Not unexpectedly, considerable variability was observed between human serum samples with those from patient 2 and 3 having the most dramatic reduction in the ability to detect biofilm cell lysates. The opposite effect was observed with sera obtained from biofilm-immunized mice. Mouse antisera strongly recognized proteins in the biofilm cell lysates and was weakly reactive with cell lysates from planktonic pneumococci (Figure 2B). These findings demonstrate that the humoral immune response developed against one growth phenotype is indeed poorly reactive against the other due to
altered protein production. Figure 2 Human convalescent sera has diminished reactivity Cisplatin ic50 against proteins from biofilm pneumococci. Whole cell lysates from biofilm (BF) and planktonic (PK) pneumococci were separated by 1DGE and transferred to nitrocellulose. Membranes were probed using A) convalescent sera from humans recovered from confirmed pneumococcal pneumonia or B) sera from mice immunized with biofilm pneumococci. Identification of proteins produced during biofilm growth that are recognized by convalescent sera As antigenic proteins produced during biofilm formation may represent novel targets for intervention, we identified pneumococcal proteins enhanced
Acalabrutinib concentration during biofilm growth that were also reactive with human convalescent sera. To do so, planktonic and biofilm whole cell lysates were separated by 2DGE and Western blotting was performed with pooled convalescent sera. Consistent with our previous immunoblots, 2DGE-transferred membranes with biofilm cell lysates were less immunoreactive than those loaded with planktonic cell
lysates when probed with the convalescent human sera (Figure 3A). Figure 3 Identification of immunogenic proteins enhanced during pneumococcal biofilm growth. A) Immunoblots of planktonic Baricitinib and biofilm S. pneumoniae cell lysates separated by 2DGE and probed with pooled human convalescent sera. B) Coomassie blue stained 2DGE gel of biofilm proteins showing the 20 immunogenic protein spots (circled in red) selected for analysis by MALDI-TOF. The corresponding spots detected with convalescent sera are circled in the biofilm immunoblot in panel A. By comparing the biofilm 2DGE immunoblots to their corresponding 2DGE Coomassie blue stained gels, we identified 20 protein spots enhanced during biofilm growth that were also immunoreactive (Figure 3B). These spots were excised and a total of 24 proteins were identified by Selleck BIX 1294 MALDI-TOF mass spectrometry (Table 2). Twelve of these 24 proteins had been previously observed to be produced at lower levels during biofilm growth in the analysis of whole cell lysates (Table 1); a finding reflecting the fact that multiple proteins may be present within each 2D-gel spot. Of the remaining 12 proteins only PsrP had been detected as biofilm-growth enhanced during our previous MALDI-TOF analysis (Table 1).