Allelic polymorphism shapes community function in evolving Pseudomonas aeruginosa populations

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the lungs of individuals with cystic fibrosis (CF) by forming antibiotic-resistant biofilms. Emergence of phenotypically diverse isolates within CF P. aeruginosa populations has previously been reported; however, the impact of heterogeneity on social behaviors and community function is poorly understood. Here we describe how this heterogeneity impacts on behavioral traits by evolving the strain PAO1 in biofilms grown in a synthetic sputum medium for 50 days. We measured social trait production and antibiotic tolerance, and used a metagenomic approach to analyze and assess genomic changes over the duration of the evolution experiment. We found that (i) evolutionary trajectories were reproducible in independently evolving populations; (ii) over 60% of genomic diversity occurred within the first 10 days of selection. We then focused on quorum sensing (QS), a well-studied P. aeruginosa trait that is commonly mutated in strains isolated from CF lungs. We found that at the population level, (i) evolution in sputum medium selected for decreased the production of QS and QS-dependent traits; (ii) there was a significant correlation between lasR mutant frequency, the loss of protease, and the 3O-C12-HSL signal, and an increase in resistance to clinically relevant β-lactam antibiotics, despite no previous antibiotic exposure. Overall, our findings provide insights into the effect of allelic polymorphism on community functions in diverse P. aeruginosa populations. Further, we demonstrate that P. aeruginosa population and evolutionary dynamics can impact on traits important for virulence and can lead to increased tolerance to β-lactam antibiotics.

Fig. 1. The evolutionary trajectory of P. aeruginosa during biofilm growth in SCFM.

The evolution trajectories were similar in four independent replicate lines, and the major genetic heterogeneity in the evolved populations occurred during the first 10 days of selection. a SNP calling analysis showed that the four independently evolved populations had more than 80% similarity (267 shared SNPs) in genomic changes in the populations over the 50 days of selection. b The Venn diagram shows the shared SNPs between all four replicate lines, over 50 days of selection. Numbers in brackets (i.e., 1354 for 10 days) represent the combined number of SNPs in each of the four evolved lines. The majority of genetic variation within the populations occurred within the first 10 days of selection. Each circle represents the total number of SNPs for each independently evolved line.

Fig. 2. Allele frequency of SNPs changes over the course of selection.

Emergence of nonsynonymous SNPs in genes involved in social traits (QS: lasR), oxidative respiration and DNA mismatch repair, motility, and iron chelation occurred after 20 and 30 days of selection, and fixed SNPs in genes such as elongation factor tufA, Cytochrome c oxidase subunit (ccoN2), and pyoverdine biosynthesis protein pvdJ emerged after 10 days of selection, and were fixed in the populations over 50 days (a–d represent replicate evolving lines).

Fig. 3. Phenotypes of evolved populations of PAO1.

a Increase in biofilm formation by evolved populations of PAO1 (Kruskal–Wallis, Dunn’s multiple comparison with PAO1, p < 0.05, n = 5. Errors bars indicate the standard error of the mean, SEM). b The total protease activity was reduced at 30 days of selection. c Production of 3O-C12-HSL significantly decreased at 30 days of selection (Kruskal–Wallis, uncorrected Dunn’s test multiple comparison to PAO1, p < 0.05, n = 5. Errors bars indicate the SEM), and there were no significant changes in the levels of C4-HSL. d There were no significant changes in siderophore production in evolved populations compared with the PAO1 ancestor (Kruskal–Wallis, uncorrected Dunn’s test multiple comparison to PAO1, p > 0.05, n = 5). Each dot represents the mean value of one independent experiment for each evolved population at each round of selection. All experiments were performed in three technical replicates.

Fig. 4. Loss of protease activity and a decrease in QS signal production in evolved populations can be explained by an accumulation of lasR SNPs.

A linear regression model showed that the loss of total protease activity and the decrease in 3O-C12-HSL levels can be explained by the accumulation of lasR SNPs. a There is a significant negative correlation between lasR SNP frequency and total protease activity of the evolved populations (R² = 0.7298, p = 0.04; n = 8). b There is no significant correlation between 3O-C12-HSL levels and accumulation of the lasR SNP in the evolved populations (R² = 0.5363, p = 0.1594; n = 8). However, if only changes in 3O-C12-HSL levels after 30 days of selection are considered, there is a strong correlation between emergence and accumulation of lasR SNP in evolved populations (small inset: R² = 1.0; p = 0.0034; n = 8). c There are no significant correlations between accumulation of lasR SNPs in the evolved populations and levels of C4-HSL (R² = 0.1187, p = 0.5702; n = 8). d There is no significant correlation between lasR frequency and biofilm formation (R² = 0.0004, p = 0.9062; n = 8).

Fig. 5. Increased resistance to ceftazidime and piperacillin/tazobactam in evolved populations is correlated with lasR SNP frequency.

There is an increased resistance, indicated by a decrease in the zone of inhibition (ZOI) to ceftazidime (a) and piperacillin/tazobactam (b) in evolved populations after 30 days of selection (R3). The red lines represent the ZOI breakpoint (mm) for each antibiotic (BSAC guidelines). Below, the lines indicate resistance. There is a positive correlation between increased resistance and the frequency of the lasR SNP in the evolved populations (n = 8, Pearson correlation. Each dot represents the mean value for the zone of inhibition at each round of selection in all eight independent experiments). c Complementation of a 50-day evolved isolate (BB8) containing a SNP in lasR with an intact lasR in trans, restores sensitivity to ceftazidime and piperacillin/tazobactam. Differential frequencies of B8 mixed with ancestral PAO1 significantly increased tolerance to ceftazidime (n = 5, two-way ANOVA, multiple comparison to PAO1, Fisher’s LSD test, p < 0.05. Errors bars indicate SEM).