Divide and conquer: the Pseudomonas aeruginosa two‐component hybrid SagS enables biofilm formation and recalcitrance of biofilm cells to antimicrobial agents via distinct regulatory circuits

Abstract

The opportunistic pathogen Pseudomonas aeruginosa forms antimicrobial resistant biofilms through sequential steps requiring several two-component regulatory systems. The sensor-regulator hybrid SagS plays a central role in biofilm development by enabling the switch from the planktonic to the biofilm mode of growth, and by facilitating the transition of biofilm cells to a highly tolerant state. However, the mechanism by which SagS accomplishes both functions is unknown. SagS harbors a periplasmic sensory HmsP, and phosphorelay HisKA and Rec domains. We used SagS domain constructs and site-directed mutagenesis to elucidate how SagS performs its dual functions. We demonstrate that HisKA-Rec and the phospho-signaling between SagS and BfiS contribute to the switch to the biofilm mode of growth, but not to the tolerant state. Instead, expression of SagS domain constructs harboring HmsP rendered ΔsagS biofilm cells as recalcitrant to antimicrobial agents as wild-type biofilms, likely by restoring BrlR production and cellular c-di-GMP levels to wild-type levels. Restoration of biofilm tolerance by HmsP was independent of biofilm biomass accumulation, RsmA, RsmYZ, HptB, and BfiSR-downstream targets. Our findings thus suggest that SagS likely makes use of a “divide-and-conquer” mechanism to regulate its dual switch function, by activating two distinct regulatory networks via its individual domains.

The PA3177 Gene Encodes an Active Diguanylate Cyclase That Contributes to Biofilm Antimicrobial Tolerance but Not Biofilm Formation by Pseudomonas aeruginosa

“…BrlR is a c-di-GMP responsive transcriptional regulator (35). Previous findings demonstrated BrlR to be absent or detectable at only low levels in total extracts characterized by low c-di-GMP levels, and unable to bind to its target promoters P brlR , P mexA , and P mexE , resulting in significantly increased susceptibility but reduced brlR, mexA, and mexE transcript levels (17,(33)(34)(35)(36). However, BrlR levels and BrlR-DNA binding was restored upon or overexpression of a diguanylate cyclase or the addition of c-di-GMP to the binding assay, respectively (17,35).…”

“…By analyzing biofilms by P. aeruginosa mutant strains characterized by various intracellular levels of c-di-GMP and strains overexpressing diguanylate cyclases and phosphodiesterases, Gupta et al (17) demonstrated that biofilm cells remain resistant to antimicrobial agents at c-di-GMP levels above 62 pmol/mg but not at or below 40 pmol/mg. These findings suggested a switch in susceptibility in a manner dependent on the c-di-GMP level but independent of the mode of growth or biofilm biomass accumulation (17,33). Moreover, the findings suggested a disconnect between the biofilm architecture and biofilm tolerance (33).…”

“…These findings suggested a switch in susceptibility in a manner dependent on the c-di-GMP level but independent of the mode of growth or biofilm biomass accumulation (17,33). Moreover, the findings suggested a disconnect between the biofilm architecture and biofilm tolerance (33). Additional evidence of c-di-GMP contributing specifically to biofilm drug tolerance stems from two P. aeruginosa proteins required for biofilm drug tolerance: the two-component sensory protein SagS and the transcriptional regulator BrlR.…”

“…Inactivation of sagS rendered biofilms as susceptible as planktonic cells to various antimicrobial agents, including tobramycin, norfloxacin, and hydrogen peroxide (34). SagS contribution to biofilm tolerance was dependent on c-di-GMP, with ΔsagS mutant biofilms demonstrating cellular c-di-GMP levels reminiscent of those present in planktonic cells (17,33,34). Moreover, ΔsagS mutant biofilms were characterized by significantly reduced brlR transcript and BrlR protein levels (17,33,34).…”

“…SagS contribution to biofilm tolerance was dependent on c-di-GMP, with ΔsagS mutant biofilms demonstrating cellular c-di-GMP levels reminiscent of those present in planktonic cells (17,33,34). Moreover, ΔsagS mutant biofilms were characterized by significantly reduced brlR transcript and BrlR protein levels (17,33,34). BrlR is a c-di-GMPresponsive transcriptional regulator that in turn activates the expression of several multidrug efflux pumps and ABC transporters (35)(36)(37)(38), with the ABC transport system PA1874-77 directly contributing to the drug tolerance of biofilms (38).…”