Targeting D-alanylation of teichoic acids to fight clinically-problematic Gram-positive pathogens – Dalatar

Determination of the most effective antibiotic treatment on mutant strains deficient in D-alanylation of TAs: It will be a question of constructing mutant strains by deletion of one or more genes of the dlt operon in E. faecium, SARM, SERM and C. difficile. The absence of D-alanylation is verified by quantification of D-Ala residues in TAs. The sensitivity of each mutant cultured in the presence of antibiotics in monotherapy or in combination is compared with that of the wild strain by physiological analyzes (MIC, survival and biofilm). Thus, this allows us to define the most effective antibiotic treatment to use in the presence of the D-alanylation inhibitors to be tested.
Synthesis and tests of new inhibitors of D-alanylation of TAs: The DltA inhibitor which allowed us to obtain our preliminary results presents limits of cell penetration and stability. A first series of chemical synthesis aims to resolve these difficulties, by making structural modifications of the inhibitor. Other series are being implemented to synthesize new specific inhibitors of DltA and DltC. The effectiveness of these inhibitors is tested in vitro by determining their median inhibitory concentration (IC50) value. For this, the DltA and DltC proteins of each of the pathogens studied are purified. The inhibitors having the best activity with respect to their target are selected to analyze their effectiveness on bacterial cultures.
Functional validation of new inhibitors in vivo: New physiological analyzes are carried out in wild strains in the presence of an inhibitor combined with the antibiotics selected previously. These same experiments are carried out in dlt mutants in order to confirm the specificity of the inhibitors for their target.
Efficacy of inhibitors in animal models. The LD50 of strains (except C. difficile) are determined by infecting larvae of Galleria mellonella. Caterpillars are also infected with the corresponding dlt mutants. The safety of inhibitors is determined by administering wide ranges of concentrations of the molecules of interest. In order to select the optimal combination(s) allowing the protection of infected caterpillars, ranges of concentrations of antibiotics associated or not with ranges of D-alanylation inhibitors are administered to them. The best “therapies” are then selected to be evaluated on a murine model. For each pathogen, these antibiotic / inhibitor combinations will be injected into infected mice in order to measure their effectiveness on the survival of the animal. Finally, these experiments will allow us to assess the toxicity of inhibitors but also to analyze the impact of different treatments on the murine intestinal microbiota, in particular by global sequencing approaches (metagenomics).

Strains deficient in D-alanylation of TAs have been obtained from clinical isolates of the pathogens E. faecalis, MRSA and C. difficile. The absence of D-alanylation for the mutants was confirmed by quantification of the D-alanine (D-Ala) esters contained in TAs. In addition, the sensitivity of the mutated and complemented strains towards different antibiotics was analyzed in vivo (MIC, survival and biofilm), in order to select the most effective treatment to use in the presence of an inhibitor to resensitize the pathogens. Thus, the antibiotics selected are a combination of amoxicillin / cefotaxime for enterococci, imipenem for methicillin-resistant S. aureus (MRSA) and bacitracin for C. difficile. Regarding the synthesis of D-alanylation inhibitor molecules, the standard inhibitor (DLT-1) that targets DltA from Bacillus subtilis was the first to be produced. Structural modifications (either in the sugar part or in the bridge between alanine and adenosine) have been made to improve the cellular penetration of DLT-1. The synthesis of DltC inhibitors is in progress by modifying DLT-1 by introducing a thiol trap, the purpose of which is to bind to CoA. In addition, the DltA and DltC proteins from E. faecalis and C. difficile were purified. Determination of the IC50 of the 24 molecules synthesized to date show that seven of them exhibit an inhibitory activity against DltA of E. faecalis. Regarding the functional validation of inhibitors in vivo, the most notable results were obtained with the inhibitor DLT-1. In enterococci, this decreases survival in the presence of an amoxicillin / cefotaxime combination. In MRSA, the inhibitor sensitizes several clinical strains to several ß-lactams, mainly imipenem, by decreasing MIC, survival and biofilm formation. Note that the phenotypes observed with the inhibitor are similar to those obtained with the dltA mutants. In addition, we have shown in these pathogens that the inhibitor causes a sharp decrease in D-Ala residues contained in TAs. Overall results suggested poor stability and / or penetration of the 7 molecules targeting DltA in vitro. The first stability analyzes in culture medium show that the reference inhibitor is stable. However, this shows poor penetration into the bacterial cell. Finally, we tested the efficacy of DLT-1 on the survival of Galleria mellonella. Thus, associated with an amoxicillin / cefotaxime combination, the inhibitor significantly improves the survival of caterpillars following infection with clinical strains of enterococci (E. faecalis and E. faecium). The same effect of the inhibitor was seen in the presence of imipenem in larvae infected with strains of MRSA.

This project focuses on pathogens presenting a serious threat level (enterococci and staphylococci) and emergency (C. difficile). The development of multidrug resistance to antibiotics has evolved more rapidly than the introduction of new drugs, considerably limiting the treatment options. This could compromise the ability to fight infectious diseases, especially in vulnerable patients. Therefore, the development of new treatment strategies would have significant economic and societal benefits. With this in mind, the Dalatar project assess the potential of new innovative drugs that inhibit D-alanylation of TAs as combined agents to sensitize or “resensitize” major Gram-positive pathogens to antibiotics. The prospects concern, on the one hand, the mutagenesis of the Dlt system of S. epidermidis and, on the other hand, the determination of the most effective antibiotic / inhibitor combination in this pathogen. In parallel, the characterization of the ?dlt mutants of C. difficile will be continued and the various inhibitors associated with the antibiotics detected during the first period will be tested in this pathogen. At the same time, the synthesis of new variants of the DltA inhibitor and that of inhibitors of the DltC protein will be undertaken. Ultimately, our results could be exploited by filing patents protecting new molecules as well as the most effective inhibitor / antibiotic treatment combinations.

- Coupri, D., Budin-Verneuil, A., Hartke, A., Benachour, A., Léger, L., Lequeux, T., Pfund, E., & Verneuil, N. (2019). Genetic and pharmacological inactivation of d-alanylation of teichoic acids sensitizes pathogenic enterococci to ß-lactams. The Journal of antimicrobial chemotherapy, 74(11), 3162–3169. doi.org/10.1093/jac/dkz322
- Coupri, D., Verneuil, N., Hartke, A., Liebaut, A., Lequeux, T., Pfund, E., & Budin-Verneuil, A. (2021). Inhibition of d-alanylation of teichoic acids overcomes resistance of methicillin-resistant Staphylococcus aureus. The Journal of antimicrobial chemotherapy. doi.org/10.1093/jac/dkab287
- Fontenelle, C., Thierry, T., Laporte, R., Pfund, E., & Lequeux, T. (2020). Selective preparation of tetrasubstituted fluoroalkenes by fluorine-directed oxetane ring-opening reactions – Special issue D. O’Hagan., Beilstein J. Org. Chem. 16, 1936-1946. doi:10.3762/bjoc.16.160.

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.