A genetic screen identifies a role foroprFinPseudomonas aeruginosabiofilm stimulation by subinhibitory antibiotics

Abstract

Biofilms are surface-associated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentrations (sub-MIC) of antibiotics induce biofilm formation, indicating a potential defensive response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unclear. We show that treatment ofPseudomonas aeruginosawith multiple classes of sub-MIC antibiotics with distinct targets induces biofilm formation. Further, addition of exogenous eDNA or cell lysate failed to increase biofilm formation to the same extent as antibiotics, suggesting that the release of cellular contents by antibiotic-driven bacteriolysis is insufficient. Using a genetic screen to find stimulation-deficient mutants, we identified the outer membrane porin OprF and the extracytoplasmic function sigma factor SigX as important for the phenotype. Similarly, loss of OmpA - theEscherichia coliOprF homologue - prevented sub-MIC antibiotic stimulation ofE. colibiofilms. The C-terminal PG-binding domain of OprF was dispensable for biofilm stimulation. Our screen also identified the periplasmic disulfide bond-forming enzyme DsbA and a predicted cyclic-di-GMP phosphodiesterase encoded by PA2200 as essential for biofilm stimulation. The phosphodiesterase activity of PA2200 is likely controlled by a disulfide bond in its regulatory domain, and folding of OprF is influenced by disulfide bond formation, connecting the mutant phenotypes. Addition of the reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, we show that activation of a c-di-GMP-responsive promoter follows treatment with sub-MIC antibiotics in the wild-type but not anoprFmutant. Together, these results show that antibiotic-induced biofilm formation is likely driven by a signalling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP.

Metabolic connections between folate and peptidoglycan pathways inPseudomonas aeruginosainform rational design of a dual-action inhibitor

“…Following up on our discovery that a P. aeruginosa oprF mutant is hypersensitive to TMP 27 , we found that this mutant is also hypersensitive to SUL ( Fig 2a ). We next tested the ability of TMP to compromise envelope integrity using the classic method of exposing cells to hyper- or hypo-osmotic stresses.…”

“…We previously discovered that a P. aeruginosa oprF mutant was hypersensitive to TMP, suggesting a possible link between folate and PG metabolism 27 . OprF is a major outer membrane porin with a C-terminal PG-binding domain that anchors the outer membrane to the cell wall 28 .…”