Choline metabolism modulates cyclic-di-GMP signaling and virulence of Pseudomonas aeruginosa in a macrophage infection model

Abstract

Background: Bacterial pathogens frequently encounter host-derived metabolites during their colonization and invasion processes, which can serve as nutrients, antimicrobial agents, or signaling molecules for the pathogens. The essential nutrient choline (Cho) is widely known to be utilized by a diverse range of bacteria and may undergo conversion into the disease-associated metabolite trimethylamine (TMA). However, the impact of choline metabolism on bacterial physiology and virulence remains largely unexplored.

Methods: Here, we employed an in vitro infection model to investigate the role of Cho in intracellular survival and virulence of Pseudomonas aeruginosa (P. aeruginosa). Additionally, a comprehensive RNA-seq based transcriptomic analysis and various phenotypic assays were performed to elucidate the impacts of Cho on P. aeruginosa.

Results: We observed that the Cho metabolite glycine betaine (GB) effectively reduced intracellular levels of cyclic-di-GMP (c-di-GMP). Supplementation of Cho or GB in P. aeruginosa had thus affected c-di-GMP regulated phenotypes, such as pyoverdine production, biofilm formation, and mobility. Depletion of Cho metabolism through knockout of the betAB operon resulted in compromised intracellular survival of P. aeruginosa. Notably, the P. aeruginosa betAB mutant elicited a more robust protective inflammatory response compared to the wild-type strain.

Conclusion: Our study showed that P. aeruginosa Cho metabolism not only interferes host nutritional immunity, but also directly affect multiple virulence phenotypes through modulation of c-di-GMP signaling.

Keywords: Pseudomonas aeruginosa; Biofilm; Choline metabolism; Cyclic-di-GMP; Virulence.

Fig. 1

Impact of Cho metabolism on P. aeruginosa survival and virulence. Effect of betAB deletion on the virulence (lactate dehydrogenase release, LDH) and intracellular survival (colony forming units, CFU) on P. aeruginosa (A-B). Effect of gbcAB deletion on the virulence (LDH) and intracellular survival (CFU) on P. aeruginosa (C-D). Statistical analysis was done using One way-ANOVA. Significance * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001. n.s. = not significant. The data represent the mean ± SD of four biological replicates

Fig. 2

Effects of GB on the intracellular survival and virulence of P. aeruginosa. Raw264.7 cells were infected with mid-log phase of PAO1 that cultured with multiple concentrations of GB for 3 h. The virulence and intracellular survival rate were evaluated by LDH release (A) and CFU (B) respectively. Statistical analysis was performed by One-way ANOVA. Significance * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.The data represent the mean ± SD of three biological replicates

Fig. 3

Transcriptomic analysis for the impact of GB on P. aeruginosa. Volcano chart of changed genes between 0.1mM GB treated group and blank control (A). Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the significantly changed genes between 0.1mM GB treated group and blank control (B)

Fig. 4

Cho and GB exhibited down-regulatory effects on c-di-GMP related phenotypes. RT-PCR varify the effects of GB on the expression of c-di-GMP-related gene relative to rpsL (A). The impact of Cho and GB on the expression of the c-di-GMP reporter gene PcdrA-gfp in both PAO1 and betAB mutant strains (B). Congo red colony morphology assay revealed the inhibitory effect of 0.1 mM Cho and 0.1 mM GB on c-di-GMP production in both PAO1 and betAB mutant (C). Statistical analysis was conducted by One-way ANOVA. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.The data represent the mean ± SD of three biological replicates

Fig. 5

Cho and GB interfere c-di-GMP regulated phenotypes in P. aeruginosa. Biofilm formation of PAO1 and betAB mutant after cultured overnight with 0.1mM Cho or GB in ABTG medium (A). Poverdine production of PAO1 and betAB mutant after cultured overnight with 0.1mM Cho or GB in ABTG medium (B). Swarming motility of PAO1 and betAB mutant on BM2 plates supplemented with 0.1 mM Cho or GB, significance evaluation was listed on the right (C). Data was presented as mean ± SD of four biological replicates. Statistical analysis was conducted by One Way-ANOVA, ** Indicates p < 0.01, *** Indicates p < 0.001

Fig. 6

Deletion of betAB gene in P. aeruginosa enhances the production of IL-1β and IL-18 during macrophage infection. Raw264.7 macrophages were infected with log-phase PAO1 or betAB mutant for 6 h. Subsequently, the supernatant was collected and analyzed for IL-1β and IL-18 levels. The data shown are from one representative of three independent experiments. ** Indicates p < 0.01, *** Indicates p < 0.001

Fig. 7

Model for Cho regulating the virulence of P. aeruginosa