Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis Shweta Fulsundar a , Heramb M. Kulkarni b , Medicharla V. Jagannadham b , Rashmi Nair a , Sravani Keerthi b , Pooja Sant a , Karishma Pardesi c , Jayesh Bellare d , Balu Ananda Chopade c, e, * a Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune 411007, India b Centre for Cellular and Molecular Biology, Hyderabad 500007, India c Department of Microbiology, Savitribai Phule Pune University, Pune 411007, India d Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai 400076, India e Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 004, India article info Article history: Received 23 January 2015 Received in revised form 27 April 2015 Accepted 28 April 2015 Available online 1 May 2015 Keywords: Acinetobacter radioresistens OMV Biofilm augmentation Cytotoxicity Apoptosis abstract Acinetobacter radioresistens is an important member of genus Acinetobacter from a clinical point of view. In the present study, we report that a clinical isolate of A. radioresistens releases outer membrane vesicles (OMVs) under in vitro growth conditions. OMVs were released in distinctive size ranges with diameters from 10 to 150 nm as measured by the dynamic light scattering (DLS) technique. Additionally, proteins associated with or present into OMVs were identified using LC-ESI-MS/MS. A total of 71 proteins derived from cytosolic, cell membrane, periplasmic space, outer membrane (OM), extracellular and undeter- mined locations were found in OMVs. The initial characterization of the OMV proteome revealed a correlation of some proteins to biofilm, quorum sensing, oxidative stress tolerance, and cytotoxicity functions. Thus, the OMVs of A. radioresistens are suggested to play a role in biofilm augmentation and virulence possibly by inducing apoptosis. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Outer membrane vesicles (OMVs) are formed by bulging of the outer membrane (OM). This phenomenon makes the neck of OM so constricted that a membrane sac is formed followed by pinching off from cell surface [1]. The process of releasing OMVs is referred to as vesiculation and is not due to the bacterial cell lysis [2,3]. It occurs in both growth and stationary phase [3]. Stress can induce vesic- ulation as the level of protein accumulation in the periplasm gives rise to a periplasmic pressure [4] followed by stress in cell envelope [5] that subsequently increases vesicle production and also affect the dimensions of the released OMVs [6]. Naturally produced ves- icles ranges in size from 50 to 250 nm [5], and often strain dependent [7,8]. OMVs are bilayered [9], the OM of bacterial cell becomes OM of vesicles and periplasm as lumen. Vesicles contain OMPs, periplasmic proteins, phospholipids, LPS, enzymes, genetic material, as well as toxins and other virulence associated factors [8]. OMVs when released in planktonic cells, perform various func- tions such as horizontal transfer of nucleic acid which is an effective way for bacterial cells to acquire resistance genes against antibi- otics [6], defence by delivery of toxins, transfer of nutrients [9], as well as adsorption of antibiotic peptides [10,11] for the survival of bacterial population [11]. They have been detected in the matrix of Helicobacter pylori and found enhancing biofilm [12]. OMVs that are released from gram negative bacteria, possess cytotoxic properties also when present in vivo, hence contributing to pathogenesis [13]. As OMVs play multifaceted roles, they have been studied for their protein content in many bacterial spp. [14], for instance Pseudo- monas syringae [15], Pseudomonas aeruginosa [16], Escherichia coli [17], Brucella melitensis [18], Staphylococcus aureus [19], Man- nheimia haemolytica [20], Xanthomonas campestris pv. campestris [21], as well as Acinetobacter baumannii [22e24]. Acinetobacter is commonly found in the environment and hos- pitals [25,26]. It is one of the Gram negative bacteria found also on healthy human skin microbiome [27e30] as well as upper * Corresponding author. Dr. Babasaheb Ambedkar Marathwada University, Aur- angabad 431 004, India, and Department of Microbiology, University of Pune, Pune 411 007, Maharashtra, India. E-mail addresses: vc@bamu.ac.in, bachopade@gmail.com (B.A. Chopade). Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath http://dx.doi.org/10.1016/j.micpath.2015.04.005 0882-4010/© 2015 Elsevier Ltd. All rights reserved. Microbial Pathogenesis 83-84 (2015) 12e22