Transcriptome-based design of antisense inhibitors potentiates carbapenem efficacy in CRE Escherichia coli

Abstract

In recent years, the prevalence of carbapenem-resistantEnterobacteriaceae(CRE) has risen substantially, and the study of CRE resistance mechanisms has become increasingly important for antibiotic development. Although much research has focused on genomic resistance factors, relatively few studies have examined CRE pathogens through changes in gene expression. In this study, we examined the gene expression profile of a CREEscherichia coliclinical isolate that is sensitive to meropenem but resistant to ertapenem to explore transcriptomic contributions to resistance and to identify gene knockdown targets for carbapenem potentiation. We sequenced total and short RNA to analyze the gene expression response to ertapenem or meropenem treatment and found significant expression changes in genes related to motility, maltodextrin metabolism, the formate hydrogenlyase complex, and the general stress response. To validate these findings, we used our laboratory’s Facile Accelerated Specific Therapeutic (FAST) platform to create antisense peptide nucleic acids (PNAs), gene-specific molecules designed to inhibit protein translation. PNAs were designed to inhibit the pathways identified in our transcriptomic analysis, and each PNA was then tested in combination with each carbapenem to assess its effect on the antibiotics’ minimum inhibitory concentrations. We observed significant PNA–antibiotic interaction with five different PNAs across six combinations. Inhibition of the geneshycA,dsrB, andbolApotentiated carbapenem efficacy in CREE. coli, whereas inhibition of the genesflhCandygaCconferred added resistance. Our results identify resistance factors and demonstrate that transcriptomic analysis is a potent tool for designing antibiotic PNA.

Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria

“…Further, in contrast with previous research that showed a higher degree of antibiotic synergy between antisense oligonucleotides and small molecule drugs when the treatments targeted related pathways 46,47 , our results indicate that unrelated mechanisms of inhibition can also result in combination effects in MDR pathogens 27 . This phenomena has also been shown by our previous works highlighting how multiplexed perturbations have a compounding effect on bacterial fitness 48 and that fitnessneutral gene perturbations can have synergistic effects across similar or different pathways 49 .…”

“…In this study we applied the FAST platform 27 for the creation of PNA antibiotics against five MDR Enterobacteriaceae clinical isolates: two Escherichia coli isolates, two Klebsiella pneumoniae isolates, and one Salmonella enterica serovar Typhimurium isolate. To design the set of candidate sequences, the Get Sequences tool was used with a list of 296 essential and 4090 nonessential E. coli genes, as well as the genome assembly and annotations for reference strain E. coli MG1655 (Fig.…”

“…In addition, the inefficient transport of PNA across cell membranes, as well as the lack of a systematic effort to target non-traditional antibiotic pathways, has limited the development of PNA as useful antibiotics. We address these limitations through the introduction of Facile Accelerated Specific Therapeutic (FAST) 27 , a semi-automated platform for the quick and efficient design, synthesis, testing, and delivery of PNA antisense antibiotics (Fig. 1).…”

Silencing Antibiotic Resistance with Antisense Oligonucleotides

“…A recent approach to designing antisense compounds was recently reported. It consists of a novel semiautomated pipeline for the design, synthesis, and testing of antisense PNAs (Facile Accelerated Specific Therapeutic, FAST) [ 95 ]. This methodology is not only rapid, but it results in highly specific antisense sequences.…”

“…Genes that showed significant expression levels in response to the presence of the antibiotic were selected as targets for FAST design of antisense PNA compounds, which were conjugated to a CPP for testing. Antisense-mediated inhibition of the genes hycA (regulatory protein in the formate hydrogenlyase system), dsrB (unknown function), and bolA (transcriptional regulator of several genes) potentiated carbapenem efficacy while inhibition of the genes flhC (transcriptional regulator of motility related functions) and ygaC (uknown function) resulted added resistance [ 95 ].…”