Molecular mechanism of nitric oxide-mediated regulation of intracellular cyclic-di-GMP in Pseudomonas aeruginosa biofilms

Abstract

Biofilms are defined as multicellular communities encased by a self-produced extracellular matrix. It is now well understood that most bacteria found in natural, clinical and industrial settings preferentially attach to surfaces or adhere to each other to grow as biofilms, causing serious problems due to their tolerance to conventional antibiotics. Previous studies have shown that low dose nitric oxide (NO) can trigger Pseudomonas aeruginosa biofilm dispersal by modulating the level of the intracellular secondary messenger cyclic dimeric guanosine monophosphate (c-di-GMP). Diguanylate cyclase (GGDEF motif) and phosphodiesterase (EAL/HD-GYP motif) activities are responsible for the synthesis and hydrolysis of c-di-GMP, respectively. Various sensor domains have been found to link environmental cues to modulation of GGDEF and EAL/HD-GYP activities, of which PAS and MHYT domains were of our interest due to their potentials to bind NO. In P. aeruginosa PAO1, a total of 14 proteins containing either PAS-DGC+/PDE or MHYT-DGC+/PDE were thought to be responsible for the NO-induced biofilm dispersal and were selected as our targets for investigation of their relationships between NO responses and biofilm phenotypes.

To investigate the response of P. aeruginosa biofilms to NO, a range of NO donors were first tested for their efficacies, of which 250µM Spermine NONOate (S150) showed outstanding results in dispersing ~60% batch cultured PAO1 biofilms within only 2 hrs. S150 was further applied to some cystic fibrosis P. aeruginosa clinical isolates (CF PA) biofilms in vitro. The results showed it successfully triggered the dispersal of surface-attached biofilms formed by 14 out of 17 CF PA strains tested, implying its potential for wide applications in clinical settings. However, S150 failed to disperse the non-attached cell aggregates formed by 4 CF PA in aqueous medium, suggesting a different mechanism might exist for clinical isolates to defend the drugs that requires much more attention. In order to facilitate future studies, a quantifying index, Concentration Coefficient, was proposed to evaluate the degree of cell aggregation by simply using one stacked CLSM image for a certain planktonic culture. This index may become widely applied for researchers to compare the cell aggregates more easily and accurately.

For mechanism studies, gene deletion was applied to 14 candidates for phenotypic analysis of mutants. An efficient gene knockout technique that minimizes cloning steps was developed and allowed for rapid generation of mutants. Phenotypic assays for 14 mutants suggested that PA0861 (RbdA) and PA5017 (DipA) play central roles in P. aeruginosa PAO1 for reducing intracellular c-di-GMP levels, enhancing swarming motility and triggering biofilm dispersal in response to NO. PA0847 and PA4601 (MorA) are involved in the regulation of biofilm dispersal and swarming/swimming motility, which were suspected to come from localized c-di-GMP pools due to their altered motility phenotypes without changes in intracellular levels. PA0285 and PA4959 (FimX) are responsible for both twitching and swimming motility, which contribute greatly to the 3D structures of the biofilms formed. Deleting either of these two proteins led to much enhanced biofilm dispersal upon NO treatment, providing insight that the deficiency in both Type IV pili and flagella functions may facilitate the elimination of P. aeruginosa biofilms.

In summary, evaluation of a broad range of commercially available NO donors showed that S150 was the most effective in dispersing laboratory and clinical P. aeruginosa strains in our experimental system. By using S150 and gene-deleted mutants, our research suggested several models whereby different proteins may be responsible for either coarse-tuning on intracellular c-di-GMP pools or fine-tuning on localized ones in response to NO, which collaboratively regulate the motility and biofilm dispersal in PAO1. This work has enhanced our understanding of the NO-c-di-GMP-swarming-dispersal pathway and its regulators in PAO1, shedding light on NO signaling mechanisms and providing potential new targets for therapeutic drug design.

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Identifiers

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

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