Characterization of B.cenocepacia BCAM0223 Mutant Figure 1. Gene organization of the TAA gene cluster in B. cenocepacia K56-2. A) Genomic organization and operon mapping of the TAA gene cluster. The regions amplified by RT-PCR are indicated by filled arrows and the respective primers location is shown in the genes. The regions co-transcribed are identified with dashed arrows. Hyp – hypothetical protein, HK- histidine kinase, TAA – trimeric autotransporter adhesin, OMP – outer membrane protein, RR – response regulator. B) Results from the RT-PCR analysis, showing that no amplification was obtained for regions c), d) and f). Regions a), b) and e) showed amplification indicating that genes are co-trasncribed. Genomic DNA was used as positive control (+). A negative control was performed with RNA without reverse transcriptase, and the result was always negative. M, molecular mass marker. doi:10.1371/journal.pone.0041747.g001 Effect of the BCAM0223 Mutation on Serum Resistance of B. cenocepacia K56-2 Bacterial TAAs are often associated with resistance to comple- ment-mediated serum killing, which represent the first line of innate defense against pathogens [42,43,44]. This, together with the preceding results showing that the BCAM0223 mutant has reduced ability to bind vitronectin (Fig. 3_A), a regulator of the complement system [45], prompt us to investigate the functional consequences of the BCAM0223 mutation on serum resistance. Serum sensitivity assays of B. cenocepacia K56-2 and BCAM0223 mutant were performed using 30% commercial Normal Human Serum (NHS). As shown in Fig. 4, after 1 h of incubation at 37uC, the wild-type strain B. cenocepacia K56-2 is highly resistant to killing by NHS (97% survival; P,0.001), whereas the BCAM0223 mutant strain was killed to a significant extent (54% survival). Very little killing was observed in the control assays prepared with heat- inactivated human serum (hNHS) (Fig. 4). The activation of the complement system can occur via three distinct routes, namely the classical, the mannose binding lectin (MBL) pathway and the alternative pathway. Despite the different activation, classical and lectin pathways have an equivalent progression [46]. To further clarify the role of BCAM0223 in serum resistance, we have conducted additional bactericidal experiments using three different sera, namely, normal human serum containing MgCl2 and EGTA to block both the lectin and classical pathways (here abbreviated to LC-NHS), a C1q-depleted serum (C-NHS) (selectively blocks classical pathway) and factor B- depleted serum (B-NHS) that selectively block the alternative pathway. As shown in Fig. 4, when the wild-type and the BCAM0223 mutant were incubated with LC- NHS little or no killing was observed for both strains. In contrast, the use of a factor B-depleted serum, that selectively blocks the alternative pathway, restored its killing activity against the BCAM0223 mutant (59% survival; P,0.001) but had no effect on the wild-type strain. In PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e41747