Rabbit polyclonal GapA-1 antiserum was used for immunoblot analysis of whole cell proteins from different clinical isolates of known MLST-type. These strains were representatives from lineages commonly

causing invasive meningococcal disease. This showed that they all express GapA-1 suggesting that GapA-1 is constitutively-expressed in N. meningitidis. A GapA-1 knock-out mutant was created in N. meningitidis strain MC58 to facilitate studies of the potential role of GapA-1 in the pathogenesis of meningococcal disease. www.selleckchem.com/products/DAPT-GSI-IX.html The GapA-1 mutant grew at the same rate (in broth culture and on solid media) as the wild-type and the complemented mutant strains, demonstrating that GapA-1 is not required for growth of the meningococcus under in vitro conditions. No differences in either colony or bacterial cell morphology (using light microscopy) were observed. In a previous study, Grifantini et al. used microarrays to show that expression of gapA-1 was up-regulated in meningococcal strain MC58 (4.8-fold) following contact for 30 min with human 16HBE14 epithelial cells [27]. Subsequent flow cytometry experiments showed that GapA-1 could be detected on the cell Selleck JNK inhibitor surface of free grown

and adherent meningococci [27]. However, the methodology used involved a pre-treatment of cells with 70% ethanol to permeabilize the capsule layer, thus making it unclear if GapA-1 is antibody-accessible in encapsulated meningococci. In our study, GapA-1 could only be detected on the meningococcal cell surface in mutants lacking capsule, suggesting that GapA-1 is usually masked by this structure. In our adhesion experiments using siaD-knockout meningococci, the GapA-1 mutant strain

exhibited a similarly significantly reduced capacity to adhere to host cells compared to the GapA-1 mutant in an encapsulated strain suggesting that the presence of capsule does not affect the role of GapA-1 in the adhesion process. It is not obvious why the influence of GapA-1 on adhesion is not itself modulated by the presence of masking capsule buy Y-27632 since the removal of capsule does increase the ability of meningococci to bind host cells via outer membrane adhesins [4]. In our adhesion experiments the binding of strains lacking capsule was approximately two-fold higher than the cognate encapulsulated strains (Figure 4 &5). This agrees with previous studies comparing the adherence of encapsulated and non-capsulated serogroup B meningococci to macrophages and buccal epithelial cells, where four-fold and less than two-fold increases, respectively, in adhesion were seen when capsule production was abolished [40, 41]. Thus, it is possible that the influence of surface-localised GapA-1 on adhesion to host cells is indirect, possibly involving its enzymatic activity, and that a direct interaction of GapA-1 with the host cell surface is not required.