prototype for this family of proteins [16], BadA from Bartonella henselae [17], NadA from Neisseria meningitidis [18], Hia from Haemophilus influenza [19], an IgD-binding protein from Moraxella catarrhalis [20], AipA and TaaP from Proteus mirabilis [21], BpaA from Burkholderia pseudomallei [22], SadA from Salmonella enterica [23] and Cha from Haemophilus cryptic genospecies [24]. All TAAs share a common domain architecture, where each monomer comprises an integral membrane-anchored C-terminal formed by four beta strands and a surface exposed passenger domain consisting of a neck, a stalk and an N-terminal head [25]. Contrary to the stalk and the head, the C-terminal translocator domain is highly conserved among TAAs and therefore is used as the defining element of the family [26]. Although it is poorly understood, the trimerization of TAAs is a multiple-step process, in which a C-terminal beta barrel membrane pore is formed, followed by the translocation of the passenger domain to the extracellular space, via a type V protein secretion pathway (T5SS) [27]. The passenger domains confer the functionalities of the TAAs; they contain repetitive sequence motifs and are variable in sequence and lengths [27]. We have previously conducted a computational analysis of the genome sequence of B. cenocepacia strain J2315 (ET12 lineage) and we identified a novel cluster of genes encoding 3 potential trimeric autotransporter adhesins (TAAs) (BCAM0219, BCAM0223 and BCAM0224), 1 outer membrane protein (BCAM0220), 2 sensor histidine kinases (BCAM0218, BCAM0227) and 3 transcriptional regulators (BCAM0221, BCAM0222, and BCAM0228) [13,28]. This cluster, here designated TAA cluster, is located within a region that corresponds to an extension of the cci island previous described [29]. In addition, we have identified BCAM0224, a gene encoding a TAA that plays a role in adhesion and virulence [28]. In the present study, we intend to elucidate the function of the BCAM0223 gene that is located near BCAM0224 and encodes another predicted TAA. For that purpose, we have constructed a BCAM0223-negative B. cenocepacia strain and characterized its ability to adhere to ECM proteins, to form biofilm, to resist to human serum, to hemagglutinate red blood cells and to cause disease in the G. mellonella model. Moreover, the role of BCAM0223 in adherence to or invasion of human CF and non-CF airway epithelial cell lines was also investigated. Taken together the findings indicate that BCAM0223, from the epidemic strain B. cenocepacia K56-2, is a novel multifunctional TAA that has hemagglutination activity, anti-complement activity and simultaneously is required for virulence and maximal host cell adherence. Materials and Methods Bacterial Strains and Growth Conditions B. cenocepacia clinical isolate K56-2 was kindly provided by Prof. J. J. LiPuma (University of Michigan, USA). Bacteria were routinely cultured in Luria–Bertani (LB) broth (Conda, Pronadisa), at 37uC with orbital agitation 250 rpm. When appropriate, media was supplemented with 150 mg trimethoprim mg/L (for B. cen- ocepacia BCAM0223::Tp mutant). For functional studies, B. cenoce- pacia and mutant were grown in 24-well polystyrene microplates (Greiner Bio-One), for 17 h under limited oxygen supply (,5% oxygen), in LB media containing NaCl (300 mM) and H2O2 (10 mM) (initial OD640 0.2) at 37uC and 60 r.p.m. Cell Lines and Cell Culture Two human bronchial epithelial cell lines were used; 16HBE14o- cells, which are normal lung cells expressing a functional CF transmembrane conductance regulator, and Characterization of B.cenocepacia BCAM0223 Mutant CFBE41o- cells, which are homozygous for the delta F508 mutation corresponding to a CF airway. Both cell lines were immortalized and characterized by Dr. Gruenert and co-workers [30,31] and kindly provided for this work. Cells were routinely maintained in fibronectin/vitrogen coated flasks in Minimum Essential Medium with Earle’s salt (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Lonza), 0.292 g/L L- Glutamine (Sigma) and Penicillin/Streptomycin 100 U/mL (Gibco) in a humidified atmosphere at 37uC with 5% CO2. Total RNA Isolation Total RNA was isolated from B. cenocepacia K56-2 grown under the conditions described above. Cells were harvested after 17 h of growth and treated with RNAprotect Bacteria reagent (Qiagen) following the manufacturer’s instructions. Bacterial lysis was achieved by enzymatic lysis with lysozyme and proteinase K (Qiagen). Total RNA was purified from bacterial lysate using RNeasy mini kit (Qiagen), according the manufacturer’s protocol. To avoid contamination with genomic DNA, RNA was treated with RNase-free DNA digestion kit (Qiagen) in column during the purification process, for 1 h at room temperature. After the RNA isolation a second step of DNase (Qiagen) treatment was performed (RNase-free DNA digestion kit), using 1 mL DNase for 1.5 mg of RNA to be treated, during 1 h at 37uC, followed by inactivation for 5 min at 65uC, according the manufacturer’s instructions. All steps described above were executed using RNase- free material. Total RNA concentration was estimated using a UV spectrophotometer (ND-1000 UV-Vis, NanoDrop Technologies, USA). Reverse Transcription PCR Reverse transcription PCR (RT-PCR) was carried out using the OneStep RT-PCR kit (Qiagen), with primers listed in Table S1, following the manufacturer’s protocol. Briefly, 800 ng of total RNA were used and combined with the reverse transcriptase/ polymerase mix. The first step of amplification was carried for 30 min at 50uC to perform the reverse transcription. The next steps allows the PCR amplification and we performed 35 cycles using annealing temperatures of 54uC (primers 219–220, 223–224, 225–227) and 58uC (primers 216–218, 220–221, 224–225). The amplified RT-PCR products were run in 0.8% agarose gel. For each PCR, the appropriate controls with water and RNA in the absence of reverse transcriptase and presence of polymerase were included to ensure that the amplifications obtained were a result of cDNA and not of contaminating genomic DNA. Construction of a BCAM0223 -Negative Mutant A 1.8-kb BCAM0223 fragment was amplified by PCR (Platinium taq DNA polymerase, Invitrogen) from B. cenocepacia K56-2, using primers 223fF1 and 223fR1 (Table S1), containing KpnI and HindIII restriction sites, respectively. The PCR product was digested and cloned into pDrive (Qiagen), creating the plasmid pDM7. The 1010 bp PstI fragment from pUC-Tp [32] containing the trimethoprim (Tp) cassette was inserted in the single PstI restriction site of the BCAM0223 fragment cloned in pDM7, in the same orientation as the BCAM0223 gene, creating the plasmid pDM8. This plasmid was introduced into B. cenocepacia K56-2 by electroporation. Transformants were selected on LB agar supplemented with 150 mg/L trimethoprim for 48 h at 37uC. To distinguish between single-and double-crossover mu- tants, trimethoprim-resistant colonies were screened by replica plating for kanamycin sensitivity. The candidate insertion mutants were further characterized by PCR using primers, 223tF2 and 223tR2 (Table S1), which allowed identification of the PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41747