Characterization and Serologic Analysis of the Treponema pallidum Proteome

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease characterized by widespread tissue dissemination and chronic infection. In this study, we analyzed the proteome of T. pallidum by the isoelectric focusing (IEF) and nonequilibrating pH gel electrophoresis (NEPHGE) forms of two-dimensional gel electrophoresis (2DGE), coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis. We determined the identity of 148 T. pallidum protein spots, representing 88 T. pallidum polypeptides; 63 of these polypeptides had not been identified previously at the protein level. To examine which of these proteins are important in the antibody response to syphilis, we performed immunoblot analysis using infected rabbit sera or human sera from patients at different stages of syphilis infection. Twenty-nine previously described antigens (predominantly lipoproteins) were detected, as were a number of previously unidentified antigens. The reactivity patterns obtained with sera from infected rabbits and humans were similar; these patterns included a subset of antigens reactive with all serum samples tested, including CfpA, MglB-2, TmpA, TmpB, flagellins, and the 47-kDa, 17-kDa, and 15-kDa lipoproteins. A unique group of antigens specifically reactive with infected human serum was also identified and included the previously described antigen TpF1 and the hypothetical proteins TP0584, TP0608, and TP0965. This combined proteomic and serologic analysis further delineates the antigens potentially useful as vaccine candidates or diagnostic markers and may provide insight into the host-pathogen interactions that occur during T. pallidum infection.Syphilis is a multistage progressive disease caused by the spirochete Treponema pallidum subsp. pallidum and is characterized by localized, disseminated, and chronic stages. Manifestations include the development of a localized lesion called a chancre during the primary stage and disseminated skin lesions and meningovascular syphilis during the secondary stage, followed by a period of latency lasting from months to decades. Chronic, debilitating symptoms develop during the tertiary stage, including granuloma-like lesions called gummas, neurosyphilis, and cardiovascular syphilis (38). Although syphilis can be successfully treated by antibiotics, it remains a significant public health problem, with an estimated 12 million new cases per year worldwide (41).Continued improvement of diagnostic tests (particularly point-of-care tests) as well as the development of an effective vaccine for syphilis would aid greatly in the control of syphilis (4, 6). T. pallidum research, including the identification of antigens, has been hindered by the inability to culture the bacterium continuously in vitro, necessitating the propagation of organisms by experimental rabbit infection (28). In addition, the fragility and low protein content of the T. pallidum outer membrane have complicated the identification of surface proteins potentially useful in vaccines (5, 28).The T. pallidum genome sequence (15) provides an additional tool for the analysis of potential antigens. The 1.14-Mb T. pallidum chromosome contains 1,039 open reading frames (ORFs) encoding predicted protein products, a smaller number than for any other spirochete genome sequenced to date (15). The average size of predicted proteins is 37,771 Da, ranging from 3,235 to 172,869 Da. Analysis of the translated genome of T. pallidum predicts an unusually basic proteome, with a mean pI of 8.1 and median pI of 8.5, with 66% of proteins having pIs of >7.0 (23). Small genome size and a predominance of basic proteins are more common in parasitic microorganisms, and the latter is thought to facilitate interaction of the organism with its host (20). Other pathogenic spirochetes also tend to have basic proteins; for example, the proteome of Borrelia burgdorferi has a mean pI of 8.36 and median pI of 9.03 (14, 29a), and 69% of Leptospira interrogans serovar Lai strain 56601 proteins have pIs greater than 7.0 (24, 33). A recent analysis of the T. pallidum genome indicates the presence of 46 putative lipoproteins, many fewer than the 127 predicted for B. burgdorferi (34).The availability of the genome sequence made it possible to examine predicted T. pallidum ORFs for potential suitability as diagnostic or immunization tools. McKevitt et al. (22) and Brinkman et al. (3) created a protein expression library of 900 of the 1,039 T. pallidum proteins predicted from the genome sequence and examined the serologic reactivity of these proteins by enzyme-linked immunosorbent assays (ELISAs). They identified 106 antigens reactive with rabbit sera and 34 antigens reactive with sera from syphilis patients. This set of antigens was termed the T. pallidum immunoproteome. This approach permits identification of low-abundance T. pallidum antigens, since they may be expressed as recombinant proteins in much larger quantities. Conversely, proteins that are poorly expressed in Escherichia coli or do not fold correctly may not be detected, leading to false-negative results.To provide a complementary set of data regarding the T. pallidum immunoproteome, we have performed proteomic analysis of T. pallidum proteins expressed during experimental rabbit infection. We used isoelectric focusing (IEF) and nonequilibrating pH gel electrophoresis (NEPHGE) forms of two-dimensional gel electrophoresis (2DGE) coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis to identify T. pallidum polypeptides. Immunoblotting was subsequently used to identify antigens reactive with infected rabbit sera (IRS) and with human sera obtained at different stages of syphilis. This approach may permit identification of antigens that are not expressed well in E. coli and provides a more accurate picture of the level of protein expression in the intact organism. We have thereby characterized most of the major T. pallidum proteins expressed in infected tissue and identified a set of antigens reactive at all stages of infection, which could potentially be useful for the development of improved immunodiagnostic tests or for vaccines.