Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

Abstract

The cell envelope of the Gram-negativeBurkholderia cepaciacomplex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals, such as those living with cystic fibrosis. To systematically identify cell envelope-associated resistance and susceptibility determinants at the genome level, we constructed a high-density, randomly-barcoded transposon mutant library in the clinical isolateB. cenocepaciaK56-2 and exposed it to a panel of more than twenty cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profiled over a hundred new chemical-genetic interactions and identified novel mediators of antibiotic susceptibility in the Bcc cell envelope. We first reveal new connections between broad-spectrum β-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism, highlighting a β-lactam critical vulnerability associated with sharing this common lipid intermediate. We then show that the clinically relevant synergy of the β-lactam/ β-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase, over more than twenty other putative β-lactamases. Importantly, avibactam more strongly potentiates the activity of aztreonam and meropenem than ceftazidime in a panel of Bcc clinical isolates. Finally, we characterize for first time in the Bcc the iron and receptor-dependent activity of the novel siderophore cephalosporin antibiotic, cefiderocol. Overall, our work has implications for antibiotic target prioritization, and for using additional combinations of β-lactam/ β-lactamase inhibitors that can extend the utility of our current clinical arsenal of antibacterial therapies.

CRISPRi-Mediated Silencing of Burkholderia O-Linked Glycosylation Systems Enables the Depletion of Glycosylation Yet Results in Modest Proteome Impacts

“…Recently, it was highlighted within B. cenocepacia that O-linked glycosylation appears to impact antibiotic resistance, with transposon mutants demonstrating altered susceptibility to several antibiotics, yet validation of these observations using CRISPRi knockdowns of pglL failed to recapitulate this phenotype. 81 Our observations here reconcile these findings and provide a rationale for why CRISPRi silencing of pglL does not mirror the phenotypes of ΔpglL strains and reveals that Burkholderia O-linked glycosylation possesses a previously unrecognized level of robustness.…”

“…While many bacterial glycosylation studies have explored the proteomic impacts of abolishing glycosylation using mutagenesis, ,,,,, few studies have used CRISPRi silencing to modulate glycosylation, and importantly, no study has explored the impact of silencing on glycosylation levels. Our observation that glycosylation persists even with silencing of PglL below the limits of detection supports the notion that within Burkholderia species, even low levels of oligosaccharyltransferase capacity maybe sufficient to prevent the defects observed in glycosylation mutants.…”