Signal Transduction proteins in Streptococcus pneumoniae and Vibrio cholerae

Abstract

[eng] The aim of this project is to structurally characterize proteins involved in bacterial signal transduction systems by applying X-ray crystallography.

Bacteria use signal transduction systems to react in response to any environmental changes detected. Bacterial signal transduction is divided into two categories, one- component systems and two-component systems. Two-component systems are composed by a Response Regulator (RR) and a Histidine Kinase (HK); the Histidine Kinase auto phosphorylates an inner domain, and soon after, it phosphorylates the receiver domain on the Response Regulator, activating the output domain; usually producing a physiological effect in the cell by activating a specific gene. While in one-component systems, one protein has both, a sensory and an output domain. An example of the one-component systems would be ToxR, and an example of the two- component systems would be ComD-ComE.

Bacterial transformation is a type of Horizontal Gene Transfer (HGT), which is a rapid evolutive mechanism in which entire genes can be transferred among bacterial cells. HGT is commonly deemed responsible for the appearance of antibiotic resistance, virulence factors and serotype switching.

Competence for genetic transformation in Streptococcus pneumoniae is a transient physiological state whose development is coordinated by a peptide pheromone (Competence Simulating Peptide or CSP) and its receptor, which activates transcription of two downstream genes, comX and comW, and 15 other “early” genes. ComD (HK) and ComE (RR) are involved in the quorum-signaling pathway that synthesizes the CSP. They help modulate the mechanism in which bacterial transformation occurs, by allowing the inclusion of naked DNA from the environment. We have successfully formed the binary (ComE+DNA) and ternary (ComD+ComE+DNA) complexes and characterized them in the attempt of obtaining crystals to solve their 3-D structure.

On the other hand, to elaborate on one-component systems, like ToxR we can discuss cholera. Cholera is caused by the causative agent Vibrio cholerae. It is estimated that there are from 1.3 to 4.0 million cases out of which up to 143,000 result in cholera deaths annually. After ingesting the V. cholerae, it travels to the small intestine

colonizing it and producing the cholera toxin. ToxR is a membrane-localized transcription factor that regulates the toxT promoter. The activation of the toxT promoter triggers the virulence cascade that leads to the secretion of toxin-coregulated pilus (TCP) and the expression of cholera toxin (CTX).

In recent years, our lab solved the crystal structure of the cytoplasmic domain of ToxR+20DNA, proposing molecular interactions between ToxR and the toxT promoter (Simone Pieretti’s PhD Thesis). In this study, we want to determine the crystallographic structure of three mutants of the cytoplasmic domain of ToxR bound to the toxT promoter. According to biochemical data from our collaborator (Professor Eric Krukonis, from the university of Detroit), these mutations down regulate the activation of ToxR and we aim to analyze the structural changes that these mutants suppose. Using X-ray crystallography we solved the structure of three complexes; ToxRQ78A+20DNA, ToxRS81A+20DNA and ToxRP101A+20DNA at 2.55, 2.95 and 2.95 Å resolution, respectively.

We have compared the final mutant structures with the wildtype, unveiling how the structural changes result in the decrease in activation of the toxT promoter.