Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Research paper The role of TonB-dependent copper receptor in virulence of Acinetobacter baumannii Sajad Abdollahi a , Iraj Rasooli a,b, ⁎ , Seyed Latif Mousavi Gargari a a Department of Biology, Shahed University, Tehran, Iran b Molecular Microbiology Research Center, Shahed University, Tehran, Iran ARTICLE INFO Keywords: Acinetobacter baumannii TonB-dependent copper receptor Virulence ABSTRACT Acinetobacter baumannii is an opportunistic gram negative pathogen that can adhere to different surfaces and cause different nosocomial infections. To investigate the role of TonB-dependent copper receptor, an outer membrane protein, in virulence of A. baumannii, we deleted this receptor from A. baumannii chromosome. There was a significant decrease in biofilm formation by copper receptor deficient mutant strain. Similarly, the ad- herence to human epithelial cell and the hydrophobicity were declined. The survival rate of the mutant strain in human sera was reduced while no change was observed in motility of strains. In murine pneumonia model, the bacterial lethal dose 0 (LD 0 ), LD 50 and LD 100 were increased for mutant strain. Moreover, in vivo and in vitro experiments revealed changes in growth rate and dissemination of mutant strain; so that the bacterial load of the mutant was significantly reduced in the spleen and lung. The findings suggest a critical role for TonB-dependent copper receptor in virulence of A. baumannii. 1. Introduction Acinetobacter baumannii is a strictly aerobic, catalase positive, oxi- dase negative and opportunistic gram negative pathogen that can ad- here to the biotic and inanimate surfaces and can survive for long time. This bacterium causes different nosocomial and harmful infections like pneumonia and urinary tract, septicaemia, wound sepsis and meningitis (Behnia et al., 2014; Potron et al., 2015). Mortality rates of A. bau- mannii infections ranges from 19 to 54% (Russo et al., 2013) and for its pneumonia is about 35–70% (Vila and Pachón, 2008). Different strains of A. baumannii are resistant to most antibiotics (Peleg et al., 2008; Qureshi et al., 2015). This bacterium is thus classified as one of the most important multidrug-resistant nosocomial microorganisms in the world. Beside the human pathogenicity of A. baumannii, it has also been described as animal pathogen. Several reports have satisfied existence of A. baumannii strains in different wild and domestic birds and animals (Zordan, 2011; Belmonte et al., 2014; Molenaar and van Engelen, 2015). Moreover, mortality rate of 47% was observed in pets that have urinary, respiratory, wound and bloodstream infections associated with A. baumannii (Francey et al., 2000). Many of the virulence proteins like outer membrane protein A (OmpA) (Choi et al., 2005), Omp33 (Smani et al., 2013), biofilm-as- sociated protein (Bap) (Loehfelm et al., 2008), K1 capsular poly- saccharide (Russo et al., 2010), sensor kinase BfmS (Liou et al., 2014), TonBs (Zimbler et al., 2013), Phospholipase D (Jacobs et al., 2010), lipopolysaccharide (Luke et al., 2010) and antimicrobial resistance proteins (Marchand et al., 2004; Bonomo and Szabo, 2006), have been identified to be involved in virulence of A. baumannii. Beside them, about 21 TonB dependent outer membrane receptors have been iden- tified in A. baumannii (Antunes et al., 2011). TonB dependent outer membrane receptors belong to Outer Membrane Proteins (OMPs). These proteins have different roles in bacteria, so that they are involved in transport of siderophore, heme, maltose, vitamin B 12 , nickel and other essential ions into the bacterium cell (Lewis et al., 1997; Chimento et al., 2003; Neugebauer et al., 2005; Schauer et al., 2007; Krewulak and Vogel, 2011). Energy needed for activity of these re- ceptors provided by ExbB-ExbD-TonB system (Postle and Kadner, 2003; Postle, 2007). TonB is a periplasmic protein which interacts with TonB- dependent outer membrane receptors via its C-terminal domain (Postle and Kadner, 2003; Chu et al., 2007). One of TonB dependent outer membrane receptors identified in A. baumannii is TonB-dependent copper (Cu) receptor (previously described as outer membrane re- ceptor, mostly Fe transport with GenBank accession No. gi184156512). It was recently reported that TonB-dependent copper receptor can bind to fibronectin and mediate the attachment of A. baumannii to host cell (Smani et al., 2012). This receptor being the most overexpressed among all other proteins in biofilm form of A. baumannii (Shin et al., 2009), has not been widely studied. In the present study we deleted the TonB- https://doi.org/10.1016/j.meegid.2018.03.001 Received 3 August 2017; Received in revised form 11 February 2018; Accepted 1 March 2018 ⁎ Corresponding author at: Biology Department, Shahed University, Tehran-Qom Express Way, Tehran 3319118651, Iran. E-mail address: rasooli@shahed.ac.ir (I. Rasooli). Infection, Genetics and Evolution 60 (2018) 181–190 1567-1348/ © 2018 Elsevier B.V. All rights reserved. T