Characterization of B.cenocepacia BCAM0223 Mutant Figure 2. Hemagglutinin motifs in the BCAM0223 protein and effect of the BCAM0223 mutation on hemagglutination ability. A) Schematic illustration of the domain organization and coiled-coil prediction of BCAM0223 protein from B. cenocepacia J2315 using data program (http://toolkit.tuebingen.mpg.de/dataa). The protein comprises the typical membrane-anchor domain of TAAs at the C-terminal, a signal peptide (SP) at the N-terminal, two YadA-like heads domains and several connector domains (Neck and DALL). Using Pfam database (http://pfam.sanger.ac.uk/) numerous Hep-Hag repeats and hemagglutinin domains (Him) were found in BCAM0223 protein. Pfam also predicted the YadA-like C-terminal corresponding to the membrane-anchor and an extended SP. B) Hemagglutination ability of the wild type B. cenocepacia K56-2 and BCAM0223::Tp mutant, using 3% sheep red blood cells. PBS was used as a negative control. The highest dilution giving hemagglutination was taken as the titre. doi:10.1371/journal.pone.0041747.g002 addition, to distinguish between the classical and lectin pathways, we conducted assays using the C-NHS serum. No or minimal killing was observed for both strains (Fig. 4). Taken together, these data suggest that the killing of the serum-sensitive BCAM0223 mutant is mediated through the classical complement pathway but not the mannose binding lectin or alternative pathway. Effect of the BCAM0223 Mutation on Adherence to and Invasion of Cultured Bronchial Epithelial Cells by B. cenocepacia K56-2 As their name suggest, TAAs such as YadA, HadA, NadA, YadBC and ApiA have been directly associated with bacterial adherence to and invasion of mammalian host cells [14,18,47,48,49]. Therefore, we have used a CFU-based quanti- fication assay to evaluate the in vitro capacity of the mutant B. cenocepacia K56-2 (BCAM0223::Tp) to adhere to monolayers of two human bronchial epithelial cell lines (16HBE14o- and CFBE41o-) which have respectively a non-Cystic Fibrosis (CF) and CF phenotype [30,31]. As shown in Fig. 5_A, the BCAM0223-negative mutant adhered less efficiently than the wild-type strain, partic- ularly to the non-CF 16HBE14o- cell line (approximately 50% reduction; P,0.01). Analysis of the stained cells by confocal immunofluorescence microscopy confirm this result, revealing that, compared with the wild type, the B. cenocepacia K56-2 (BCAM0223::Tp) mutant is impaired in its adherence to bronchial epithelial cells (Fig. 5_A, bottom panel). Thus, these in vitro results suggest that the TAA BCAM0223 is needed to maximize adhesion to human bronchial epithelial cells. Furthermore, we have also evaluated by an antibiotic protection assay, the ability of the B. cenocepacia K56-2 (BCAM0223::Tp) mutant to invade host epithelial cells. Surprisingly, it was observed that in contrast to the adherence, the BCAM0223-negative mutant, compared with the wild-type strain B. cenocepacia K56-2, displayed an enhanced invasive capacity on both cell lines, particularly to the non-CF 16HBE14o- cell line (approximately 2 fold increase; P,0.001) (Fig. 5_B). Taken together, our results suggest that the TAA BCAM0223 is required for B. cenocepacia maximal adherence to but not for invasion of cultured human respiratory epithelial cells. The Mutant B. cenocepacia K56-2 (BCAM0223::Tp) Exhibits Reduced Virulence in a Galleria Mellonella Model of Infection Finally, in order to evaluate the effect of the BCAM0223 mutation on B. cenocepacia virulence, we compared the ability of the wild-type and the BCAM0223::Tp mutant to kill the larvae of the insect model Galleria mellonella. As shown in Fig. 6, 96 h following infection, the BCAM0223::Tp exhibited attenuated (20%) killing PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e41747