Unraveling the thermoregulation of biofilm formation in pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a ubiquitous Gram-negative opportunistic pathogen. The unusually large genome size and diverse genotype confer this species extraordinary adaptability to inhabit various environments and infect a great number of hosts. Compounding the burden, P. aeruginosa has a strong capability of forming biofilms, a state of matrix encased multicellular aggregation, which protects bacteria from various physical, chemical, and therapeutic treatments. During infection, P. aeruginosa frequently encounters temperature fluctuations as it must transfer from the hospital environment (room temperature, RT, 20–25°C) to the human host (37°C). However, currently, little is known about the effect of temperature on the capacity of biofilm formation in P. aeruginosa. In this study, I first screened the biofilm formation capabilities of 54 clinical P. aeruginosa strains and found that 63% of them formed more than 2-fold higher biofilm biomass at RT (21°C) than 37°C, suggesting that the temperature-responsive biofilm formation represents a previously unrecognized, common pathoadpation strategy in clinical P. aeruginosa. I then set out to investigate this clinically significant phenomenon and its underlying regulatory mechanisms using P. aeruginosa ATCC 27853, which displayed 9.6-fold higher biofilm biomass at RT than 37°C in the crystal violet staining-based assay, as a paradigm. Scanning electron microscopy (SEM), gene and protein expression, and immunostaining analysis showed the enhanced production of the extracellular matrix Psl was primarily responsible for the enhanced biofilm formation in ATCC 27853 at RT. To investigate the underlying regulatory mechanism, I deleted fleQ, a c-di-GMP effector transcriptional factor that regulates psl expression, and found that psl transcription and biofilm formation at RT was significantly decreased in the resulting strain. LC-MS/MS detected a 4-fold higher c-di-GMP level at RT than at 37°C in ATCC 27853, suggesting that the c-di-GMP/FleQ pathway is crucial for temperature-responsive biofilm formation. RNA-seq analysis showed that expression of 17 diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) encoding genes, which are involved in c-di-GMP homeostasis, were differentially expressed (DE) at the two temperatures. I then constructed clean, single-gene deletion of 15 DE and 4 more genes containing GGDEF, GGDEF-EAL hybrid, EAL, and HD-GYP domains in ATCC 27853. Dynamic biofilm formation of the resulting cells showed a strong dependence of temperature-responsive biofilm formation on SiaD. SiaD encodes a DGC protein which activity is triggered by the binding of unphosphorylated SiaC in the SiaABCD pathway dependent on the reversible activity of the phosphatase SiaA and the kinase SiaB. Consistently, ΔsiaA, ΔsiaC, or overexpression siaB led to 50–90% reduction of biofilm formation at RT in ATCC 27853. Phos-tag SDS-PAGE demonstrated that SiaC exists as an unphosphorylated state at RT and a phosphorylated state at 37°C, confirming the activation of the SiaABCD pathway at RT which leads to activation of the DGC SiaD and enhanced biofilm formation through the c-di-GMP/FleQ/Psl pathway. Applying of a membrane-sensitive PI-BactD probe showed that temperature downshift from 37°C to RT resulted in a dramatic change on the membrane features of ATCC 27853 as opposed to a minimal membrane perturbation in the reference strain PAO1, suggesting that the hyper-sensitivity of the membrane of ATCC 27853 to temperature shift may be responsible for the activation of the SiaABCD pathway. Together, these studies not only provide insights into the previously unrecognized thermoregulation of biofilm formation in P. aeruginosa but also provide molecular basis for controlling biofilm formation through temperature shift.