of T. pallidum [13,14]. Some antigens of T. pallidum were reactive with both sera, including a trio of 37, 35 and 33 kDa [13] relative to M r standards and values identical to those we used.
144
Thus, the 37-kDa polypeptide was certainly P5. Clearly then, P5 is an important antigen, and antibodies against it are prominent in early infection.
3.4. Cross-reactions with other treponemal species The Reiter treponeme is the most-used 'model' for studies of treponemal immunochemistry. It has been hoped that such work would yield knowledge of the antigens of T. pallidum, primarily because the Reiter treponeme was known to cross-react antigenically with T. pallidum, and the organism has indeed been used as a source of both diagnostic antigen for the Reiter protein complement fixation test and of 'sorbent' for enhancing the specificity of immunofluorescence tests for T. pallidurn antibody. The Reiter treponeme has been very thoroughly investigated by Strandberg-Petersen and co-workers [32-39] who have extensively analysed the antigens of the Reiter treponeme by 2-dimensional immunoelectrophoresis using both homologous antiserum and rabbit and human syphilis antibody. While this technique does not necessarily separate antigens as individual polypeptides, and is thus not strictly analogous to the immunoblotting technique used in most of the work described so far, it has the advantage that native antigenic species can be analysed and compared. These authors have identified a number of immunoprecipitates formed by antibodies common to T. pallidum and the Reiter treponeme [32,33], and from these common immunoprecipitates they have characterized axial filaments [34], a heat-stable antigen, possibly a polysaccharide [35], protein antigens with subunits of 66 [36], 70 [37] and 48 kDa [38], and an RNA antigen [391. The protein of 48 kDa [38] relative to ovalbumin at 43 kDa may be analogous to P3. We have also identified the immunoprecipitate of the Reiter treponeme formed by axial filaments [40], confirming the observations of StrandbergPedersen et al. [34], and we have shown an identical immunoprecipitate to be formed by T. pallidum axial filaments [6]. We have discussed above the literature concerning axial filament polypeptides, whether or not shared with the Reiter treponeme, and we have evidence from studies with monoclonal antibodies and other data (M.J. Bailey and
A. Cockayne, unpublished observations) that P1 and P3, in addition to P5, may have antigenically or functionally related equivalent polypeptides in the Reiter treponeme which differ significantly in M r between the 2 species. Cross-species comparisons should therefore be interpreted with caution in the context of the immunochemistry of T. pallidum. We believe that the relevance of Reiter treponeme antigens as potential diagnostic reagents and otherwise, is in theory limited in comparison to antigens of T. pallidum which may have unique, possibly dominant epitopes of greater importance in the human antibody response. Comparisons of T. pallidum with a number of other pathogenic spirochaetes have also been made, using Western blotting of T. pallidum antigens with antisera against Treponema paraluiscuniculi, Treponema hyodysenteriae, Borrelia herrnsii and Leptospira interrogans [22]. T. pallidum antigens of 80, 69, 48, 37 and 30 kDa (relative to Bio-Rad markers with the manufacturer's values) were frequently detected by heterologous sera. It was assumed that where polypeptides of T. pallidum were reactive with heterologous antisera, identical polypeptides would be found in the heterologous species, although, as we have seen in the case of axial filaments, such reactions may result simply from shared epitopes on polypeptides differing in
Mr. 3.5. Studies of the treponemal surface Surface properties are fundamental in determining the interaction between bacteria and the host's immune and non-specific defences, and several groups have attempted to identify important surface components by surface labelling or receptor-ligand binding experiments. Baseman and colleagues [3,14,41-45] have made extensive use of these procedures, but comparison of their work with that of others is often difficult, due to heavy exposure of autoradiographs of gel profiles, which obscures the pattern of prominent polypeptides we have described. Also, use of lower concentrations of acrylamide in gels than those used by most groups, and often of M r markers of undefined origin, makes comparison difficult. A polypeptide profile somewhat similar to our standard pattern was initially published [3] but no M r information
145 was given. Following surface iodination of density-gradient-purified, washed treponemes polypeptides of 89.5, 29.5, 25.5 and 20 kDa, and to a lesser extent 59 and 42.5 kDa (markers undefined), were labelled and assumed to be outer membrane components. We cannot identify these with certainty relative to P1-P7, although release of polypeptides 2, 5 and 6 from the treponeme by octyl glucoside suggests that 6 could possibly be the Triton-extractable P3. Radioimmunoprecipitation identified a number of other proteins, making a total of 11 surface-exposed, 'outer-membrane' proteins. Our own experience with surface iodination of intact, 'viable' treponemes [7] and a comparison of lactoperoxidase- and iodogen-mediated iodination [23], in both of which minimal iodination was achieved under mild conditions, suggests that some damage to the treponemal surface or penetration of label may have led to labelling of internal polypeptides in the above work. Receptor binding studies using formalinised HEp-2 cells and SDS-Triton-solubilised treponemes, showed that the proteins designated 1, 2 and 3 [411 bound strongly to cells [42]. In radioimmunoprecipitation experiments these proteins were prominent antigens. Mr markers and their values were not specified, and we cannot identify these proteins. Subsequently, these authors analysed the antibody response in rabbits to polypeptides 1-6 by radioimmunoprecipitation and demonstrated early responses to 2, 3 and 6; 29.5, 25.5 and 42.5 kDa, respectively. If these values had been higher, the polypeptides of 29.5 and 42.5 kDa could have been P5 and P3, respectively. Further studies on binding of plasma proteins by the surface of T. pallidum showed that fibronectin was particularly avidly bound, and proteins 1, 2 and 3 interacted strongly and specifically with fibronectin on affinity chromatography [45]. While the M r of these proteins were given as 89, 29 and 25 (relative to ovalbumin at 43) and all marker proteins were specified, uncertainty remains as to their identity. In a recent paper from this group [14], similar M r markers were used, but the values assigned to them were different. The protein 6 was clearly located above the ovalbumin marker at 44 kDa and could not have had the value of 42.5 kDa claimed previously. Similarly,
protein 2 was about mid-way between the xchymotrypsinogen marker at 27 kDa and ovalbumin at 44 kDa, and could not have had the value of 29.5 kDa previously claimed. It therefore appears possible that protein 6 could be identical to P3, and protein 2 to P5. It remains unclear, however, whether identical polypeptides were being studied throughout the series of papers published by this group. Surface iodination has been thoroughly investigated by Norris and Sell [23], who showed that, particularly with lactoperoxidase-catalysed iodination, penetration of iodine radicals through surface layers appeared to occur, so that the results should be interpreted with caution. Our own studies [7] showed that, using intact treponemes, viable until addition of iodination reagents, little label could be incorporated into any treponemal proteins in comparison with presumed host-derived albumin. However, iodination of washed treponemes [23] under mild conditions preferentially labelled a 39-kDa protein (relative to ovalbumin at 43 kDa) which appears to have been P4. This accords with our own observation of partial extraction of P4 by Triton correlating with removal of the outer membrane [7]. A most useful development is the use of 2-dimensional gel electrophoresis of proteins [23]. Combined with surface labelling or other identification procedures, this promises to assist in unequivocal identification of treponemai proteins. We were, however, unable fully to correlate 2-dimensional gel patterns with 1-dimensional protein profiles, the latter as shown by these authors correlating well with our standard pattern (Fig. 1).
3.5. Treponemal antigen gene cloning Several groups have now reported the successful cloning in E. coli of genes from T. pallidum encoding antigens reactive with human or animal syphilitic sera [16,18,19,24,46,47]. Polypeptides of 46, 43, 38, 24, 23, 20 and 18.5 kDa were revealed by reaction with human secondary syphilitic serum [18], and were said to correspond with antigenic polypeptides of T. pallidum described subsequently by Hanff and colleagues [9,11,12]. Thus, the cloned 48-kDa polypeptide might have been identical with P3. We have already discussed the
146 cloned polypeptides of 46 [16] and 44 k D a [19], which might be fragments of P3. If all these cloned antigens are P3-related it appears that this antigen is particularly easy to clone, possibly as a result of multiple gene copies or promoters which function strongly in E. coil The nature of most other cloned antigens is less easy to assess. Cloned polypeptides of 39, 35 and 25 k D a relative to Bio-Rad standards [47] c a n n o t be clearly identified relative to a total treponemal protein profile since none is shown, and it appears unlikely that any of these is P5 since they did not react strongly with primary h u m a n syphilitic sera. It should of course be remembered that cloned antigens will not necessarily be identical in M r to their counterparts in T. pallidum, due for example to the expression of incomplete genes or differences in post-translational processing between species, and indeed, antigenically related but physically disparate polypeptides might occur in different recombinant clones derived from the same T. pallidum genes [47]. The best-characterised cloned antigen at present is a protease-resistant ' 4 D ' antigen of 190 k D a [24] which was strongly antigenic. Boiling in SDS broke down this antigen to 19-kDa fragments, which were not antigenically reactive with h u m a n or rabbit syphilitic sera, but to which antib o d y could be induced by immunisation with the cloned, purified antigen. Immunisation with the intact 4 D antigen also induced treponemal immobilising antibody, but the serum was not reactive with commercially available T. pallidum preparations in immunofluorescence tests.
4. C O N C L U S I O N It now appears possible to impart some order to the diverse and confusing range of observations that have been m a d e on the antigenic and other polypeptides of T. pallidum. M a n y problems remain in the interpretation of data from various laboratories, and because we remain ignorant of the functional identity of most of the polypeptides of interest, it would be desirable that in future reports these be located as far as possible within the patterns we have recognised in S D S - P A G E
profiles. By comparing and correlating the work of different groups, faster progress can be made towards a full understanding of the structure and antigenicity of this pathogen.
ACKNOWLEDGEMENTS We thank Dr. J. Stephen for a critical reading of the manuscript and helpful comments. The work was supported by the Wellcome Trust and by the British Technology G r o u p .
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