The Nutritional Environment Is Sufficient To Select Coexisting Biofilm and Quorum Sensing Mutants of Pseudomonas aeruginosa

Abstract

The evolution of bacterial populations during infections can be influenced by various factors including available nutrients, the immune system, and competing microbes, rendering it difficult to identify the specific forces that select on evolved traits. The genomes of Pseudomonas aeruginosa isolated from the airways of people with cystic fibrosis (CF), for example, have revealed commonly mutated genes, but which phenotypes led to their prevalence is often uncertain. Here, we focus on effects of nutritional components of the CF airway on genetic adaptations by P. aeruginosa grown in either well-mixed (planktonic) or biofilm-associated conditions. After only 80 generations of experimental evolution in a simple medium with glucose, lactate, and amino acids, all planktonic populations diversified into lineages with mutated genes common to CF infections: morA, encoding a regulator of biofilm formation, or lasR, encoding a quorum sensing regulator that modulates the expression of virulence factors. Although mutated quorum sensing is often thought to be selected in vivo due to altered virulence phenotypes or social cheating, isolates with lasR mutations demonstrated increased fitness when grown alone and outcompeted the ancestral PA14 strain. Nonsynonymous SNPs in morA increased fitness in a nutrient concentration-dependent manner during planktonic growth and surprisingly also increased biofilm production. Populations propagated in biofilm conditions also acquired mutations in loci associated with chronic infections, including lasR and cyclic di-GMP regulators roeA and wspF. These findings demonstrate that nutrient conditions and biofilm selection are sufficient to select mutants with problematic clinical phenotypes including increased biofilm and altered quorum sensing. IMPORTANCE Pseudomonas aeruginosa produces dangerous chronic infections that are known for their rapid diversification and recalcitrance to treatment. We performed evolution experiments to identify adaptations selected by two specific aspects of the CF respiratory environment: nutrient levels and surface attachment. Propagation of P. aeruginosa in nutrients present within the CF airway was sufficient to drive diversification into subpopulations with identical mutations in regulators of biofilm and quorum sensing to those arising during infection. Thus, the adaptation of opportunistic pathogens to nutrients found in the host may select mutants with phenotypes that complicate treatment and clearance of infection.

Keywords: biofilm; cystic fibrosis; nutrition; quorum sensing.

FIG 1

Propagation of P. aeruginosa in nutrients present in the CF airway rapidly selects for mutations in regulators of quorum sensing and cyclic di-GMP. Mutations were inferred from whole genome sequencing of evolved populations after 12 days of selection. All mutations detected by population WGS are indicated and genes that acquired multiple mutations are labeled. Details of detected variants are in the supplemental material. Allele frequency for each mutation is indicated by symbol size, mutation type by symbol shape, and function of the impacted loci by symbol color. Mutations detected by new junction evidence, which include insertions, large deletions, and structural rearrangements, are indicated at the position of the most upstream side of the junction.

FIG 2

Convergent evolution of mutations in lasR and morA reveal sites under strong selection. (A) Two large deletions that include the lasI and lasR genes were detected as well as many nonsynonymous SNPs and missense mutations. (Top) The extent and overlapping region of these deletions; (bottom) evolved lasR mutations by relative position. (B) Mutations in morA were also frequently selected, primarily within the diguanylate cyclase (DGC) domain and linker region between the DGC and phosphodiesterase (PDE) domains (This Study). We also identified nonsynonynous morA mutations in other evolution experiments in our laboratory and other studies (Laboratory Isolates) (23, 44). Identical mutated sites have been identified in clinical isolates from patients with cystic fibrosis (Clinical Isolates) (2). Mutations are shaded by domain in which they occur.

FIG 3

Mutations in lasR and in multiple cyclic di-GMP regulators produce coexisting subpopulations with distinct biofilm and motility phenotypes (A and B). Biofilm production by isolates from planktonic lineages (A) and biofilm lineages (B); (C and D) swimming motility by isolates from planktonic lineages (C) and biofilm lineages (D). Strains are labeled by genotype. Data points represent the average of technical replicates from at least three independent experiments and are shown with mean and 95% CI. Data were analyzed by one-way ANOVA [biofilm, F(16,34) = 32.76, P < 0.0001; swimming motility, F(16,41) = 72.67, P < 0.0001] with Tukey’s multiple-comparison test. Groups labeled with the same letter are not statistically different (P < 0.05).

FIG 4

Environment-specific fitness advantages of lasR and morA mutants. A marked ancestral strain was competed against the ancestral strain (A), an isolate with a morA SNP (E1153K) and an intergenic SNP between a tRNA and the tufB gene (B), an isolate with a SNP in lasR (R216Q) (C), and an isolate with a large deletion encompassing lasR and lasI (D). Competitions were performed in the medium used for the evolution experiment (All) as well as medium containing only a subset of the carbon sources (Glucose, Amino Acids and Lactate). The morA mutant was also competed in media in which the concentration of nutrients was doubled or halved to determine the effect of nutrient concentration on fitness (0.5X All, 0.5X Glucose and 2X Amino Acids). Data points represent fitness measurements from independent competitions spread across at least three batches of assays. Mean and 95% CI are shown. Within each genotype, statistical differences in fitness in each media were determined using ANOVA [ancestor, F (3, 41) = 2.680, P = 0.0594; morA tRNA/tufB, F (6, 92) = 15.20, P < 0.0001; lasR R216Q, F (3, 35) = 8.064, P = 0.0003; ΔPA14_45920.PA14_46440, F (3, 38) = 10.07, P < 0.0001] with Tukey’s multiple-comparison test. Groups labeled with the same letter are not statistically different.

FIG 5

Relative fitness of lasR and morA mutants is greater when in the minority. We performed direct competitions at the starting concentrations indicated and measured relative fitness at 48 h. Genotypes were differentiated using a lac-marked ancestral strain for the ancestral competitions and by colony morphology for the lasR mutant versus morA mutant competitions. The first competitor listed is genotype 1; the second is genotype 2. Data was analyzed by linear regression, shaded area represents 95% CI of the regression. Ancestor versus ancestor, slope = −0.2060; lasR mutant versus morA mutant, slope = −0.6760; difference between the slopes was statistically significant F = 4.195, DFn = 1, DFd = 83, P = 0.0437.