A Strategic Target Rescues Trimethoprim Sensitivity in Escherichia coli

Trimethoprim, a preferred treatment for urinary tract infections, is becoming obsolete owing to the rapid dissemination of resistant E. coli. Although direct resistance mechanisms such as overexpression of a mutant FolA and dfr enzymes are well characterized, associated alterations that drive or sustain resistance are unknown. We identify the repertoire of resistance-associated perturbations by constructing and interrogating a transcriptome-integrated functional interactome. From the cross talk between perturbations in stress-response and metabolic pathways, we identify the critical dependence on serine hydroxymethyltransferase (GlyA) as an emergent vulnerability. Through its deletion, we demonstrate that GlyA is necessary to sustain high levels of resistance in both laboratory-evolved resistant E. coli and a multidrug-resistant clinical isolate. Through comparative evolution, we show that the absence of GlyA activity decelerates the acquisition of resistance in E. coli. Put together, our results identify GlyA as a promising target, providing a basis for the rational design of drug combinations.