Characterization of B.cenocepacia BCAM0223 Mutant Figure 5. Effect of the BCAM0223 mutation on adherence to and invasion of cultured bronchial epithelial cells. A) Adherence to 16HBE14o- (non-CF) and CFBE41o- (CF) epithelial cell lines by BCAM0223::Tp mutant (open bars) expressed as ratio of B. cenocepacia K56-2 (black bars) adherence. BCAM0223-negative mutant adhered less efficiently than the wild-type strain, particularly to the non-CF cell line (*P,0.01). Fluorescence confocal microscopy images of B. cenocepacia K56-2 and BCAM0223::Tp mutant adhered to 16HBE14o- (non-CF) and CFBE41o- (CF) epithelial cell lines. Left and right panels correspond to representative images obtained from 0.4 mm confocal slices of cells monolayers. The green color reflects the eukaryotic cells plasma membranes labeled with Alexa FluorH 488 anti-mouse E-cadherin (green) while the blue color represents the nucleus stained with DAPI. The red color represents the bacteria labeled with DsRed. B) Invasion of 16HBE14o- (non-CF) and CFBE41o- (CF) epithelial cell lines by BCAM0223::Tp mutant (open bars) expressed as ratio of B. cenocepacia K56-2 (black bars) internalized. The BCAM0223-negative mutant displayed an enhanced invasive capacity on both cell lines, particularly to the non-CF cell line (*P,0.001), when compared with the wild-type strain B. cenocepacia K56-2. All the results are from three independent experiments. doi:10.1371/journal.pone.0041747.g005 ability in comparison to the wild-type B. cenocepacia K56-2 (P,0.01), indicating a role of BCAM0223 in virulence. Discussion Our work has focused on one category of virulence determi- nants, the trimeric autotransporter adhesins (TAAs). To date, very little is known about their mode of action and their contribution for the overall virulence of Bcc members. In a previous study, we have identified in silico seven TAA-encoding genes in the genome of the epidemic strain B. cenocepacia J2315 [13,28]. Among those, three TAA-encoding genes (BCAM0219, BCAM0223 and BCAM0224) are located within a cluster (named TAA cluster) on chromosome 2 of B. cenocepacia J2315. The TAA cluster span approximately 30 kb and includes, beyond the three TAA- encoding genes, six other genes respectively coding for one lipoprotein, two sensor histidine kinases, and three response- regulators [28]. Moreover, it has been suggested that this cluster might represent a prototype of a two-component signal trans- duction cascade, where the two histidine kinases enable bacteria to sense specific environmental stimulus thereby transmitting the signal to response regulators that ultimately control the expression of the TAA-encoding virulence genes [28]. This observation is consistent with recent findings that characterize the sensor kinase BCAM0227 as a novel sensor of the quorum-sensing signal molecule cis-2-dodecenoic acid (BDSF) [50]. Figure 6. Kaplan-Meier graph of Galleria mellonella survival after injection (10 CFU/larvae) with wild-type B. cenocepacia K56-2 and the BCAM0223::Tp mutant (P,0.01, for comparison of the wild-type and the mutant). Uninfected larvae injected with NaCl 0.9% were used as control. Results represent means of three independent determinations for 10 animals per treatment. doi:10.1371/journal.pone.0041747.g006 In this work, we have continued the characterization of the TAA cluster. Firstly, we have used RT-PCR to determine the transcriptional organization of the cluster. The nine clustered genes were shown to be organized in six transcriptional units. The four virulence effector genes of the cluster were grouped in two divergent sub-clusters, grouping respectively, two TAAs (BCAM0224 and BCAM0223) and one TAA together with a lipoprotein (BCAM0219 and BCAM0220) (Fig. 1). We hypoth- esized that these putative virulence genes are expressed under certain external environmental conditions sensed by the two histidine kinases. Interestingly, in a comparative transcriptomic analysis of two clonal variants of B. cenocepacia (IST439 and IST4113), BCAM0219 and BCAM0223 are proposed to be up- regulated in IST4113, the clinical isolate associated with a multiple resistance phenotype and long-term persistence [51]. In addition, a study by Bernier and Sokol [52] reveals that the overexpression of BCAM0223 enhances survival of B. cenocepacia in the lungs of a CF rat model. Taken together, these findings prompted us to undertake this study, in which we functionally analyzed and compared wild-type with mutated B. cenocepacia K56-2 (BCAM0223::Tp), aiming to investigate the role of this annotated TAA-encoding gene in the overall virulence of B. cenocepacia J2315. Unfortunately, despite many efforts, we failed to clone the entire BCAM0223 gene and hence we were unable to perform the mutant complementation analysis. However, as shown in figure 1, BCAM0223 is the downstream gene in a bicistronic operon with BCAM0224, so its disruption should not have a polar effect on the expression of the downstream gene, BCAM0222. RT-PCR assays using mRNA isolated from the B. cenocepacia K56-2 (BCAM0223::Tp) mutant confirm the lack of polarity (data not shown). Hemagglutination is one of the various functions attributed to TAAs [53,54]. Here we demonstrate that the BCAM0223 mutation caused a marked decrease in the hemagglutination- positive phenotype of B. cenocepacia K56-2. This finding is consistent with the in silico analysis of BCAM0223 in which various hemagglutinin motifs (8) were predicted (Fig. 2). It will be interesting to see whether these motifs have immunogenic properties making them useful candidates to develop a protective vaccine against B. cenocepacia. Next we analyzed the impact of the BCAM0223 mutation on adherence to immobilized extracellular matrix proteins (ECM). Among the five ECM proteins tested, the BCAM0223::Tp mutant showed only a significantly reduced adhesion to vitronectin (Fig. 3_A). Further work is needed to prove direct association between vitronectin and BCAM0223 and ultimately map the BCAM0223 region(s) involved in such interaction(s). Notably, vitronectin has been described to participate in the adhesion of Gram-negative and Gram-positive pathogens to host cells and exists in high amounts in an insoluble form in lungs tissues [55]. Moreover, vitronectin has two separate binding sites for pathogens and host epithelial cells, thereby serving as a bridge to connect host and bacterial cells [56–58]. It has been demonstrated that other PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e41747