ThePseudomonas aeruginosaWsp pathway undergoes positive evolutionary selection during chronic infection

Abstract

Running title: Evolution of P. aeruginosa RSCVs in a chronic infection model 22Author Contributions: ESG and CWM performed the experimental work. JSH 23 performed the colony PCR. SBC infected and sampled the porcine burn wounds. DS 24 generated the sequence library. MW and GRL quantified the strain frequency in the 25 wounds. ESG, CWM, MW, VSC, and DJW conceptualized the project and wrote the 26 manuscript. 27Chronic infections are those that persist despite extensive treatment. These 55 persistent infections are often attributed to difficult to eradicate biofilms, which are 56 communities of adhered microorganisms encased in an extracellular polymeric 57 substance (EPS) 1,2 . Complicating chronic infections is the high likelihood that 58 bacterial populations adaptively evolve, producing persistent phenotypes with 59 increased fitness. 60 61One of the most understood bacterial adaptive responses to chronic infection is that 62of Pseudomonas aeruginosa to the cystic fibrosis (CF) lung 3 . CF patients exhibit 63 mucus accumulation, where P. aeruginosa biofilms commonly colonize the mucus 64 lining and establish persistent pulmonary infections 4-6 . Evolved phenotypic variants 65 of this organism are routinely isolated from CF patient sputum samples, and several 66 of these variants are often associated with worsening patient prognosis 7 . Of 67 particular interest are the rugose small-colony variants (RSCVs), which are isolated 68 from up to 50% of P. aeruginosa-positive CF sputum samples 8,9 . When isolated, 69 their frequencies range drastically between 0.1 − 100% 9,10 . In contrast there is little-70 to-no reports of the frequency of P. aeruginosa adapted variants from other chronic 71 infections, such as chronic wound infections. 72 73 Common to RSCVs are mutations in pathways that lead to elevated cyclic 74 diguanylate monophosphate (c-di-GMP) 7 . C-di-GMP is a messenger molecule that 75 signals the transition from planktonic to biofilm lifestyle in many bacteria 11 . In P. 76 aeruginosa, increased c-di-GMP, among many responses, leads to overproduction of 77 exopolysaccharides, Psl and Pel, and matrix proteins 12,13 . As a result, RSCVs have 78 hyper-biofilm phenotypes 13,14 , increased tolerance to antimicrobials 10 , and enhanced 79 resistance to immunity 15,16 . P. aeruginosa RSCVs also evolve from in vitro grown 80 biofilms 17,18 , suggesting that there is strong selection for ecological diversification in 81 both in vivo and in vitro biofilms. 82 defined by a matte, rugose colony morphology that stained intensely by the two dyes, 132 indicating exopolysaccharide overproduction, were isolated from all three timepoints 133 (Fig 2A). Two RSCV sub-populations were observed; one that had a pink, rugose 134 phenotype and a second that had an orange, textured phenotype (Fig 2A). 135 136The RSCV abundance in the wounds was quantified by expressing their frequency 137 as a percentage of the total P. aeruginosa burden. The RSCV frequency was low in 138

EvolvingSTEM: a microbial evolution-in-action curriculum that enhances learning of evolutionary biology and biotechnology

“…This result not only simulates the process of adaptive radiation often illustrated using Darwin's finches in textbooks (Fig. 2), but also reproduces the selection for traits associated with adherence that often occurs during biofilm-associated infections (Traverse et al 2013;Cooper et al 2014;O'Rourke et al 2015;Gloag et al 2018). The "wrinkly" colony morphologies that evolve in our model are genetically and functionally identical to those commonly isolated from infections of the related species Pseudomonas aeruginosa in the airways of cystic fibrosis patients and in chronic skin wounds (Starkey et al 2009;Gloag et al 2018).…”

“…2), but also reproduces the selection for traits associated with adherence that often occurs during biofilm-associated infections (Traverse et al 2013;Cooper et al 2014;O'Rourke et al 2015;Gloag et al 2018). The "wrinkly" colony morphologies that evolve in our model are genetically and functionally identical to those commonly isolated from infections of the related species Pseudomonas aeruginosa in the airways of cystic fibrosis patients and in chronic skin wounds (Starkey et al 2009;Gloag et al 2018). Students can therefore connect their classroom experiments to recent findings at the interface of evolutionary biology and medicine to see how basic biological research impacts their everyday lives.…”

“…(Cooper 2018)) that underlie the adaptive mutant phenotypes, supporting a greater understanding of inheritance and trait variation (NGSS HS-LS3). Previous research in our lab indicates that many commonly identified mutations are found in the wsp (wrinkly spreader phenotype) gene cluster (Cooper et al 2014;Gloag et al 2018), which coordinates bacterial surface recognition with increased biofilm production (Hickman and Tifrea 2005). Students are likely to identify wsp mutants in their classroom experiments and can therefore connect how changes in DNA can result in changes in protein structure and intracellular signaling that lead to increased biofilm production and changes to colony morphology, supporting a greater understanding of DNA, protein structure, and cellular function (NGSS HS-LS1).…”

Evolutionary Divergence of the Wsp Signal Transduction Systems in Beta- and Gammaproteobacteria

“…Mutations in wsp genes are frequently identified in clinical specimens and are associated with increased biofilm formation 22,24,25,[73][74][75][76][77][78] , whether or not this is achieved through elevated production of cyclic di-GMP. Genetically deregulating the Wsp system appears to be a significant but transient adaptive strategy in a dynamic and overcrowded ecosystem.…”