Role of the Vibrio cholerae Matrix Protein Bap1 in Cross-Resistance to Antimicrobial Peptides Marylise Duperthuy, Annika E. Sjo ¨ stro ¨ m, Dharmesh Sabharwal, Fatemeh Damghani, Bernt Eric Uhlin, Sun Nyunt Wai* Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea ˚ University, Umea ˚, Sweden Abstract Outer membrane vesicles (OMVs) that are released from Gram-negative pathogenic bacteria can serve as vehicles for the translocation of effectors involved in infectious processes. In this study we have investigated the role of OMVs of the Vibrio cholerae O1 El Tor A1552 strain in resistance to antimicrobial peptides (AMPs). To assess this potential role, we grew V. cholerae with sub-lethal concentrations of Polymyxin B (PmB) or the AMP LL-37 and analyzed the OMVs produced and their effects on AMP resistance. Our results show that growing V. cholerae in the presence of AMPs modifies the protein content of the OMVs. In the presence of PmB, bacteria release OMVs that are larger in size and contain a biofilm-associated extracellular matrix protein (Bap1). We demonstrated that Bap1 binds to the OmpT porin on the OMVs through the LDV domain of OmpT. In addition, OMVs from cultures incubated in presence of PmB also provide better protection for V. cholerae against LL-37 compared to OMVs from V. cholerae cultures grown without AMPs or in presence of LL-37. Using a bap1 mutant we showed that cross-resistance between PmB and LL-37 involved the Bap1 protein, whereby Bap1 on OMVs traps LL-37 with no subsequent degradation of the AMP. Citation: Duperthuy M, Sjo ¨ stro ¨ m AE, Sabharwal D, Damghani F, Uhlin BE, et al. (2013) Role of the Vibrio cholerae Matrix Protein Bap1 in Cross-Resistance to Antimicrobial Peptides. PLoS Pathog 9(10): e1003620. doi:10.1371/journal.ppat.1003620 Editor: Craig R. Roy, Yale University School of Medicine, United States of America Received March 21, 2013; Accepted July 30, 2013; Published October 3, 2013 Copyright: ß 2013 Duperthuy 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: This work was supported by the Swedish Research Council (https://www.vr.se/; Grant number: 2010-3073,2010-3031, 2007-8673 UCMR Linnaeus, and 2006-7431 MIMS) and the Faculty of Medicine, Umea ˚ University (http://www.medfak.umu.se/). 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: sun.nyunt.wai@molbiol.umu.se Introduction V. cholerae is the causative agent of the disease cholera, which remains a significant public health problem, causing large numbers of infections and deaths annually in the world [1]. During the infection, V. cholerae colonizes the surface of the small intestine where it secretes virulence factors [2,3]. Gram-negative bacteria constitutively release lipid bilayer vesicles during normal growth. These outer membrane vesicles (OMVs) range in size from 20–200 nm in diameter. As the vesicles are extruded from the surface of the bacterial cells, some underlying periplasmic components become entrapped inside the vesicles [4,5]. OMVs possess outer membrane proteins, lipopolysaccharide (LPS), phospholipids, and some periplasmic constituents. They have been suggested to play diverse roles in bacterial pathogenesis, including involvement in bacterial communication through OMV-associated signaling molecules, as virulence factors, and in genetic transformation [6,7,8,9,10,11,12]. Antimicrobial peptides (AMPs) contribute to the innate human defense against bacterial infections, as well as in the defense employed by some bacteria against predators [13]. The intestinal epithelium is the site of synthesis of many AMPs, including defensins and cathelicidins such as LL-37, whose expression can be constitutive or induced by microorganisms [14]. Similarly, AMPs stored in Paneth cells are delivered to the lumen of the small intestine in response to bacterial stimulation [15]. Sub-inhibitory concentrations of AMPs are commonly encountered by pathogens on the epithelial surfaces, or in the environment [16]. The most common mechanism of action of AMPs is alteration of bacterial membrane permeability, by pore formation and/or lipid modifi- cations to alter the charge of the outer membrane [17]. Resistance to AMPs is now recognized as an important virulence phenotype in many human pathogenic bacteria. Gram-negative bacteria have developed a wide range of mechanisms to overcome AMPs, such as production of proteases that degrade the peptides, production of secretory proteins that bind the AMP before it reaches its target, efflux systems that actively extrude AMPs if they access the bacterial cytoplasm, modification of the bacterial envelope in order to reduce its net anionic charge and subsequently to decrease the affinity of the cationic AMP for the outer membrane, and regulation of host AMP production [18]. In this study we examined the potential role for OMVs in resistance to AMPs of V. cholerae El Tor O1 strain A1552. To address this question, we isolated OMVs from V. cholerae cultures grown in presence of AMPs (PmB and LL-37) and compared their OMV protein profiles and AMP resistance with those of OMVs from cultures grown without AMPs. Our data suggest that OMVs from V. cholerae are involved in AMP cross-resistance. This cross- resistance is mediated by a biofilm-associated extracellular matrix protein (Bap1) associated with OMVs of V. cholerae grown with PmB. PLOS Pathogens | www.plospathogens.org 1 October 2013 | Volume 9 | Issue 10 | e1003620