Research:
Dr. Centurion focuses on the mechanisms
of immune evasion in T. pallidum in collaboration with
Drs. Sheila Lukehart and Wes Van Voorhis
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.
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 and establish correlations with mRNA
levels at different time points during infection.
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” homopolymeric repeats in the promoter
regions.
In collaboration
with Dr. Christina Marra, we are currently studying the genetic
markers of T. pallidum strains isolated during the syphilis
epidemic in Seattle.
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