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 airway of patients 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 alone sufficient to select mutants with problematic clinical phenotypes including increased biofilm and altered quorum sensing.

Evolution of the quorum sensing systems in Pseudomonas aeruginosa can involve both loss of regulon function and network modulation

“…As an important human pathogen, the evolution of Pseudomonas aeruginosa populations has been studied in a myriad of context, with extensive genetic adaptation being repeatedly observed in diverging environments such as human cystic fibrosis (CF) lungs, animal infection models, as well as in the natural environments and in vitro experimental evolutions (Rumbaugh et al ., 2009; Jansen et al ., 2015; Feltner et al ., 2016; Granato et al ., 2018; Groleau et al ., 2021; Scribner et al ., 2021; Smalley et al ., 2022). The quorum sensing (QS) regulon, a global three-unit regulatory system that controls the expression of up to 10% of the genes in P. aeruginosa , many of which include virulence factors, is often among the most mutated pathways.…”

“…We suggest that the difference in our findings is driven by the fact that we predominantly found large-scale deletions of the Las system, where the signal synthase ( lasI ), repressor ( rsaL ) and regulator ( lasR ) are deleted, therefore, leading to a loss of QS function. There is increasing evidence that large-scale deletions of the Las system are common in in vitro experimental evolution (O’Brien et al ., 2017; Scribner et al ., 2021; Tostado-Islas et al ., 2021), but might have been overlooked in the past due to computational challenges of identifying them in draft genomes produced by short-read sequencing. Given that these large-scale deletions occurred many times independently, they must have an adaptive advantage.…”

“…Given that these large-scale deletions occurred many times independently, they must have an adaptive advantage. Similar large-scale deletions have previously been associated with both cheating and loss due to disuse in the process of medium adaptation (O’Brien et al ., 2017; Scribner et al ., 2021; Tostado-Islas et al ., 2021). In our case, the QS mutants emerged from wild type populations that had been experimentally evolved in casamino acid medium, predominantly consisting of digested amino acids, an environment in which classic QS-regulated traits such as proteases and rhamnolipids are not needed.…”

“…P. aeruginosa isolates from chronically infected CF patient lungs frequently contain mutations in the master transcriptional regulator, LasR, thereby influencing the activity of all three QS systems (Feltner et al ., 2016; Chen et al ., 2019). While initially interpreted as a specific adaptation to the CF lung environment, it has become clear that lasR mutants are also selected for under many other conditions, including chronic wounds (Vanderwoude et al ., 2020), corneal infections (Preston et al ., 1997; Hammond et al ., 2016), ventilator-associated pneumonia (Köhler, Buckling and Van Delden, 2009), infections in Caenorhabditis elegans (Jansen et al ., 2015; Granato et al ., 2018), as well as in the absence of a host such as in natural environments (Groleau et al ., 2021) and in experimental evolutions (Wilder, Diggle and Schuster, 2011; Kostylev et al ., 2019; Scribner et al ., 2021; Smalley et al ., 2022). Why then, are lasR mutants consistently favored across these difference environments?…”