The role of filamentous hemagglutinin adhesin in adherence and biofilm formation in Acinetobacter baumannii ATCC19606 T Shakiba Darvish Alipour Astaneh a , Iraj Rasooli a, b, * , Seyed Latif Mousavi Gargari b a Department of Biology, Shahed University, Opposite Imam Khomeini's Shrine, Tehran-Qom Express Way, Tehran, Iran b Molecular Microbiology Research Center, Shahed University, Tehran, Iran article info Article history: Received 22 April 2014 Received in revised form 14 June 2014 Accepted 21 July 2014 Available online 30 July 2014 Keywords: TPS system FHA-like Acinetobacter baumannii Adhesion Biofilm abstract Filamentous hemagglutinin adhesins (FHA) are key factors for bacterial attachment and subsequent cell accumulation on substrates. Here an FHA-like Outer membrane (OM) adhesin of Acinetobacter baumannii ATCC19606 T was displayed on Escherichia coli. The candidate autotransporter (AT) genes were identified in A. baumannii ATCC19606 T genome. The exoprotein (FhaB1) and transporter (FhaC1) were produced independently within the same cell (FhaB1C1). The fhaC1 was mutated. In vitro adherence to epithelial cells of the recombinant FhaB1C1 and the mutant strains were compared with A. baumanni ATCC19606 T . A bivalent chimeric protein (K) composed of immunologically important portions of fhaB1 (B) and fhaC1 (C) was constructed. The mice vaccinated with chimeric protein were challenged with A. baumannii ATCC19606 T and FhaB1C1 producing recombinant E. coli. Mutations in the fhaC1 resulted in the absence of FhaB1 in the OM. Expression of FhaB1C1 enhanced the adherence of recombinant bacteria to A546 bronchial cell line. The results revealed association of FhaB1 with bacterial adhesion and biofilm for- mation. Immunization with a combination of recombinant B and K proteins proved protective against A. baumanni ATCC19606 T . The findings may be applied in active and passive immunization strategies against A. baumannii. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Acinetobacter baumannii is harboring a number of virulence factors. These factors include the ability to obtain essential nutri- ents such as iron, the adhesion to and subsequent destruction of epithelial cells, and the ability in some strains to produce gelatinase and proteinase that damage host tissues. A. baumannii also has the ability to form biofilm, which may play a role in the process of colonization [1]. Biofilm helps the bacteria resist disinfection while allowing the participating cells to acquire resistance genes [2] further facilitating the persistence of the pathogen [3]. Nowadays, scientists have focused on surface antigens [4e8]. Serological methods and fatty acid analysis indicated immunogenicity of lipopolysaccharide purified from A. baumannii [9]. Siderophores are secreted in the external melieu where they compete with host iron binding proteins to capture iron by forming iron-siderophore complex. This gets internalized through the specific outer mem- brane protein receptors, termed as Iron Regulated Outer Membrane Proteins (IROMPs) [10]. Vaccination with outer membrane proteins (OMPs) and antibody-based therapies may be valuable approaches to prevent the morbidity and mortality caused by this pathogen [5,11]. The rOmpA vaccine has been shown to protect mice from lethal infection caused by extreme-drug-resistant (XDR) A. bau- mannii. The rOmpA vaccine resulted in an enhanced Type 2 im- mune response, accompanied by substantial IL-4-inducing T cell epitope spreading and restricted IFN-g einducing epitopes [12]. The refinement of genomic and proteomic techniques are signifi- cant prospects for the development of recombinant vaccines. The investigators showed that killed but metabolically active (KBMA) vaccines stimulate strong immune responses [13]. In order to identify a candidate antigen in A. baumannii, the immunodominant targets from A. baumannii membrane protein preparations were determined following systemic infection [14]. Further to these studies new approaches are needed to expand our understanding of the basic features of this organism essential to control the spread of A. baumannii infections which will ultimately end up at development of effective means to prevent and/or treat this harmful pathogen. To gain greater insight into A. baumannii virulence factors, our search of A. baumannii ATCC 19606 T genome revealed three open reading frames (ORFs) encoding putative protein autotransporter of Two-partner secretion system (TPS) types. * Corresponding author. Biology Department, Shahed University, Tehran-Qom Express way, Tehran 3319118651, Iran. Tel.: þ98 21 51212200; fax: þ98 21 51212201. E-mail addresses: rasooli@shahed.ac.ir, irasooli@yahoo.com (I. Rasooli). Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath http://dx.doi.org/10.1016/j.micpath.2014.07.007 0882-4010/© 2014 Elsevier Ltd. All rights reserved. Microbial Pathogenesis 74 (2014) 42e49