Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli

Abstract

Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.

Keywords: CRISPR/Cas9; acquired resistance; deoxyxylulose phosphate reductoisomerase; fosmidomycin; malaria; sequence to activity mapping.

Fig. 1.

A schematic representation of the experimental procedure. A, an individual CREATE cassette includes a homology arm, harboring both the target site and adjacent mutations, and the corresponding gRNA. B, 660 such cassettes were synthesized on an array and were inserted into a plasmid backbone in a single multiplex cloning reaction (C). D, Plasmid library was transformed in triplicate into E. coli cells with the transcription induction of Cas9 and the recombineering machinery. Following 2 hours of recovery (E), cells were either plated on FSM-containing plates (F), or cultured overnight with (G) or without (H) the presence of FSM. Samples for deep sequencing were taken from E, G, and H. For a more detailed CREATE cassette, please refer to Fig. S1.

Fig. 2.

A, the DXR target sites. The DXR structure is shown in gray (PDB #1q0l). Red residues represent mutated sites enriched following FSM incubation, while the green amino acids represent the rest of the library. FSM is colored orange, and NADPH is in blue. B, eight different mutations were isolated from FSM-resistant colonies (step F in Fig.1). The CREATE plasmids were isolated, retransformed into fresh E. coli cells and treated with FSM. Optical density was measured following 24 hours of incubation. Data represent three independent experiments. C. library cells growth in the presence of FSM. Optical density was measured 16 hours following FSM addition. Results correspond to steps G and H in Fig. 1. Control cells were edited in the E. coli galK gene which is not related to growth in LB or FSM resistance.

Fig. 3.

Enrichment analysis of library cells at the genomic level. A, enrichment histogram of all three repeats. B-D, Enriched mutants from repeats 1–3, respectively. The X-axis represents the DXR sequence. The dashed line represents the average of all data points, and the solid lines are two standard deviations from the average. Mutants with enrichment values exceeding two standard deviations are labeled. For clarity, the randomly selected internal control (G14) was omitted and can be found in Fig. S5. A similar analysis was also performed using the CREATE reporter plasmid and can be found in Fig. S4.

Fig. 4.

Saturation data of the P274 residue. A, The X-axis represents the final amino acids replacing the wild-type proline and are ordered according to the average value of the three repeats. Y-axis represents the enrichment values of each mutant. B, Growth response curve to FSM of the P274K, P274M, and P274R mutants relative to wild-type DXR and the previously reported S222T mutant by Armstrong et al. The data points and error bars represent mean ± standard error of the mean (SEM). Data shown represents three independent replicates. EC₅₀ values derived from these curves are shown in Table S2.