Elevated levels of the second messenger c‐di‐GMP contribute to antimicrobial resistance of Pseudomonas aeruginosa

Abstract

Biofilms are highly structured, surface-associated communities. A hallmark of biofilms is their extraordinary resistance to antimicrobial agents that is activated during early biofilm development of Pseudomonas aeruginosa and requires the regulatory hybrid SagS and BrlR, a member of the MerR family of multidrug efflux pump activators. However, little is known about the mechanism by which SagS contributes to BrlR activation or drug resistance. Here, we demonstrate that ΔsagS biofilm cells harbor the secondary messenger c-di-GMP at reduced levels similar to those observed in wild-type cells grown planktonically rather than as biofilms. Restoring c-di-GMP levels to wild-type biofilm-like levels restored brlR expression, DNA binding by BrlR, and recalcitrance to killing by antimicrobial agents of ΔsagS biofilm cells. We likewise found that increasing c-di-GMP levels present in planktonic cells to biofilm-like levels (≥55 pmol/mg) resulted in planktonic cells being significantly more resistant to antimicrobial agents, with increased resistance correlating with increased brlR, mexA, and mexE expression and BrlR production. In contrast, reducing cellular c-di-GMP levels of biofilm cells to ≤40 pmol/mg correlated with increased susceptibility and reduced brlR expression. Our findings suggest that a signaling pathway involving a specific c-di-GMP pool regulated by SagS contributes to the resistance of P. aeruginosa biofilms.

Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics

“…The same group identified BrlR as an activator of mexAB-oprM and mexEF-oprN (79) and the two-component hybrid protein SagS as a possible upstream regulator of BrlR (80). Recently, SagS was shown to contribute to BrlR activation and tolerance toward antibiotics through an increase of the level of the second messenger cyclic di-GMP (c-di-GMP) (81).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…Together, SagS, BrlR, and c-di-GMP form an important signaling network related to the susceptibility-resistance switch and are all required for elevated expression in biofilm cells of the MexAB-OprM and MexEFOprN pumps, which contribute to resistance in both biofilm and planktonic cells. It should be noted that similar to those in biofilm cells, increased levels of c-di-GMP in planktonic cells also confer elevated resistance to antibiotics to the cells, and this phenotype correlates well with the hyperexpression of brlR, mexA, and mexE (823). Additionally, a PA1875-PA1877-encoded efflux system in P. aeruginosa is composed of an OM protein (OpmL), an ABC exporter, and a membrane fusion protein (HlyD homolog) and contributes to biofilm-specific resistance to ciprofloxacin, gentamicin, and tobramycin (897).…”

“…Unlike the MerR-type regulators (e.g., BltR and BmrR) that function in the regulation of MDR pumps of Gram-positive bacteria (896), brlR expression is not induced by the pump substrates. The inactivation of SagS correlates with a reduced level of c-di-GMP, and thus, c-di-GMP is positively regulated by SagS (823). c-di-GMP further positively affects the production of BrlR and also enhances the binding of BrlR to the promoters of BrlR target genes (821,823).…”

“…The inactivation of SagS correlates with a reduced level of c-di-GMP, and thus, c-di-GMP is positively regulated by SagS (823). c-di-GMP further positively affects the production of BrlR and also enhances the binding of BrlR to the promoters of BrlR target genes (821,823). Together, SagS, BrlR, and c-di-GMP form an important signaling network related to the susceptibility-resistance switch and are all required for elevated expression in biofilm cells of the MexAB-OprM and MexEFOprN pumps, which contribute to resistance in both biofilm and planktonic cells.…”

Biofilms 2015: Multidisciplinary Approaches Shed Light into Microbial Life on Surfaces

“…Continuing the theme of antibiotic tolerance, Olga Petrova (Binghamton University, Binghamton, NY) presented results suggesting that P. aeruginosa biofilm formation and biofilm tolerance to antibiotics are regulated by distinct signaling cascades, both involving the transcriptional regulator FleQ and the sensor-regulator hybrid SagS, but with discrete c-di-GMP requirements and protein interaction partners. Separating the factors involved in biofilm formation and biofilm tolerance will allow biofilm control strategies by targeting of the two distinct pathways (22).…”