Christa Castro

Opsonic Potential, Protective Capacity, and Sequence Conservation of the Treponema pallidum subspecies pallidum Tp92

By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%-100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.

The tprK gene is heterogeneous among Treponema pallidum strains and has multiple alleles.

ABSTRACT We have previously shown that the TprK antigen of T. pallidum, Nichols strain, is predominantly expressed in treponemes obtained 10 days after infection and that the hydrophilic domain of TprK is a target of opsonic antibodies and confers significant protection against homologous challenge. The T. pallidum genome sequence reported the presence of a single copy of the tprK gene in the Nichols strain. In the present study we demonstrate size heterogeneity in the central portions of the TprK hydrophilic domains of 14 treponemal isolates. Sequence analysis of the central domains and the complete open reading frames (ORFs) of the tprK genes confirms this heterogeneity. Further, multiple tprK sequences were found in the Nichols-definedtprK locus in three isolates (Sea 81-4, Bal 7, and Bal 73-1). In contrast, only a single tprK sequence could be identified in this locus in the Nichols strain. Alignment of the DNA and deduced amino acid sequences of the whole tprK ORFs shows the presence of seven discrete variable domains flanked by highly conserved regions. We hypothesize that these heterogeneous regions may be involved in antigenic heterogeneity and, in particular, evasion of the immune response. The presence of different tprK alleles in the tprK locus strongly suggests the existence of genetically different subpopulations within treponemal isolates.

Multiple Alleles of Treponema pallidum Repeat Gene D in Treponema pallidum Isolates

Two new tprD alleles have been identified in Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in one T. pallidum subsp. pertenue isolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating that tprD2 is expressed during infection.

Comparison of methods for the detection of treponema pallidum in lesions of early syphilis.

Background: The clinical diagnosis of primary and secondary syphilis can be difficult because of the wide variability of lesions. The available laboratory tests (dark‐field microscopy and direct fluorescent antibody) require specialized microscopes and skilled technicians, and serologic tests are insensitive in early infection. Methods: Dark‐field microscopy and monoclonal antibody staining were compared to a new solid‐phase enzyme‐linked immunosorbent assay (Visuwell test) for detection of T. pallidum in lesion exudate of 188 patients with genital lesions. Results: Sixty‐four patients (34%) had lesions of early syphilis diagnosed by either dark‐field, monoclonal antibody staining, or both. The Visuwell test and dark‐field examination were positive in 52 (81.3%) and 55 (85.9%) of the 64 patients, respectively, whereas the monoclonal antibody staining technique demonstrated the presence of T. pallidum in 59 (92.2%) of the 64 patients. The Visuwell test gave a negative result in 111 of 124 patients who had negative dark‐field and direct fluorescent antibody test results (89.5% specificity). Conclusions: The Visuwell test is an alternative method for evaluating genital ulcers but is less sensitive and specific than existing tests.

Mechanisms of clearance of Treponema pallidum from the CSF in a nonhuman primate model

Objectives: To establish a model of CNS invasion by Treponema pallidum and to use it to investigate the immune mechanisms responsible for clearance. Methods: Four macaques were intrathecally inoculated with 0.6 to 2.1 × 108 T. pallidum and underwent clinical examinations and blood and CSF collections every 1 to 2 weeks for 12 to 13 weeks. The following were determined: serum Venereal Disease Research Laboratory (VDRL) and microhemagglutination-T. pallidum reactivities, CSF-VDRL, CSF white blood cell (WBC) count, and the presence of viable T. pallidum in CSF by the rabbit infectivity test (all animals), as well as the presence of T. pallidum in CSF by reverse-transcriptase (RT)-PCR, WBC phenotype by fluorescence-activated cell sorter, WBC cytokine production by RT-PCR, and brain MRI at 10 weeks (two animals). Results: All animals became systemically infected and developed CSF pleocytosis that resolved after 8 weeks. CSF T. pallidum was detected from 2 to 8 weeks. CSF T lymphocytes were predominantly CD4+. Interferon-gamma (IFN-γ) mRNA was consistently detected in CSF WBCs, but interleukin (IL)-4 and IL-5 were not. All animals remained clinically well. MRIs were normal. Conclusions: In this model, T. pallidum is cleared from the CNS just as in most humans with early syphilis. Local production of IFN-γ likely participates in this process. This model could be used to clarify the effect of retrovirus-induced immunodeficiency on clearance of T. pallidum from the CNS and on the local CNS immune response.