A gacS Deletion in Pseudomonas aeruginosa Cystic Fibrosis Isolate CHA Shapes Its Virulence Khady Mayebine Sall 1,2,3,4 , Maria Guillermina Casabona 1,2,3,4 , Christophe Bordi 5 , Philippe Huber 1,2,3,4 , Sophie de Bentzmann 5 , Ina Attre ´e 1,2,3,4 , Sylvie Elsen 1,2,3,4 * 1 INSERM, UMR-S 1036, Biology of Cancer and Infection, Grenoble, France, 2 CNRS, ERL 5261, Bacterial Pathogenesis and Cellular Responses, Grenoble, France, 3 UJF- Grenoble 1, Grenoble, France, 4 CEA, DSV/iRTSV, Grenoble, France, 5 Laboratoire d’Inge ´nierie des Syste `mes Macromole ´culaires, UMR 7255 CNRS - Aix Marseille University, Marseille, France Abstract Pseudomonas aeruginosa, a human opportunistic pathogen, is capable of provoking acute and chronic infections that are associated with defined sets of virulence factors. During chronic infections, the bacterium accumulates mutations that silence some and activate other genes. Here we show that the cystic fibrosis isolate CHA exhibits a unique virulence phenotype featuring a mucoid morphology, an active Type III Secretion System (T3SS, hallmark of acute infections), and no Type VI Secretion System (H1-T6SS). This virulence profile is due to a 426 bp deletion in the 39 end of the gacS gene encoding an essential regulatory protein. The absence of GacS disturbs the Gac/Rsm pathway leading to depletion of the small regulatory RNAs RsmY/RsmZ and, in consequence, to expression of T3SS, while switching off the expression of H1- T6SS and Pel polysaccharides. The CHA isolate also exhibits full ability to swim and twitch, due to active flagellum and Type IVa pili. Thus, unlike the classical scheme of balance between virulence factors, clinical strains may adapt to a local niche by expressing both alginate exopolysaccharide, a hallmark of membrane stress that protects from antibiotic action, host defences and phagocytosis, and efficient T3S machinery that is considered as an aggressive virulence factor. Citation: Sall KM, Casabona MG, Bordi C, Huber P, de Bentzmann S, et al. (2014) A gacS Deletion in Pseudomonas aeruginosa Cystic Fibrosis Isolate CHA Shapes Its Virulence. PLoS ONE 9(4): e95936. doi:10.1371/journal.pone.0095936 Editor: Min Wu, University of North Dakota, United States of America Received January 9, 2014; Accepted March 30, 2014; Published April 29, 2014 Copyright: ß 2014 Sall et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: KM Sall is a PhD student supported by the University of Grenoble. This work was in part supported by grants from Fondation innovations en infectiologie (FINOVI), Alliance nationale pour les sciences de la vie et de la sante ´ (AVIESAN), Commissariat a ` l’e ´ nergie atomique et aux e ´nergies alternatives (CEA), Centre national de la recherche scientifique (CNRS) and Institut national de la sante ´ et de la recherche me ´ dicale (INSERM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sylvie.elsen@cea.fr Introduction Pseudomonas aeruginosa is an opportunistic Gram negative bacterium able to trigger either severe acute or chronic human infections, depending on the environmental signals it encounters. The persistence of the bacterium during decades in the lungs of individuals with cystic fibrosis (CF) is associated with massive and inefficient inflammation contributing to airway epithelium de- struction, decline of lung function and death due to respiratory failure [1]. During chronic infection, P. aeruginosa has to survive and adapt to the stressful environment encountered in the CF lungs where it is continuously exposed to antibiotics, oxidative and osmotic stresses as well as active host immune system. The CF respiratory mucus has been shown to directly impact bacterial gene transcription [2,3]. A CF mucoid strain was reported to trigger, in response to the CF niche, the synthesis of enzymes protecting the bacteria against oxidative stress and the activation of genes encoding the HSI-I Type VI Secretion System (H1- T6SS), known to play a role in bacterial competition [4,5,6]. In addition, the alginate production was unexpectedly repressed, and the expression of two small RNAs (PA2G_05393.1 and PA2G_03487.1) with putative regulatory roles was observed, pointing out the major effect of contact with CF mucus on bacterial physiology [3]. Besides these immediate adaptive responses, P. aeruginosa is also prone to accumulate point mutations and/or significant genomic rearrangements induced by extrinsic and intrinsic factors associ- ated with CF chronic disease [1,7]. Mutations in global regulatory genes, such as lasR, vfr, rpoS, or other regulatory genes such as mucA, are frequently observed in CF isolates and these mutations profoundly affect virulence gene expression [1,8]. The most obvious phenotypic changes of P. aeruginosa observed during infection in CF disease are conversion to mucoidy, emergence of Small Colony Variants (SCV), acquisition of antibiotic multi- resistance, loss of motility and shutdown of quorum sensing (QS) system [7,9]. Conversion to mucoidy is proposed to be a major survival mechanism promoting the persistence of the bacterium in CF lungs; it mostly results from the over-expression of the alginate polysaccharide by the alternative sigma factor AlgU, deseque- strated from its anti-sigma partner MucA due to mutations in mucA [10,11]. More aggressive virulence factors secreted by Type II (T2SS) and Type III (T3SS) secretion machineries are switched off, thus leading to CF-adapted strains usually less virulent in animal models than the primary infecting ones [9]. However, while sharing some generally-accepted traits, adaptive evolution and resulting phenotypes of CF isolates can be extremely diverse, even within isolates from the same CF patient sputum sample [1,9,12,13]. PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e95936