Identification of essential genes that support fitness ofAcinetobacter baumanniiefflux pump overproducers in the presence of fluoroquinolone

Abstract

Acinetobacter baumanniiis a nosocomial pathogen often associated with multidrug resistance (MDR) infections. Fluoroquinolone resistance (FQR) due to drug target site mutations and elevated expression of RND drug transporters is common among clinical isolates. We describe here a CRISPRi platform that identifies hypomorphic mutations that preferentially altered drug sensitivity in RND pump overproducers. An sgRNA library against essential genes ofA. baumanniiwas constructed with single and double nucleotide mutations that produced titratable knockdown efficiencies and introduced into multiple strain backgrounds. Other thannusGdepletions, there were few candidates in the absence of drug treatment that showed lowered fitness specifically in strains overexpressing clinically relevant RND efflux pumps AdeAB, AdeIJK, or AdeFGH. In the presence of ciprofloxacin, the hypomorphs causing hypersensitivity were predicted to result in outer membrane dysfunction, to which the AdeFGH overproducer appeared particularly sensitive. Depletions of either the outer membrane assembly BAM complex, LOS biogenesis proteins, or Lpt proteins involved in LOS transport to the outer membrane caused drug hypersensitivity in at least two of the three pump overproducers. On the other hand, depletions of translation-associated proteins, as well as components of the proton-pumping ATP synthase pump resulted in fitness benefits for at least two pump-overproducing strains in the presence of the drug. Therefore, pump overproduction exacerbated stress caused by defective outer membrane integrity, while the efficacy of drug resistance in efflux overproducers was enhanced by slowed translation or defects in ATP synthesis linked to the control of proton movement across the bacterial membrane.

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens

“…4A; Dataset S4). We tested the effects of no treatment, 100 µM P2-56-3 only, 0.7 µg/mL RIF only, and 0.7 µg/mL RIF with 100 µM P2-56-3 (P2-56-3/RIF) in the presence of stoichiometric CRISPRi-based gene depletions – also known as hypomorphic gene depletions – in A. baumannii ATCC 17978 (31, 32). We looked for hypomorphic gene depletions that induced hypersensitivity to RIF, P2-56-3, and P2-56-3/RIF treatments using the following treatment comparisons: RIF only treatment relative to no treatment, P2-56-3 only treatment relative to no treatment, and P2-56-3/RIF treatment relative to RIF only treatment.…”

“…CRISPRi depletion pools were generated as previously described in A. baumannii ATCC 17978 (32). To briefly summarize, the CRISPRi library contained 12,000 guides targeting 406 essential genes, including one perfect match and 23 mismatched guides per gene and included 2,000 scrambled, non-targeting negative controls (32).…”

“…CRISPRi depletion pools were generated as previously described in A. baumannii ATCC 17978 (32). To briefly summarize, the CRISPRi library contained 12,000 guides targeting 406 essential genes, including one perfect match and 23 mismatched guides per gene and included 2,000 scrambled, non-targeting negative controls (32). The mismatched guides were designed with 1 or 2 mismatches at varying distances from the PAM site to allow for varying fitness defects and thus stoichiometric gene depletions.…”

“…We acquired an A. baumannii ATCC 17978 with an aTc inducible dCas9 gene inserted at the chromosomal att Tn-7 site downstream of glmS locus (32). sgRNA inserts were designed to be 24 nucleotides with TAGT (top) and AAAC (bottom) sticky ends.…”