Dalatar

This project addresses the important problem of the worldwide increase of antibiotic resistance. It aims to develop adjuvant drugs potentiating the action of conventional antibiotics to restore susceptibility of resistant pathogens to these drugs. Our extensive preliminary results show that the D-alanylation system is a promising target in this context. This system comprises 4 genes (dltABCD) encoding enzymes implicated in the decoration of teichoic acids (TAs) with D-alanine. TAs are abundant polymers of the cell wall of many Gram-positive bacteria. Genetic or pharmacological prevention of this modification of TAs led to re-sensitize drug resistant enterococci and Staphylococcus aureus to ß-lactams. The inhibitor used in these experiments has high affinity for its target in vitro but has limitations in vivo. The consortium of this project will synthesize and test new D-alanylation inhibitors to select drugs with improved in vivo performances. These new adjuvant drugs will be tested on 5 clinically critical Gram-positive pathogens, Enterococcus faecalis, E. faecium, S. aureus, S. epidermidis and Clostridium difficile. The project is organized into 4 Tasks. In Task 1 we will construct markerless D-alanylation deficient deletion strains and test their sensitivities to ß-lactams (MIC determinations and time-kill curves) in order to define the most effective antibiotic treatments to inhibit or kill these mutants. Absence of D-alanylation will be verified by 14C D-alanine labelling of TAs. In Task 2, several series of new inhibitors will be chemically synthesized. The first series will focus on the structural modification of the DltA inhibitor used in the preliminary experiments to enhance its stability and/or lipophilic character to increase penetration across the cell membrane. Two other series will be developed to get mimics of the transition states and stable analogues of nucleosides. Affinity of these new inhibitors for their respective purified targets will be tested. In Task 3, the inhibitors with the best in vitro properties will be tested on wild-type strains of all 5 pathogens. MIC and time-kill curves will be determined using the most efficient antibiotics defined in Task 1. Comparison with the results obtained with the D-alanylation deficient mutants will allow to asses to which degree the new drugs phenocopies the antibiotic sensitivities of these strains. Molecules with the best in vivo efficiencies will be tested for their penetration, stability in biofluids and cell extracts, and inhibition of D-alanylation. The lead compounds will be produced on a larger scale necessary for Task 4. In this last task we will conduct experiments to asses if the new adjuvant drugs are effective to rescue animals or prevent their colonization after infection with the 5 pathogens. Two models will be used, the insect model Galleria mellonella and mice. The mice experiments will also give first indications on the toxicity of the chemical inhibitors in mammals. Furthermore, the impact of the new drugs on the mice microbiota will be assessed by 16S RNA analysis. The results gathered in this project will be of fundamental and applied interest. They will help to understand why the absence of D-alanylation restores the susceptibility to ß-lactam antibiotics in bacteria resistant to these drugs. Moreover, the project has the potential to be a translational research project since the new adjuvant drugs could be valorised by the deposition of patents and by entrance of the project into a maturation state to evaluate clinical applications.