title: Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels.
abstract: The host immune system offers a hostile environment with antimicrobials and reactive oxygen species (ROS) that are detrimental to bacterial pathogens, forcing them to adapt and evolve for survival. However, the contribution of oxidative stress to pathogen evolution remains elusive. Using an experimental evolution strategy, we show that exposure of the opportunistic pathogen Pseudomonas aeruginosa to sub-lethal hydrogen peroxide (H2O2) levels over 120 generations led to the emergence of pro-biofilm rough small colony variants (RSCVs), which could be abrogated by l-glutathione antioxidants. Comparative genomic analysis of the RSCVs revealed that mutations in the wspF gene, which encodes for a repressor of WspR diguanylate cyclase (DGC), were responsible for increased intracellular cyclic-di-GMP content and production of Psl exopolysaccharide. Psl provides the first line of defence against ROS and macrophages, ensuring the survival fitness of RSCVs over wild-type P. aeruginosa Our study demonstrated that ROS is an essential driving force for the selection of pro-biofilm forming pathogenic variants. Understanding the fundamental mechanism of these genotypic and phenotypic adaptations will improve treatment strategies for combating chronic infections.
authors.affiliation: Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 639798 sl.chua@ntu.edu.sg.|Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.
authors.forename: Song Lin
authors.identifier: 0000-0001-7047-7451
authors.initials: SL
authors.lastname: Chua
authors.affiliation: Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.|Interdisciplinary Graduate School, Nanyang Technological University, Singapore 637551.
authors.forename: Yichen
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.|Interdisciplinary Graduate School, Nanyang Technological University, Singapore 637551.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Cai
authors.affiliation: Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.|Integrative Microbiology Research Centre, South China Agricultural University, Guangzhou 510642, People's Republic of China.
authors.forename: Jianuan
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.|Department of Biological Sciences, National University of Singapore, Singapore 117543.|NUS Environmental Research Institute, National University of Singapore, Singapore.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Swarup
authors.affiliation: Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.
authors.forename: Daniela I
authors.initials: DI
authors.lastname: Drautz-Moses
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 639798.|Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.
authors.forename: Stephan Christoph
authors.initials: SC
authors.lastname: Schuster
authors.affiliation: Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.|Center for Marine Bio-Innovation and School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia.
authors.forename: Staffan
authors.initials: S
authors.lastname: Kjelleberg
authors.affiliation: Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.|Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, 2200 København N, Denmark.
authors.forename: Michael
authors.initials: M
authors.lastname: Givskov
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 639798 yangliang@ntu.edu.sg.|Singapore Centre for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore 637551.
authors.forename: Liang
authors.initials: L
authors.lastname: Yang
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D018441:Biofilms / Q000187:drug effects; D005075:Biological Evolution; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005978:Glutathione / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D017382:Reactive Oxygen Species; C062025:bis(3',5')-cyclic diguanylic acid; D006861:Hydrogen Peroxide; D005978:Glutathione; D006152:Cyclic GMP
keywords: Pseudomonas aeruginosa; adaptive evolution; biofilms; c-di-GMP; reactive oxygen species; rough small colony variants