Discovery of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases in Diverse Environmental Settings Suggests an Evolutionary Advantage Unrelated to Antibiotic Resistance

Abstract

Type B dihydrofolate reductases (DfrB) are intrinsically highly resistant to the widely used antibiotic trimethoprim, posing a threat to global public health. The ten known DfrB family members have been strongly associated with genetic material related to the application of antibiotics. Several dfrB genes were associated with multidrug resistance contexts and mobile genetic elements, integrated both in chromosomes and plasmids. However, little is known regarding their presence in other environments. Here, we investigated the presence of dfrB beyond the traditional areas of enquiry by conducting metagenomic database searches from environmental settings where antibiotics are not prevalent. Thirty putative DfrB homologues that share 62 to 95% identity with characterized DfrB were identified. Expression of ten representative homologues verified trimethoprim resistance in all and dihydrofolate reductase activity in most. Contrary to samples associated with the use of antibiotics, the newly identified dfrB were rarely associated with mobile genetic elements or antibiotic resistance genes. Instead, association with metabolic enzymes was observed, suggesting an evolutionary advantage unrelated to antibiotic resistance. Our results are consistent with the hypothesis that multiple dfrB exist in diverse environments from which dfrB were mobilized into the clinically relevant resistome. Our observations reinforce the need to closely monitor their progression.

From a binding module to essential catalytic activity: how nature stumbled on a good thing

“…In contrast, the only conserved feature of the ∼20-residue, unstructured N-terminus of the DfrB protomer is its length. 15,23,44 Consistent with this, the creation of all possible point substitutions in the enzyme through a deep mutational scanning approach has demonstrated that substitutions in the SH3 core of DfrB1 are detrimental to the enzyme fitness, whereas the N-terminus is largely tolerant to sequence alteration. 45 Small-angle neutron scattering, MD simulations and 19 F NMR have determined that the N-terminus adopts a globally compact shape and interacts with Trp45 at the monomer–monomer interface, providing 2.6 kcal mol −1 of stability to the assembly.…”

“…We therefore hypothesized that the DfrB in these environmental organisms were selected for a function other than to provide TMP resistance; whether they procure an evolutionary advantage by serving as an additional dihydrofolate reductase or have another, yet undiscovered function remains to be determined. 44…”

“…2A). 44 Both above and below the center of the tunnel, two pairs of Gln67 and Tyr69 form a hydrogen bonding network that shapes the narrower tunnel centre. 8 NADPH is the first to bind to the enzyme, with a K D of 2.5 mM.…”

“…More recently, we reported an additional set of ten DfrB genes identified in proteobacteria from environments not directly associated with antibiotic use, such as different types of soil, growing the number of confirmed DfrB from eight (prior to 2019) to 20 (in 2022). 44 In most cases, the genes identified alongside the DfrB are unrelated to antibiotic resistance. An independent study found that DfrB genes are abundant in the maize rhizosphere (microbes associated with the roots of corn), suggesting that DfrB are intrinsic to this environment.…”

“…We therefore hypothesized that the DfrB in This journal is © The Royal Society of Chemistry 2023 these environmental organisms were selected for a function other than to provide TMP resistance; whether they procure an evolutionary advantage by serving as an additional dihydrofolate reductase or have another, yet undiscovered function remains to be determined. 44 A factor contributing to the fact that their native function is unknown is that the DfrB are evolutionarily unrelated to any characterized protein. Interestingly, we have identified proteins of unknown function from bacteriophages and proteobacteria that contain a DfrB domain -with all sequence features necessary to yield an active enzyme -within larger proteins of length ranging from 167 to 463 residues.…”