Baby steps towards unraveling transcriptional regulation in the unculturable syphilis spirochete

I 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

Role of the second messenger cyclic diguanylate (c-di-GMP) in the Lyme disease spirochete

Second messengers are the intracellular intermediaries that transmit the signals received from the environment (first messenger) to the cellular machinery that generates the appropriate response. Well known examples of second messengers in mammalian cells include cyclic AMP, cyclic GMP, calcium ion, and inositol triphosphate. A second messenger unique to bacteria is cyclic diguanylate, abbreviated c-di-GMP. First described in the 1980s, c-di-GMP is only now attracting wide interest among those who study signal transduction in bacteria.

Cyclic di-GMP is created from two GTP molecules by diguanylate cyclase and destroyed by phosphodiesterases. Genes encoding the opposing enzymatic activities can be identified by the conserved GGDEF motif in diguanylate cyclases and an EAL or HD-GYP motif in phosphodiesterases. Bacteria modulate the intracellular concentration of c-di-GMP by controlling the amounts and activities of the diguanylate cyclases and phosphodiesterases in response to changes in environmental conditions.

Cyclic di-GMP is best known for promoting the formation of biofilms. Biofilm assembly requires the synthesis and secretion of the special polysaccharides that make up the biofilm matrix and the down-regulation of motility. Both polysaccharide synthesis and the inhibition of motility are modulated by c-di-GMP. The molecule can also affect virulence functions. In many cases, the mechanistic details of how c-di-GMP exerts its effects remain unknown. The molecular target of c-di-GMP includes proteins with the "PilZ" domain (see figure). However, not all proteins bound by c-di-GMP possess the PilZ domain. In some bacteria, c-di-GMP can also bind specific sequences found within the 5' untranslated region of several mRNAs to modulate gene expression.

Figure 1 from Tamayo et al., 2007. DGC, diguanylate cyclase; PDEA, phosphodiesterase A; PDE, phosphodiesterase

The Borrelia burgdorferi gene rrp1 encodes the only protein in the Lyme disease spirochete containing the GGDEF motif. The Rrp1 protein consists of a receiver domain and the GGDEF domain, whose diguanylate cyclase activity requires phosphorylation of the receiver domain. A paper in the March issue of Molecular Microbiology revealed the genes whose expression is affected by Rrp1. The authors compared the transcript profiles (transcriptome) of a B. burgdorferi wild-type and an rrp1 deletion mutant by microarray analysis. It turned out that most of the genes affected by the mutation encode what the authors call the "core" cellular functions of Borrelia burgdorferi. The core functions allow the spirochete to seek out and capture nutrients from the environment, synthesize the building blocks necessary for assembling cellular parts, and extract energy from nutrients to fuel its activities. All bacteria, whether or not they cause disease, possess these core functions. Most transcripts from core genes were increased in the wild-type B. burgdorferi strain relative to the rrp1 mutant. The impaired growth of the rrp1 mutant compared to wild type is consistent with the importance of Rrp1 on the expression of the core functions of B. burgdorferi.

The investigators also found that the rrp1 transcript levels increased 6 fold when ticks harboring B. burgdorferi took a blood meal from mice. High levels of rrp1 mRNA were also maintained in B. burgdorferi growing in culture medium. These observations suggest that more rrp1 transcript is made when the spirochete is awash in nutrients, whether in blood or culture medium. Under these conditions, c-di-GMP signals B. burgdorferi to turn on genes necessary to acquire and metabolize the nutrients.

The protein that directly or indirectly senses changes in nutrient availability is likely to be the histidine kinase encoded by hpk1, the gene that lies immediately upstream of rrp1. The Hpk1 and Rrp1 proteins form a phosphorelay in which phosphate groups swiped from ATP molecules are transferred to the target Rrp1 protein in response to some signal in the environment.

In summary, Rrp1 diguanylate cyclase activity is enhanced at two levels when nutrients become abundant. First, rrp1 transcript levels are increased, leading to more Rrp1 protein being made. Second, the Rrp1 diguanylate cyclase activity is activated by phosphorylation. Increased c-di-GMP levels is the result. The c-di-GMP stimulates increased levels of transcripts emanating primarily from genes encoding the core cellular functions of B. burgdorferi. The question that remains unexplored is how c-di-GMP causes transcript levels to increase. One protein in B. burgdorferi harbors the PilZ domain, but as I mentioned earlier, PilZ is not the only protein domain capable of binding c-di-GMP.

Finally, what does this study reveal about the role of Rrp1 and c-di-GMP in Lyme disease? One possibility is that Rrp1 is involved in tick-to-human transmission and the early stages of the infection:

• As I already mentioned, Rrp1 upregulation in B. burgdorferi residing in a tick taking a blood meal may prepare the spirochete to metabolize the nutrients found in the blood as they are transmitted into the skin of the human victim.
• Transcripts expressed from several genes encoding factor H-binding proteins (some of the few non-core genes affected by the rrp1 knock out) were at higher levels when Rrp1 was present. Factor H is an inhibitor of the complement system found in our bloodstream. As such, binding of factor H by the spirochete may protect it from being killed by the host complement system. Indeed, the authors demonstrated that the rrp1 mutant was more sensitive to human serum than the wild-type B. burgdorferi strain. However, the significance of this observation is unclear as Borrelia garinii, another agent of Lyme disease, was just as sensitive as the B. burgdorferi rrp1 mutant to human serum. Additionally, earlier studies have shown that factor H is not necessary for successful B. burgdorferi infections (at least in the mouse model of Lyme disease).
• The ospC gene, which encodes another protein that may impair immune function during the early stages of infection, was also upregulated by Rrp1.
• Several transcripts expressing motility and chemotaxis functions are expressed at higher levels when Rrp1 is present. Motility and chemotaxis are considered to be core functions, but they may also be necessary for B. burgdorferi to establish infection in humans. Note that the proposed effect of c-di-GMP on B. burgdorferi motility is opposite of that found in other bacteria (see figure above).
  

The obvious experiment to perform is to test whether the rrp1 mutant can cause infection in the mouse model of Lyme disease. Unfortunately, the effect of rrp1 on virulence could not be tested as the authors were unable to knock out the rrp1 gene in an infectious strain of B. burgdorferi.

Featured paper

Rogers, E.A., Terekhova, D., Zhang, H.-M., Hovis, K.M., Schwartz, I., & Marconi, R.T. (2009). Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions Molecular Microbiology, 71 (6), 1551-1573 DOI: 10.1111/j.1365-2958.2009.06621.x

Image source

Tamayo R., Pratt J.T., and Camilli, A. (2007). Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annual Review of Microbiology 61:131-148. DOI: 10.1146/annurev.micro.61.080706.093426