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 a 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.

Predicting Drug Resistance Using Deep Mutational Scanning

“…Following the construction of the genome-edited library, the cells were incubated in the presence of FSM to enrich for mutations that confer resistance, then were deep-sequenced to identify the mutations. Indeed, several mutations that induce FSM resistance were identified [40]. Importantly, thanks to the conserved nature of dxr, the identified sites also exist in other organisms, including P. falciparum and P. vivax strains (Figure 4C).…”

“…Examples of such approaches involving the genomic incorporation of synthetic libraries utilizing CRISPR/Cas9 systems as a selection tool are CRISPR-Enabled Trackable Genome Engineering (CREATE) and HI-CRISPR, among others [35][36][37][38][39]. Using the CREATE technology, we were able to search for point mutations in the ispC/dxr gene that confer resistance to the antimalarial fosmidomycin (3-(N-formyl-N-hydroxyamino)propylphosphonic acid (FSM), uncovering mutations not elucidated in previous error-prone PCR strategies (see below) [40,41].…”

“…While complete saturation mutagenesis, allowing access to the entire amino acid repertoire, may be necessary for basic studies and protein engineering efforts, for the study of naturally-occurring resistance phenotypes, it may only be required to restrict the library to single base changes, thus reducing downstream labor and cost. Another approach to reducing library size is to rationally select the targets for mutagenesis rather than systematically mutating the complete gene [40]. Such rationally designed libraries are only possible when a large enough body of knowledge is present, as described above.…”

Potential repurposing of four FDA approved compounds with antiplasmodial activity identified through proteome scale computational drug discovery and in vitro assay

“…Hydrogen mass repartitioning (HMR) was applied to the proteins and ligands: masses of hydrogens bound to heavy atoms were repartitioned allowing an accelerated 4-fs time step. HMR has been shown to accelerate MD simulations without loss of accuracy [71][72][73][74] . Ligand topologies were generated using ACPYPE 75 with their charges obtained from Discovery Studio Visualizer V1.7, also used to analyse protein-ligand interactions.…”

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

“…Finally, each gRNA is coupled to a different synonymous PAM mutation (SPM) and synonymous mutations can lead to significant fitness effects, especially in essential genes (Lind et al, 2010;Agashe et al, 2013;Lajoie et al, 2013a). Because of these limitations, CREATE experiments have largely been limited to finding mutants with large fitness effects in the presence of strong selective pressures (Bassalo et al, 2018;Pines et al, 2018).…”