A Strategic Target Rescues Trimethoprim Sensitivity in Escherichia coli

Abstract

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.

Keywords: Microbiology; Multi-Drug Resistant Organisms; Transcriptomics.

Graphical abstract

Figure 1

Laboratory-Evolved TMP-Resistant E. coli Exhibit Multiple Transcriptomic Changes (A) Evolution of TMP-resistant 32xR E. coli: TMP-sensitive WT E. coli were adapted to TMP in a stepwise manner over 2.5 days. The adaptation was initiated by growing WT in sub-inhibitory (0.25xMIC) TMP concentration of 0.125 μg/mL to A₆₀₀ ~ 0.6 (green filled circle) followed by inoculation in 2x TMP (0.25 μg/mL). This was done iteratively by doubling the concentration in each step till E. coli adapted to 16 μg/mL TMP. Line at the bottom indicates time after which the culture reached A₆₀₀ ~ 0.6 in a particular concentration. (B) log₂FC values of 397 DEGs in biological replicates of 4xR and 32xR E. coli. The FC of a gene is the mean of the FC of biological replicates of 4xR or 32xR. In general, FC is seen to be higher in 32xR as compared with 4xR. (C) Common DEGs in 4xR and 32xR: 75 and 46 genes were commonly downregulated (D) and upregulated (U), respectively. (D) Gene Ontology (GO) Biological Process enrichment of DEGs in 32xR E. coli: Biofilm formation, response to pH, and SOS response were significantly enriched in upregulated DEGs (red), whereas motility and amino acid biosynthesis were enriched in downregulated DEGs (blue).

Figure 2

Cross Talk between Processes Perturbed by TMP Is Identified from 32xTopNet (A) Flow chart showing steps involved in extraction of 32xTopNet from 32xNet. (B) 32xTopNet: filled diamonds (DEGs) and circles (non-DEG genes) represent nodes and lines connecting them are knowledge-based functional interactions. The nodes are colored based on FC. (C) Cross talk between processes perturbed by TMP viz. GASR, motility, biofilm, and folate metabolism inferred from the 32xTopNet. Genes belonging to each process are shown in a different shape. Like in (B), node colors signify extent of upregulation or downregulation.

Figure 3

Comparative Evolution Shows Slower Adaptation to TMP in Absence of glyA (A) Comparative evolution experiment schematic: Dilutions of TMP were prepared in a 96-well plate and inoculated with overnight cultures of BW25113 or BW25113:ΔglyA. Culture from well with the highest TMP concentration was used for inoculating plate for the next day provided growth in that well was comparable with growth in the absence of TMP (A₆₀₀ ≥ A₆₀₀ of corresponding well without TMP). (B) Adaptation trajectories to TMP in six biological replicates of BW25113 (blue) and its corresponding BW25113:ΔglyA (red) over ~180 generations are shown. Each point for a particular number of generations for a particular replicate represents the maximum TMP concentration at which satisfactory growth (A₆₀₀ ≥ A₆₀₀ of corresponding well without TMP) was observed. Adaptation trajectory of each replicate is shown in a dotted line connecting the points for that replicate across all generations. The mean adaptation trajectory for BW25113 or BW25113:ΔglyA is shown in a solid line. (C) Plot shows the mean resistance gained at a particular number of generations for BW25113 or BW25113:ΔglyA. For each replicate, the ratio of concentration at which it grows after a particular number of generations and the concentration at which it grew on the first day (after ~12 generations) is calculated. Thus, each ratio represents the fold increase in resistance. Six ratios are obtained per strain and the mean ± SD of these ratios is shown for a particular number of resistant. Since the number of generations completed every 12 h is roughly the same for the two strains, for the purpose of comparison, the BW25113 ratios have also been plotted using the number of generations obtained for BW25113:ΔglyA. Between ~100 and 140 generations (~8–10.5 days), BW25113 (blue) is significantly more resistant to TMP than BW25113:ΔglyA (red) (p value < 0.05; indicated by ∗).