Identification of Lactobacillus Transcriptional Regulators Responsive to Small Molecules

NON-TECHNICAL SUMMARY: Live bacterial vectors are very promising tools in the development of cheaper and safer vaccines. However, better inducible expression systems that can circumvent toxicity problems are required to enable precise temporal and spatial control of antigen expression in vivo. Our long-term goal is to increase the efficacy with which vaccine antigens can be delivered using Lactobacillus vectors and to regulate the antigen expression in the live vector using a Federal Drug Administration (FDA) approved drug. The finding of a regulatory system that could be triggered by a drug that is already approved by the FDA would constitute an enormous advantage to reach human trials. The objective of this project, is to identify transcriptional regulators in the MerR, LysR, TetR, MarR, and IclR families that respond in vitro to small molecules approved by the FDA. We plan to achieve this objective by 1) Identifying transcription factors within the genomes of lactobacilli that are able to interact with FDA approved small molecules. We will perform a rational in silico selection of our targets and high throughput technology for the screening of small molecule libraries. 2) Determining the ability of the drug to bind the transcription factor in presence of its putative DNA binding site using electrophoretic mobility shift assays. These experiments are aimed to independently confirm the results obtained in the high throughput screen and help in the identification of true binders in contrast to unspecific binders. The affinity of the drug-protein interaction will be defined by isothermal titration calorimetry. The rationale is that, once efficacious new transcription factor/small molecule combos are identified, definitive studies designed to optimize their efficacy in animal models will be justified. The aproach is innovative because the discovery of a new protein that binds a chemical that has already been approved by the FDA for its use in humans will be a very important stepping stone in reaching the human trial phase. After completion of this proposal we will have identified new regulatory proteins in lactobacilli that respond with high affinity to FDA approved drugs. Second, by testing the interaction protein/small molecule in the presence of their native DNA binding sequence we will be able to have identified ligands that will disrupt the protein/DNA interaction in vivo. Since the families of transcriptional regulators selected for this proposal are involved in the development of multidrug resistance it is anticipated that what is learned will be equally applicable to the identification of drug targets to combat infections caused by many gram-positive pathogenic bacteria.

<P>APPROACH: 1. Specific aim #1: Identify transcription factors within the genomes of lactobacilli that are able to interact with FDA approved small molecules. We plan to achieve this objective by: 1.1 Identifying different homology/functional groups within sequenced Lactobacillus genomes. We will perform whole genome search for proteins that show similar binding motifs as the multidrug resistance associated transcriptional regulators within the genomes of lactobacilli. We anticipate that around 70 proteins will be selected. As a second selection parameter, we will analyze the genome context of the selected gene. A priority target list will be constructed based on the presence of efflux pumps in the immediate area of the transcription factor. 1.2 Cloning and purifying the target proteins. The target genes will be fused to a 6Xhis-tag by cloning them in a modified pET15b vector and will be purified by affinity chromatography. 1.3 High throughput screening for binding of FDA approved small molecules. Each transcription factor will be tested on the Prestwick chemical library by differential scanning fluorometry. This technique consists of analyzing the kinetics of isothermal denaturation of the protein in presence or absence of potential specific ligands. We will monitor the unfolding of the protein by the increase in fluorescence of the fluorophor SYPRO Orange (BioRad). The temperature shift between the melting temperature (Tm) in the presence and absence of a bound ligand will be measured. 2. Specific aim #2: Determine the ability of the small molecule to bind the transcription factor in presence of its putative DNA binding site. We plan to achieve this objective by: 2.1. Identifying the transcriptional regulator native binding region by in silico and in vitro techniques. In the abundant literature available on local regulators it has been reported that, in general, they are encoded upstream or downstream of the regulated genes. With this criterion in mind, we will analyze the genomic environment of the transcriptional regulator and select sequences (150-300 bases-long) in the surroundings that could contain the putative binding site. After the in silico identification of the putative binding site, the selected DNA regions will be tested for binding by EMSA as described in Lorca et al. (2007). 2.2. In vitro determination of the effect of small molecules on the DNA/transcription factor interaction using electrophoretic mobility shift assays (EMSA). Since, in general, transcription factors are two headed molecules (a DNA binding domain and a small molecule binding domain) there is a need to test the effect of the small molecules on the DNA/protein interaction. The interactions of selected drug compounds with the transcriptional regulators will be functionally characterized by EMSA. 2.3. Quantitative characterization of the small molecule/transcription factor interaction by isothermal titrating calorimetry (ITC). We plan to use isothermal titrating calorimetry (ITC) to specifically determine the affinity constant and the stoichiometry of the binding. Measurements will be performed on a VP-Microcalorimeter (MicroCal).