Mechanisms of action and synergies of a novel lipid IVA biosynthesis inhibitor

The development of novel antimicrobials provides additional treatment options for infectious diseases, including antimicrobial resistant infections. There are many hurdles to antimicrobial development and identifying an antimicrobial’s mechanism of action is a crucial step in progressing candidate molecules through the drug discovery pipeline. We used the genome wide screening method TraDIS-Xpressto identify genes in two model Gram-negative bacteria that affected sensitivity to three analogues of a novel antimicrobial compound (OPT-2U1). TraDIS-Xpressidentified that all three analogues targeted the lipid IVAbiosynthetic pathway inE. coliandSalmonellaTyphimurium. Specifically, we determined that the antimicrobial target was likely to be LpxD, and validated this by finding a 5 log2-fold increase in the MIC of the OPT-2U1 analogues inE. coliwhenlpxDwas overexpressed. Synergies were identified between OPT-2U1 analogues combined with rifampicin or colistin, to varying strengths, in bothE. coliandS. Typhimurium. LPS composition was a likely reason for differences betweenE. coliand S.Typhimurium, as perturbation of LPS synthesis affected synergy between antibiotics and OPT-2U1 analogues. Finally, genes involved in ATP synthesis and membrane signalling functions were also found to affect the synergy between colistin and OPT-2U1 analogues. TraDIS-Xpresshas proven a powerful tool to rapidly assay all genes (and notably, essential genes) within a bacterium for roles in dictating antimicrobial sensitivity. This study has confirmed the predicted target pathway of OPT-2U1 and identified synergies which could be investigated for development of novel antimicrobial formulations.

Data Summary

Nucleotide sequence data supporting the analysis in this study has been deposited in ArrayExpress under the accession number E-MTAB-13250. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

http://dx.doi.org/10.1101/2023.09.15.557861