Fosfomycin perception by CreBC: Novel insights into Escherichia coli's response to MurA blockage

Introduction:

Antimicrobial resistance will be a leading cause of mortality within a few decades, due to the current antibiotic discovery pipeline crisis. Thus, it remains essential to optimize the use of existing antibiotics by better understanding their broad cellular impact on bacteria. Fosfomycin (FOS), first discovered in 1969, remains active against multi-drug resistant (MDR) bacteria and its excellent pharmacological properties and unique mode of action make it ideal for synergistic therapies. Within bacteria, it covalently inhibits the enzyme MurA, blocking the first committed step of peptidoglycan synthesis. However, its clinical use against Escherichia coli often leads to high rates of resistance development and infection relapse.

Goal:

A better understanding of FOS cellular effects and off-target actions in E. coli could enable improved treatments, reducing the risk of regrowth of resistant or adapted bacteria.

Materials and methods:

Susceptibility to FOS was tested with gradient strip tests and broth dilutions in LB medium, monitoring the growth with a microplate reader. Transcriptional analyses were conducted with Illumina RNA sequencing. E. coli BW25113 and KO-mutants of the genes of interest were tested. The induction conditions of these genes of interest were further assessed with a lacZ-reporter plasmid.

Results:

E. coli alters its transcriptional state in response to FOS, shortly before drug-induced lysis. Transcriptional analyses of exponentially growing E. coli treated with FOS showed the rapid up-regulation of genes of the CreBC core regulon (cbrA, cbrB, cbrC, creD). The CreBC two-component system (TCS) is known to be responsive to carbon source changes in E. coli, but its real purpose remains unclear. Knockout mutants of these genes showed heightened sensitivity to FOS, and a similar effect was observed in strains completely lacking the CreBC TCS. The link between FOS and the upregulation of the CreBC regulon core genes was further investigated with a lacZ-reporter of creD expression.

Summary:

FOS-treated E. coli upregulated CreBC-related genes, and their role in response to FOS bactericidal action was investigated. This work advances efforts to better exploit the potential of FOS in MDR therapy, while preventing resistance development.