Biofilms Formed by Gram-Negative Bacteria Undergo Increased Lipid A Palmitoylation, Enhancing <italic toggle='yes'>In Vivo</italic> Survival

Bacterial biofilm communities are associated with profound physiological changes that lead to novel properties compared to the properties of individual (planktonic) bacteria. The study of biofilm-associated phenotypes is an essential step toward control of deleterious effects of pathogenic biofilms. Here we investigated lipopolysaccharide (LPS) structural modifications in Escherichia coli biofilm bacteria, and we showed that all tested commensal and pathogenic E. coli biofilm bacteria display LPS modifications corresponding to an increased level of incorporation of palmitate acyl chain (palmitoylation) into lipid A compared to planktonic bacteria. Genetic analysis showed that lipid A palmitoylation in biofilms is mediated by the PagP enzyme, which is regulated by the histone-like protein repressor H-NS and the SlyA regulator. While lipid A palmitoylation does not influence bacterial adhesion, it weakens inflammatory response and enhances resistance to some antimicrobial peptides. Moreover, we showed that lipid A palmitoylation increases in vivo survival of biofilm bacteria in a clinically relevant model of catheter infection, potentially contributing to biofilm tolerance to host immune defenses. The widespread occurrence of increased lipid A palmitoylation in biofilms formed by all tested bacteria suggests that it constitutes a new biofilm-associated phenotype in Gram-negative bacteria. IMPORTANCE Bacterial communities called biofilms display characteristic properties compared to isolated (planktonic) bacteria, suggesting that some molecules could be more particularly produced under biofilm conditions. We investigated biofilmassociated modifications occurring in the lipopolysaccharide (LPS), a major component of all Gram-negative bacterial outer membrane. We showed that all tested commensal and pathogenic biofilm bacteria display high incorporation of a palmitate acyl chain into the lipid A part of LPS. This lipid A palmitoylation is mediated by the PagP enzyme, whose expression in biofilm is controlled by the regulatory proteins H-NS and SlyA. We also showed that lipid A palmitoylation in biofilm bacteria reduces host inflammatory response and enhances their survival in an animal model of biofilm infections. While these results provide new insights into the biofilm lifestyle, they also suggest that the level of lipid A palmitoylation could be used as an indicator to monitor the development of biofilm infections on medical surfaces. Received 26 March 2014 Accepted 7 July 2014 Published 19 August 2014 Citation Chalabaev S, Chauhan A, Novikov A, Iyer P, Szczesny M, Beloin C, Caroff M, Ghigo J-M. 2014. Biofilms formed by Gram-negative bacteria undergo increased lipid A palmitoylation, enhancing in vivo survival. mBio 5(4):e01116-14. doi:10.1128/mBio.01116-14. Invited Editor Stephen Trent, University of Texas at Austin Editor Yves Brun, Indiana University Copyright © 2014 Chalabaev et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Jean-Marc Ghigo, jmghigo@pasteur.fr. Biofilm communities developing on medical and industrial surfaces constitute a recognized reservoir of bacterial pathogens, and control of biofilm-associated infections is a major focus of microbiology at this time (1, 2). Identification of key biofilm determinants in several pathogens led to potential antibiofilm strategies based either on prevention of initial bacterial adhesion, inhibition of biofilm maturation, biofilm dispersion, or eradication of highly antibiotic-tolerant biofilm bacteria (3, 4). Study of gene expression and physiological changes occurring during biofilm formation suggested that specific molecules might be associated with the biofilm environment (5–8). Consistently, several compounds were shown to accumulate within biofilms, including biofilm matrix components, antiadhesion molecules, and amino acids (9–11). We previously showed that formation of biofilms by certain Pseudomonas aeruginosa strains induced reversible loss of lipopolysaccharide (LPS) O antigen and alteration of lipid A that contributes to modulating the host inflammatory response to P. aeruginosa biofilms (12). LPS is a major component of all Gram-negative bacterial outer membranes, and although its structure varies in response to certain environmental stimuli (13), few studies have investigated modifications in LPS structure in biofilms (12, 14, 15). Here, we compared Escherichia coli LPS from biofilm and planktonic bacteria and identified a reversible increased LPS modification corresponding to incorporation of a palmitate acyl chain to lipid A (palmitoylation) mediated by the PagP enzyme. While the appearance of this LPS modification is correlated to the ability of the bacteria to form biofilms, it occurs progressively and only after several days in very long stationaryphase culture bacteria. We show that pagP is negatively regulated RESEARCH ARTICLE crossmark July/August 2014 Volume 5 Issue 4 e01116-14 ® mbio.asm. org 1 by H-NS, which itself is under the biofilm-regulated control of the SlyA regulator. We demonstrate that increased lipid A palmitoylation in biofilm bacteria decreases the inflammatory response and increases biofilm bacterial survival in an in vivo rat model of catheter-associated biofilm infection. Since increased lipid A palmitoylation also occurs in biofilms formed by a wide range of Gram-negative bacteria, our study therefore identified a new biofilm phenotype, which could be used as a biofilm biomarker to monitor Gram-negative bacterial biofilm-associated