Engineering a CRISPR interference system targeting AcrAB-TolC efflux pump to prevent multidrug resistance development in Escherichia coli

Abstract

Objectives We engineered a CRISPR interference (CRISPRi) system targeting the AcrAB-TolC efflux pump to prevent MDR development in Escherichia coli. Methods Nine specific single-guide RNAs (sgRNAs) were designed to target the components of the AcrAB-TolC efflux pump, namely AcrA, AcrB and TolC. A total of thirteen CRISPRi recombinant plasmids were constructed with single or clustered sgRNAs. The transcriptional levels of the target genes, MICs of multiple antibiotics and biofilm formation in each CRISPRi strain were tested. Results The CRISPRi system expressing sgRNA clusters targeting acrB and tolC simultaneously exhibited the highest inhibitory effect on AcrAB-TolC efflux pump activity in E. coli HB101, with 78.3%, 90.0% and 65.4% inhibition rates on the transcriptional levels of acrA, acrB and tolC, respectively. The CRISPRi system resulted in ∼2-, ∼8- and 16-fold increased susceptibility to rifampicin, erythromycin and tetracycline, respectively. In addition, the constructed CRISPRi system reduced biofilm formation with inhibition rates in the range of 11.2% to 58.2%. Conclusions To the best of our knowledge, this is the first report on the construction of an inducible CRISPRi system targeting the AcrAB-TolC efflux pump to prevent MDR development in E. coli. This study provides insights for future regulation and manipulation of AcrAB-TolC activity and bacterial MDR by a CRISPRi system.

RND Pump-Mediated Efflux of Amotosalen, a Compound Used in Pathogen Inactivation Technology to Enhance Safety of Blood Transfusion Products, May Compromise Its Gram-Negative Anti-Bacterial Activity

“…The magnitude of MIC reduction was similar to that observed for positive controls, clindamycin, fusidic acid, ethidium bromide, and minocycline, known efflux substrates ( 33 , 36 , 37 ). As expected, based on prior observations ( 36 ), non-effluxed, gentamicin and apramycin aminoglycosides, and meropenem; and poorly effluxed, rifampin negative controls showed minimal or no TolC-dependent change in MIC ( 38 – 40 ). Furthermore, phenylalanine-arginine β-naphthylamide (PAβN), a competitive inhibitor of RND efflux pumps ( 41 ), reduced the MICs of psoralens including amotosalen in the wild-type strain by approximately 4-fold, but without significant additional effect on the tolC mutant, also supporting a major contribution of RND efflux pumps to observed psoralen resistance in the E. coli K-12 strain background.…”

CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance

“…As seen in Table , some of the most commonly targeted genes are related to antibiotic-inactivation enzymes like beta-lactamases ( bla genes) as well as genes related to membrane structure and cell permeability ( cmr-1 gene) . The simultaneous use of multiple gRNAs has also been implemented to target complex antibiotic resistance mechanisms like the AcrAB-TolC efflux pump system ( AcrA , AcrB , and TolC genes) in Escherichia coli …”

“…The CRISPRi system was first derived from a catalytically inactive Cas9 protein, which causes transcription blockage . This approach can be repurposed to repress gene expression of important genes involved in antimicrobial resistance (AMR) and pathogenicity. , Ultimately, most of these studies have still favored the CRISPR-Cas9 mechanisms over others to design their antimicrobial system in vitro and in vivo (Table ).…”

“…43 The simultaneous use of multiple gRNAs has also been implemented to target complex antibiotic resistance mechanisms like the AcrAB-TolC efflux pump system (AcrA, AcrB, and TolC genes) in Escherichia coli. 44 The existing body of literature has described three primary mechanisms of how CRISPR-Cas can mediate antimicrobial activity, these include double-strand chromosomal cleavage, global RNA degradation, and inhibition of essential gene expression in the target bacteria. 45−47 One of the first attempts to create a CRISPR-Cas antibacterial agent used the wellknown CRISPR-Cas9 nuclease system (Type I) to induce specific chromosomal DSBs and reported potent bacterial killing by targeting diverse locations throughout the genome.…”

Effects of TolC on the pathogenicity of porcine extraintestinal pathogenic Escherichia coli

“…In addition to the TolC membrane protein, which is part of the efflux protein of the E. coli outer membrane (OEP), it plays a crucial role in the maintenance of both the structure and function of expulsion of many compounds ( 17 , 18 ). TolC is essential not only for exporting large proteins but also for the efflux of small compounds.…”