Thursday, October 8, 2009

Baby steps towards unraveling transcriptional regulation in the unculturable syphilis spirochete

ResearchBlogging.orgI would never select Treponema pallidum as my experimental model if I had to study gene regulation in a spirochete. The main problem is that no one has figured out how to grow T. pallidum in any type of culture medium. T. pallidum can be propagated only by growing the spirochete in the testes of rabbits. Consequently, investigators have not even begun to develop the genetic tools (e.g., gene knock outs, shuttle plasmids) necessary to unravel the regulatory mechanisms that control T. pallidum gene expression.

Despite the limitations imposed by T. pallidum upon those who wish to study gene regulation, a group of syphilis researchers at the University of Washington in Seattle have started to dissect the regulation of several members of the 12-gene tpr (Treponema pallidum repeat) family. No one has figured out what the Tpr proteins do, but syphilis researchers are interested in them in part because they show how the immune response battles T. pallidum infections. For example, antibodies generated against TprK during infection bind to TprK exposed on the surface of T. pallidum and mark them for destruction by macrophages. More recent studies suggest that TprK undergoes antigenic variation (a topic of a future post), which may allow T. pallidum to persist in the host.

The Seattle group's studies on gene regulation have focused on the Subfamily II tpr genes tprE, tprG, and tprJ, as reported in the journal Molecular Microbiology. The sequences upstream of their transcription start sites contain a sequence that closely matches the consensus binding sequence for the E. coli global transcriptional regulator CRP (cAMP regulatory protein), also known as CAP (catabolite activator protein). The T. pallidum genome encodes a CRP homolog designated TP0262. In E. coli and a few other Gram negatives, CRP is an integral component of the complex network of transporter, regulatory, and enzymatic proteins that allow bacteria to selectively metabolize the preferred sugar, usually glucose, from those available in the environment. When glucose is absent, the enzyme adenylate cyclase is activated and synthesizes the second messenger cAMP (cyclic AMP), which turns on CRP by allosteric activation. (Here's a nice description of the allosteric activation of CRP.) The cAMP-CRP complex then binds upstream of various promoters and activates transcription by recruiting RNA polymerase to the promoter. Additional layers of regulation ensure that the genes are transcribed only when the sugar that is to be broken down by the gene products is present.

Because it's not possible to examine gene regulation in T. pallidum, the Seattle group transferred the tpr genes to E. coli, a genetically pliable bacterium. They fused each tpr gene, including the upstream sequences containing the proposed CRP binding site and the promoter, to a gene whose product is easily measurable, green fluorescent protein (gfp). They then introduced the plasmid carrying the gene fusion into an E. coli strain missing its crp gene so that they could measure tpr-driven GFP levels in the presence and absence of a second plasmid expressing TP0262. They found that TP0262 increased tprE'-gfp and tprJ'-gfp fusion expression while decreasing trpG'-gfp expression. The ability of TP0262 to control tpr'-gfp expression was lost when the CRP binding site was removed from the fusion constructions. They also showed that control of the tprJ'-gfp fusion by TP0262 was lost when the adenylate cyclase gene in E. coli was removed, indicating that cAMP was needed to activate TP0262 (data for tprE and tprG were not presented). Their in vitro experiments demonstrated binding of purified recombinant TP0262 to the proposed CRP binding site upstream of the three tpr genes by DNase I protection and gel shift assays.

What was missing from the study, as acknowledged by the authors, were experiments to demonstrate that TP0262 does the same thing in T. pallidum. For future studies, they plan to show that TP0262 is bound upstream of the Subfamily II tpr genes in T. pallidum by chromatin immunoprecipitation, which entails determining the sequence of the segment of DNA that is bound when TP0262 is immunoprecipitated from a T. pallidum extract. Such experiments would not require genetic manipulation or the ability to cultivate T. pallidum. It would only require harvesting a large number of T. pallidum spirochetes from infected rabbits.

What signal does TP0262 respond to? Does it respond to the glucose found in the host? The insightful Commentary by Radolf and Desrosiers sheds some light on the question. They note that T. pallidum is missing the special transporter genes that in E. coli encode the components necessary to link sugar availability to cAMP and CRP. They surmise that TP0262 has thus been freed to regulate genes not related to sugar metabolism, such as the tpr genes. Since CRP is a global transcriptional regulator in other bacteria, it is likely to regulate expression of not only the Subfamily II tpr genes but also additional genes in T. pallidum.

Near the end of their commentary, Radolf and Desrosiers made one comment that stood out:
One of the most important outcomes of the present study is that it will help put to rest the pregenomic view of the syphilis spirochaete as a transcriptionally invariant organism.

Maybe I'm too young to appreciate their point, but I can't believe that there ever was a time when syphilis researchers believed that T. pallidum genes were not regulated!

Featured articles

Giacani, L., Godornes, C., Puray-Chavez, M., Guerra-Giraldez, C., Tompa, M., Lukehart, S.A., & Centurion-Lara, A. (2009). TP0262 is a modulator of promoter activity of tpr Subfamily II genes of Treponema pallidum ssp. pallidum
Molecular Microbiology, 72 (5), 1087-1099 DOI: 10.1111/j.1365-2958.2009.06712.x

Radolf, J.D., & Desrosiers, D.C. (2009). Treponema pallidum, the stealth pathogen, changes, but how?
Molecular Microbiology, 72 (5), 1081-1086 DOI: 10.1111/j.1365-2958.2009.06711.x