Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels

Abstract

The host immune system offers a hostile environment with antimicrobials and reactive oxygen species (ROS) that are detrimental to bacterial pathogens, forcing them to adapt and evolve for survival. However, the contribution of oxidative stress to pathogen evolution remains elusive. Using an experimental evolution strategy, we show that exposure of the opportunistic pathogen Pseudomonas aeruginosa to sub-lethal hydrogen peroxide (H₂O₂) levels over 120 generations led to the emergence of pro-biofilm rough small colony variants (RSCVs), which could be abrogated by l-glutathione antioxidants. Comparative genomic analysis of the RSCVs revealed that mutations in the wspF gene, which encodes for a repressor of WspR diguanylate cyclase (DGC), were responsible for increased intracellular cyclic-di-GMP content and production of Psl exopolysaccharide. Psl provides the first line of defence against ROS and macrophages, ensuring the survival fitness of RSCVs over wild-type P. aeruginosa Our study demonstrated that ROS is an essential driving force for the selection of pro-biofilm forming pathogenic variants. Understanding the fundamental mechanism of these genotypic and phenotypic adaptations will improve treatment strategies for combating chronic infections.

Keywords: Pseudomonas aeruginosa; adaptive evolution; biofilms; c-di-GMP; reactive oxygen species; rough small colony variants.

Figure 1.

Evolution assay of Pseudomonas aeruginosa in H₂O₂. (a) Percentage of rough small colony variants (RSCVs) after evolution in the presence of H₂O₂ compared with the control. (b) Resistance of RSCV isolates compared with PAO1 in H₂O₂. (c) Competitive fitness assay between RSCV isolates and PAO1 grown in H₂O₂. (d) Antioxidants (glutathione; GSH) reduced the proportion of RSCVs formed with ROS treatment, in a dose-dependent manner. Means ± s.d. from triplicate experiments are shown.

Figure 2.

Adaptation of RSCVs to H₂O₂ is dependent on induction of c-di-GMP content of isolates. (a) C-di-GMP content of RSCVs quantified by HPLC. (b) The p_cdrA-gfp expression in RSCVs. (c) Pyoverdine production in RSCVs. (d) Expression of p_lac-yhjH in RSCVs reduces their resistance to H₂O₂. Means ± s.d. from triplicate experiments are shown.

Figure 3.

Genetic sequencing for identification of mutation sites. (a) Number of mutations identified in evolved genotypes. (b) Location of mutations in the wspF gene of different RSCVs.

Figure 4.

ΔwspF mutation is important in the induction of c-di-GMP in the presence of ROS. (a) Induction of c-di-GMP in PAO1 by short term (4 h) H₂O₂ exposure (LC-MS quantification). (b) Induction of p_cdrA-gfp expression level in PAO1 by short term (4 h) H₂O₂ exposure. (c) Resistance of PAO1, ΔwspF, PAO1/p_lac-yedQ and PAO1/p_lac-yhjH strains to 4 mM H₂O₂. (d) p_cdrA-gfp expression of evolved RSCV isolates with epistatic wspR mutations to H₂O₂. (e) Resistance of evolved RSCV isolates with epistatic wspR mutations to H₂O₂. (f) Resistance of PAO1 and clinical RSCV isolates with known wspF mutations to H₂O₂ treatment. Means ± s.d. from triplicate experiments are shown; p < 0.05, one-way ANOVA.

Figure 5.

C-di-GMP-mediated exopolysaccharides were required for resistance to ROS stress. (a) Resistance of PAO1, ΔwspFΔpelAΔpslBCD and ΔpelAΔpslBCD/p_lac-yedQ to H₂O₂. (b) Psl was more influential than Pel in conferring ROS resistance. (c) Psl staining showed formation of small aggregates and synthesis of larger amounts of Psl in PAO1 treated with 0.5, 1 and 2 mM H₂O₂ compared with control PAO1 cultures. (d) Psl staining revealed synthesis of larger amounts of Psl in representative RSCVs and strains with high intracellular c-di-GMP content than PAO1. (e) Addition of l-arabinose to PAO1/p_BAD-psl increased biofilm formation in a dose-dependent manner. (f) Resistance of PAO1/p_BAD-psl to H₂O₂ increased with increasing l-arabinose concentrations. (g) Cellulase treatment abolished ROS resistance in PAO1/p_BAD-psl. Means ± s.d. from triplicate experiments are shown; p < 0.05, one-way ANOVA.

Figure 6.

Adaptation to H₂O₂ provides further benefits by conferring protection against macrophages. (a) Quantification of phagocytosed PAO1 and evolved RSCV isolates by macrophages. (b) Quantification of phagocytosed PAO1, ΔwspF and PAO1/p_lac-yhjH strains by macrophages. (c) Cytotoxicity assay of macrophages by PAO1, ΔwspF and PAO1/p_lac-yhjH strains. (d) Quantification of ROS produced by macrophages in the presence of PAO1, ΔwspF, PAO1/p_lac-yedQ and PAO1/p_lac-yhjH strains using ROS detection assay. DCF (e) Quantification of phagocytosed PAO1 and ΔpslBCD strains by macrophages. (f) Cytotoxicity assay of macrophages by PAO1 and ΔpslBCD strains. Means ± s.d. from triplicate experiments are shown; p < 0.05, one-way ANOVA.

Figure 7.

Model of Pseudomonas aeruginosa adaptation to oxidative stress. Upon repeated exposure of oxidative stress, the cells evolved to form RSCVs via wspF mutation, resulting in the induction of c-di-GMP signalling and increased Psl production. This endowed RSCVs with the ability to resist oxidative stress and phagocytes.