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