Figure 6. Pneumococcal modulate pneumolysin, CbpA, and PsrP during biofilm growth. Immunoblots comparing protein expression in whole cell lysates of planktonic and biofilm-derived bacteria. Whole cell lysates (5 mg) were confirmed to be equally loaded by Coomassie brilliant blue staining. Membranes were probed with antisera to pneumolysin (Ply), choline binding protein A (CbpA), and the pneumococcal serine-rich repeat protein (PsrP). For PsrP immunodot blot was used because PsrP is glycosylated and separates at a molecular weight of .4000 kDa, thus it does not enter SDS-PAGE gels normally used to separate proteins [26]. doi:10.1371/journal.pone.0028738.g006 As PsrP binds to surface-exposed Cytokeratin 10, which is present on A549 cells but absent in Detroit 562 cells [26], we sought to determine the sole contribution of PsrP to the hyper- adhesive biofilm phenotype. To do this, we tested the ability of biofilm and planktonic cultures of T4 DpsrP, a PsrP deficient mutant, to adhere to these cells (Figure 3A–B). As expected, planktonic T4 DpsrP failed to adhere A549 cells but bound normally to Detroit 562 cells. Importantly, biofilm-derived T4 DpsrP still adhered to A549 and Detroit cells at levels 4 to7-fold greater than its planktonic counterparts, indicating that other factors including, but possibly not limited to, ChoP and CbpA were involved in adhesion. In support of this notion, the serotype 14 clinical isolate used in Figure panel 3B, which naturally lacks psrP (i.e. as determined by comparative genomic hybridization [40]), also showed enhanced biofilm-mediated adhesion. Planktonic growth of biofilm pneumococci partially restores virulence Finally, we sought to determine the durability of the observed biofilm phenotype. We did this by testing the virulence of planktonic pneumococci derived from a mature biofilm sample. Following intranasal and intraperitoneal challenge, biofilm- derived planktonic pneumococci remained attenuated colonizing the nasopharynx normally but being unable to enter the bloodstream or rapidly kill mice, respectively (Figure 2A, 2C). In contrast, following intratracheal challenge, biofilm-derived plank- tonic pneumococci were able to establish pneumonia and present Altered Virulence of Biofilm Pneumococci in the lungs at bacterial titers equivalent to mice challenged with stock planktonic bacteria. Interestingly, biofilm-derived planktonic pneumococci remained unable to enter the bloodstream (Figure 2B), suggesting that they had an intermediate phenotype that specifically affected translocation into the bloodstream or survival therein. Discussion Intranasal and intratracheal challenge of mice with disrupted mature biofilms allowed us to directly test whether pneumococci within biofilms were in a virulent state and modeled the aspiration of bacterial aggregates that might be present in mucosal secretions from the nasopharynx or that which might be introduced during intubation [57,58]. Our results indicate that pneumococci within biofilms are highly suited for attachment to mucosal epithelial cells, but as a result are avirulent. This was unexpected as the formation of biofilms has been suggested to be a pivotal event to numerous infectious diseases [59]. One important limitation of this study is that this approach does not examine the pathogenic potential of biofilms that form in vivo and de novo, such as within the nasopharynx during normal colonization. Thus there is the possibility that in vivo biofilms might act differently. Based on our experimental results, the hyper-adhesive pheno- type of biofilm pneumococci could be attributed to: i) reduced capsule which exposes bacterial surface proteins [53], ii) selection for the transparent phenotype which carries greater amounts of ChoP that binds to the host-protein PAFr [60], iii) enhanced production of CbpA which binds to Laminin receptor [20], as well as PsrP, which binds to Keratin 10 [26]. Unconfirmed by protein analysis, but possibly also contributing to the hyper-adhesive phenotype, we observed increased expression of the gene encoding Neuraminidase A by qRT-PCR, which has been shown to enhance bacterial adhesion by cleaving sialic acid moieties and thereby exposing cryptic ligands on the host-cell surface [61]. The attenuated phenotype of biofilm pneumococci could be attributed to: i) a reduced metabolic rate that would delay its ability to respond to stressors in a novel host-environment, ii) enhanced ChoP, which would enhance opsonization by C-reactive protein [55], iii) reduced capsule, which would also facilitate phagocytosis [34], iv) a reduction in pneumolysin production [25], v) reduced PcpA and possibly type I pilus [62]. While a reduction in capsule and pneumolysin expression along with enhanced neuraminidase has been shown for S. pneumoniae biofilms [14,15,63], ours is the first study to suggest they act collectively to dramatically impact the ability of biofilm pneumococci to progress from the nasopharynx and cause invasive disease; in particular bloodstream infections. Thus, implying that pneumococci within biofilms do not directly contribute to the development of invasive disease. Using TEM to examine pneumococci within a mature biofilm structure we were surprised to determine that only a small percentage of the mature biofilm was composed of electron dense and presumably viable pneumococci. A finding that suggests robust pneumococcal biofilm formation occurred through the accumulation of dead pneumococci. Most recently, Trappetti et al. have shown that it is the opaque phase variant of S. pneumoniae that is responsible for formation of the EPM and not the transparent. As our biofilms contained both transparent and opaque S. pneumoniae, the opaque variant most likely accounts for the EPM we detected by electron microscopy. Furthermore, and in contrast to our in vivo findings, Trappetti et al. observed that opaque but not transparent biofilm-derived pneumococci, were able to translocate from the nasopharynx to the lungs and brain of mice [19]. One possible explanation for this discrepancy in results is that the PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28738