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Transcriptome-Based Design of Antisense Inhibitors Re-Sensitizes CRE E bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878389; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Transcriptome-based design of antisense inhibitors re-sensitizes CRE E. coli to 2 carbapenems 3 Thomas R. Aunins1,#, Keesha E. Erickson1,#, and Anushree Chatterjee1,2,3* 4 #These authors contributed equally 5 *Corresponding author: Email: [email protected]. Phone: (303) 735-6586, Fax: (303) 492- 6 8424 7 8 1Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder 9 Colorado, USA 10 2Biomedical Engineering Program, University of Colorado Boulder, USA. 11 3Antimicrobial Regeneration Consortium, Boulder, USA. 1 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878389; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 12 Abstract 13 Carbapenems are a powerful class of antibiotics, often used as a last-resort treatment to eradicate 14 multidrug-resistant infections. In recent years, however, the incidence of carbapenem-resistant 15 Enterobacteriaceae (CRE) has risen substantially, and the study of bacterial resistance 16 mechanisms has become increasingly important for antibiotic development. Although much 17 research has focused on genomic contributors to carbapenem resistance, relatively few studies 18 have examined CRE pathogens through changes in gene expression. In this research, we used 19 transcriptomics to study a CRE Escherichia coli clinical isolate that is sensitive to meropenem but 20 resistant to ertapenem, to both explore carbapenem resistance and identify novel gene 21 knockdown targets for carbapenem re-sensitization. We sequenced total and small RNA to 22 analyze gene expression changes in response to treatment with ertapenem or meropenem, as 23 compared to an untreated control. Significant expression changes were found in genes related to 24 motility, maltodextrin metabolism, the formate hydrogenlyase complex, and the general stress 25 response. To validate these transcriptomic findings, we used our lab’s Facile Accelerated Specific 26 Therapeutic (FAST) platform to create antisense peptide nucleic acids (PNA), gene-specific 27 molecules designed to inhibit protein translation. FAST PNA were designed to inhibit the 28 pathways identified in our transcriptomic analysis, and each PNA was then tested in combination 29 with each carbapenem to assess its effect on the antibiotics’ minimum inhibitory concentrations. 30 We observed significant treatment interaction with five different PNAs across six PNA-antibiotic 31 combinations. Inhibition of the genes hycA, dsrB, and bolA were found to re-sensitize CRE E. coli 32 to carbapenems, whereas inhibition of the genes flhC and ygaC was found to confer added 33 resistance. Our results identify new resistance factors that are regulated at the level of gene 34 expression, and demonstrate for the first time that transcriptomic analysis is a potent tool for 35 designing antibiotic PNA. 2 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878389; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 36 Keywords: Carbapenem-resistant E. coli, transcriptome, small RNA sequencing, genome 37 sequence, antibiotic resistance. 3 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878389; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 38 Introduction 39 In recent years, the emergence of carbapenem-resistant Enterobacteriaceae (CRE) has 40 been marked by the World Health Organization (WHO) as a critical priority for antibiotic 41 development.1 Resistance to carbapenems, a subclass of the cell wall-targeting β-lactams that 42 are often termed antibiotics of last resort,2 has become increasingly common in recent decades,3– 43 5 resulting in rising threats of infection mortality.6–9 In 2017, the Centers for Disease Control 44 estimated that, in the U.S., 13,100 infections and 1,100 deaths per year were caused by 45 carbapenem-resistant Enterobacteriaceae alone.10 Furthermore, the WHO has called for further 46 investment in both basic research and clinical development for CRE pathogens, as the current 47 pipeline is not sufficient to address the threat.11 Our study seeks to use transcriptomics both to 48 better understand carbapenem resistance and to design new methods to combat its development. 49 Each of these aims is facilitated by the application of our Facile Accelerated Specific Therapeutic 50 (FAST) platform, a semi-automated pipeline for the design, synthesis, and testing of antisense 51 peptide nucleic acids (PNA)—synthetic DNA analogues with peptide backbones that can inhibit 52 translation on a gene-specific basis. Here, FAST PNA allow us to validate our conclusions from 53 our transcriptomic analysis, and to restore carbapenem susceptibility to a CRE clinical isolate. 54 Structural modifications, particularly a trans-α-1-hydroxylethyl substituent at position 6, are 55 believed to endow carbapenems with higher stability against β-lactamases,12–14 as well as broader 56 observed efficacy,15,16 compared with other β-lactam drugs. However, increasing prevalence of 57 carbapenemase enzymes in Enterobacteriaceae—such as oxacillinase-48, metallo-β- 58 lactamases, and Klebsiella pneumoniae carbapenemases—provide mechanisms for the 59 development of resistance.3,5,17,18 Multiple studies have found that pathogenic bacteria without 60 carbapenemases can acquire carbapenem resistance through the combination of β-lactamases 61 and outer membrane porin deficiencies (neither factor alone was sufficient for resistance),19–23 4 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878389; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 62 through the expression of low-affinity or mutated penicillin-binding proteins,24–27 or through the 63 overproduction of efflux pumps.28,29 64 Other research has sought to understand resistance by tracking transcriptomic changes 65 in carbapenem-resistant pathogens. Two studies on the gene expression profile of carbapenem- 66 resistant Acinetobacter baumanii found significant upregulation of transposable elements, 67 recombinase, and other mutation-encouraging factors, in addition to the expected upregulation of 68 efflux pump and β-lactamase genes.30,31 Four recent studies that examined transcriptomic profiles 69 of CRE—Enterobacter cloacae, K. pneumoniae (2), and Escherichia coli—identified less 70 consistent responses, although downregulation of porin genes and upregulation of cell survival 71 and β-lactamase genes were observed.32–35 Of this prior research, only one study32 attempts to 72 evaluate the transcriptomic response of a resistant Enterobacteriaceae strain under antibiotic 73 challenge, the majority examining only the constitutive gene expression levels of an untreated 74 resistant pathogen. 75 In this study, we both explore and counter carbapenem resistance by examining the short- 76 term (<1 hr) transient transcriptomic response of a clinical isolate of multidrug resistant (MDR) E. 77 coli (referred to from here as E. coli CUS2B) to carbapenem treatment. The strain demonstrates 78 resistance to ertapenem, but sensitivity to meropenem and doripenem, and we sought to use this 79 partial carbapenem resistance phenotype to help understand the development of resistance in 80 Enterobacteriaceae. We used a combination of total and small RNA sequencing to assess the 81 response of E. coli CUS2B to either ertapenem or meropenem treatment and identify genes that 82 were potentially important to the pathogen’s resistance profile. We then used these genes, 83 together with genome sequencing of the clinical isolate, as inputs for the FAST platform.36 For 84 each target gene, we designed and synthesized PNA molecules that were predicted to inhibit 85 translation via sequence-specific binding to the mRNA translation initiation region. Growth assays 86 of PNA-carbapenem combination treatments were used to validate each gene’s relevance to the 5 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878389; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 87 resistance phenotype, and to determine whether PNA designed using transcriptomics could re- 88 sensitize E. coli CUS2B to sub-MIC carbapenem concentrations. 6 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.16.878389; this version posted December 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 89 Results 90 E. coli CUS2B: a multidrug-resistant Enterobacteriaceae with partial carbapenem resistance 91 To validate the E. coli CUS2B resistance phenotype observed in the clinic, we measured 92 the isolate’s minimum inhibitory concentrations
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