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 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.

Metabolic basis for the evolution of a common pathogenic Pseudomonas aeruginosa variant

“…The CbrB-dependent rise of LasR – strains in the complex CF mimetic medium (i.e. artificial sputum medium, ASM) alongside the positive selection observed in minimal media with CF relevant substrates ( Scribner et al, 2022 ) shown to require CbrB for LasR – strain growth enhancement suggests that the growth advantages of lasR mutants may be sufficient to overcome any potential negative selective pressures mediated by the host, neighboring microbes, or inaccessibility to nutrients like complex protein or adenosine. In addition, the loss of LasR function enables other inherent advantages that contribute to competitive fitness including resistance to lysis under conditions of high aeration, enhanced microoxic fitness, enhanced RhlR activity ( Chen et al, 2019 ; Clay et al, 2020 ; Heurlier et al, 2005 ), and altered intraspecies interactions ( Mould et al, 2020 ) which may be relevant in the complex and dynamic nutritional environment of the CF airway over the course of disease.…”

“…The numerous differences described between LasR + and LasR – strains indicate that an understanding of the factors that drive the rise and persistence of lasR mutants may be complex and are not yet well understood. While it is clear that there are many ways in which lasR LOF mutants differ from their LasR +progenitors, a common trait that promotes the rise of LasR – strains in diverse environments, even in rich and minimal laboratory media ( Heurlier et al, 2005 ; Scribner et al, 2022 ; O Brien et al, 2017 ; Luján et al, 2007 ; Qi et al, 2016 ; Robitaille et al, 2020 ; Sandoz et al, 2007 ; Wong et al, 2012 ; Yan et al, 2018 ), has not been established.…”

Evolution of Quorum Sensing in Pseudomonas aeruginosa Can Occur via Loss of Function and Regulon Modulation

“…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, leading to an overall loss of QS function. There is increasing evidence that large-scale deletions of the Las system are common in in vitro experimental evolution ( 40 , 50 , 51 ), but might have been overlooked in the past due to computational challenges of identifying them in draft genomes produced by short-read sequencing. Although we also used short-read sequencing, our coverage was high so that large deletions could be identified with high confidence ( Table S3 ).…”

“…While initially interpreted as a specific adaptation to the CF lung environment, it has become clear that lasR mutants are also selected for in a large range of infection types ( 34 ), including chronic wounds ( 35 ), corneal infections ( 36 , 37 ), ventilator-associated pneumonia ( 30 ), and infections of the nematode Caenorhabditis elegans ( 18 , 38 ). Additionally, lasR mutants are ubiquitous in environments such as hospital sink drains and wet markets ( 39 ), and spontaneously evolve under laboratory conditions on agar plates and in liquid, static, and well-mixed environments ( 33 , 38 , 40 , 41 ).…”

Adaptation and Evolution of Pathogens in the Cystic Fibrosis Lung

“…While the pervasive changes in the superficial or exposed aspects of a bacterial cell in the CF lung are likely to be tightly linked to the immune response, it is worthwhile considering other possible advantages that losses or changes in these factors may have for bacteria. Indeed, some changes associated with LPS may arise simply because of selection for biofilms and CF-like nutritional regimes [ 53 , 54 ]. Also, once established in a thriving community in the lungs, bacteria may no longer need to expend the energy for appendages that are primarily advantageous for colonization and motility.…”