TAAs are also able to bind vitronectin, such as DsrA from Haemophilus ducreyi [59], Hsf from H. influenzae type b [60] and UspA2 from Moraxella catarrhalis [61]. Besides the reduced capacity to adhere to vitronectin, the B. cenocepacia K56-2 (BCAM0223::Tp) mutant is also impaired in biofilm formation on polystyrene surfaces (Fig. 3_B). Our study thus suggests that, like many other TAAs [20,23,41], BCAM0223 may be necessary for biofilm formation. In general, the production of biofilm may contribute for long-term persistence of virulent pathogens in their host [62]. This is probably the case of Bcc long- term infection, where the establishment of biofilms in the respiratory tract of in CF patients may confer antibiotic resistance and protect bacteria from attack by the host’s immune system [6]. Several TAAs have been described to directly bind various factors of the complement system, thereby representing critical components associated with bacterial complement escape [46]. These include, among others, UspA1 and UspA2 from Moraxella catarrhalis that have been reported to bind C4b-binding protein [63], YadA from Y. enterocolitica reported to bind factor H (FH), the negative regulator of the alternative complement pathway [64] and EibD from E. coli, which binds IgG and IgA through its coiled- coil stalk domain [65]. However, unlike other bacterial pathogens, in Bcc bacteria, the mechanisms of human serum resistance are poorly understood. To date, only the O-antigen portion of LPS has been described to confer serum-resistant to B. cenocepacia K56-2 [66]. In this study, we investigated the impact of the BCAM0223 mutation on serum resistance. Our results show a marked reduction (50%; P,0.001) in the serum resistance of B. cenocepacia K56-2 (BCAM0223::Tp) mutant. Furthermore, we also present evidences that the killing of BCAM0223 mutant required the classical complement pathway but not the lectin or alternative pathways (Fig. 4). Interestingly, as stated above, it has been shown that the disruption of BCAM0223 gene caused a marked inhibition of bacterial adhesion to vitronectin. Since vitronectin, beside its function as a cell-adhesion molecule, is also a human plasma protein, acting as potent inhibitor of complement activation, we hypothesized that BCAM0223 by interacting with vitronectin can confers protection to B. cenocepacia K56-2 against the membrane attack complex (MAC) of complement. Further work is needed to elucidate the molecular details of serum resistance mediated by BCAM0223, namely the stage of the classic complement cascade that is blocked. These studies will analyze the rate and nature of complement components deposited on the bacterial cell surface of both wild type and BCAM0223 mutant. Ultimately, we intend to map the BCAM0223 region(s) involved in such interaction(s). Bcc strains were able to adhere to and invade cell culture monolayers of human respiratory epithelial cells with various efficiencies. However, many aspects about the molecular mechan- isms underlying such events, particularly the nature of bacterial ligands and their cognate host receptors, remains to be elucidated [8]. In this study, we examined the functional consequences of the BCAM0223 mutation on in vitro bacterial adherence to and invasion of two human bronchial epithelial cell lines (16HBE14o- and CFBE41o-) which have respectively a non-Cystic Fibrosis (CF) and CF phenotype [30,31]. Results obtained revealed a reduction of adherence of the BCAM0223::Tp mutant compared to the parent strain. This observation is particularly significant for host References Characterization of B.cenocepacia BCAM0223 Mutant cells with a non-CF phenotype (Fig. 5_A), suggesting that, at least in vitro and under the experimental conditions used, CF cells may have exposed on cell surface, a lower level of BCAM0223 adherence receptors. On the other hand, surprisingly, we found that while with a reduced adherence phenotype, the BCAM0223::Tp mutant invades host cells better than the wild- type strain (Fig. 5_B). The reason for this is still unclear. We hypothesized that since B. cenocepacia K56-2 has multiple multi- functional TAAs and other conventional adhesins as cell-surface components, including BCAM0219 and BCAM0224, it is perhaps not surprising that the ablation of one of them, such as BCAM0223, could further improve the function of other specialized invasion factors. Moreover, it also remains possible that the impaired cell adhesion observed in the BCAM0223::Tp mutant appeared as an indirect effect due to its increased invasion ability. In this context, further works will be required to clarify this observation and assign the function of the various B. cenocepacia TAAs in host cell invasion. Finally, we have used the host Galleria mellonella wax moth larvae as a model for assessing the in vivo role of BCAM0223 in B. cenocepacia virulence. We have observed that although moder- ated, the absence of BCAM0223 impairs the virulent phenotype of B. cenocepacia K56-2 (Fig. 6). Thus, we believe that BCAM0223 should be included in the large arsenal of virulence factors encoded by B. cenocepacia, thereby contributing to its overall pathogenicity. In conclusion, in this study we have performed the functional analysis of a TAA mutant of B. cenocepacia K56-2, which is defective in one (BCAM0223) of the seven predicted TAA genes encoded by its genome. The phenotypic analysis of this mutant strongly suggests that BCAM0223 is a multi-faceted adhesin that possesses hemagglutinin activity and represents a vitronectin- binding protein that may directly contributes to the virulence of B. cenocepacia K56-2, complement evasion and bacterial binding to epithelial host cells. Supporting Information Table S1 (PDF) Acknowledgments Oligonucleotide primers used in this study. We acknowledge Prof. J. LiPuma from University of Michigan, who kindly provided B.cenocepacia K56-2 strain. We also acknowledge Dr. A. Vergunst and Dr. D. O’Callaghan from INSERM, Universite´ de Montpellier, France, for kindly provided the plasmid encoding the red fluorescent protein DsRed (pIN29). We gratefully acknowledge Prof. M. Prieto from the Centro de Qu´ımica F´ısica Molecular, Instituto Superior Te´cnico, Portugal, for use of the confocal microscope. We thank Sandra Pinto for her assistance with the confocal microscopy. We thank Dr. D. Gruenert, California Pacific Medical Center Research Institute, San Francisco, for kindly providing us with the 16HBE14o2 and CFBE41o2 cell lines. Author Contributions Conceived and designed the experiments: AMF. Performed the experi- ments: DMH. Analyzed the data: AMF DMH. Contributed reagents/ materials/analysis tools: AMF. Wrote the paper: AMF DMH. 1. Mahenthiralingam E, Urban TA, Goldberg JB (2005) The multifarious, multireplicon Burkholderia cepacia complex. Nat Rev Micro 3: 144–156. 2. Mahenthiralingam E, Vandamme P, Campbell ME, Henry DA, Gravelle AM, et al. (2001) Infection with Burkholderia cepacia complex genomovars in patients with cystic fibrosis: virulent transmissible strains of genomovar III can replace Burkholderia multivorans. Clin Infect Dis 33: 1469–1475. 3. Drevinek P, Mahenthiralingam E (2010) Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence. Clin Microbiol Infect 16: 821–830. 4. LiPuma JJ, Dasen SE, Stull TL, Nielson DW, Stern RC (1990) Person-to-person transmission of Pseudomonas cepacia between patients with cystic fibrosis. Lancet 336: 1094–1096. PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e41747