Research in this laboratory focuses on Treponema pallidum, the etiologic agent
of syphilis. We are studying the mechanisms of immune evasion in T. pallidum
in collaboration with Drs. Sheila Lukehart and Wes Van Voorhis.: 1. Gene Conversion.
We have identified the first mechanism of antigenic variation in syphilis responsible
for the generation of tprK diversity. It involves donor cassettes, one expression
site and non-reciprocal gene conversion mechanisms. Experimental data demonstrate
sequential acquisition of sequence diversity consistent with this mechanism.
We are also studying the role of the homologous and non-homologous recombination
machinery in T. pallidum to determine their role in the generation of tprK diversity.
As a first step, we are using the E. coli recombination machinery to reproduce
recombinatorial events between donor sites and the tprK expression site. 2.
Phase Variation. We have recently identified “G” homopolymeric repeats
in the promoter regions of the tpr genes of the Tpr Subfamilies I and II. It
is likely that poly “G” tracts are involved in ON-OFF switch mechanisms
of gene expression regulation. Similar structures have been already shown to
be regulatory elements in several bacterial species. We have shown that these
potential regulatory sequences vary in length within and among T. pallidum isolates.
Due to the inability to cultivate T. pallidum in vitro, we are using in-vitro
transcription systems and reporter genes (lacZ fusions for ß-galactosidase
assays) to study promoter strength. 3. Identification of operons/single transcriptional
units of the tpr genes. We are defining the boundaries of the tpr transcriptional
units and identifying their regulatory elements (promoters, transcriptional
start sites, termination structures, repeat sequences). We have found a complex
transcriptional pattern of these genes. There is co-transcription of tprJ and
tprI as well as of tprG and tprF genes, but we have also identified monocistronic
messages in some T. pallidum isolates. The transcriptional starts of these two
operons are localized immediately downstream of the “G” homoplymeric
repeats in the promoter regions.