However, flow cytometry indicated that GapA-1 is made inaccessible to antibodies on the surface of meningococci by capsule (Figure 3). In order to determine whether capsule expression influences the role of GapA-1 in adhesion to host cells we constructed a gapA-1 deficient derivative of MC58ΔsiaD, which does not Niraparib chemical structure express a capsule. After confirming that GapA-1 expression had been abolished in MC58ΔsiaD ΔgapA-1 (Figure 2, lanes 4 & 5), we determined the capacity

of both strains to associate with HBME cells. GapA-1 deficient non-encapsulated meningococci had a significantly reduced capacity to adhere to monolayers of HBME cells compared to the parent strain (Figure 5), confirming our observation that GapA-1 is required for optimal Saracatinib in vivo host cell adhesion. However, this reduction was not enhanced in the non-encapsulated background, indicating that the role of gapA-1 in the adhesion process was not moderated by the production of capsule. In summary, these experiments show that GapA-1 plays a role in the adherence of N. meningitidis with human cells in a capsule-independent

manner. Figure 5 MC58Δ siaD Δ gapA-1 has a reduced ability to associate with HBME cells compared to MC58Δ siaD. The number of MC58ΔsiaD ΔgapA-1 cells associating was significantly lower than the capsule null (*P = 0.0008). Mean levels shown from three independent experiments, each using triplicate wells. Bars denote standard deviation. Cfu denotes colony forming units. Discussion It is now apparent that many of the classical cytoplasmic house-keeping enzymes, including enolase, FBA and GAPDH, are often localized to the surface of microbial pathogens, where they exhibit various functions, unrelated to their housekeeping roles [36–38]. Currently, there Non-specific serine/threonine protein kinase is considerable interest in identifying the additional roles of these bacterial glycolytic enzymes. In N. meningitidis, enolase was recently

shown to be a surface-localized protein, where it acts to recruit plasminogen onto the bacterial surface [28]. In addition, we have recently demonstrated that FBA is also a partially surface-localized GSK3326595 protein and is required for optimal adhesion to human cells through an unknown mechanism [29]. Furthermore, it is noteworthy that GAPDH is also a multi-functional protein in eukaryotic cells. For example, in addition to its role in central metabolic pathways, GAPDH is involved in controlling cell survival by delaying apoptosis via the inhibition of caspase-dependant proteolysis [39]. This raises the possibility that GAPDH on the surface of invasive bacterial pathogens such as N. meningitidis may influence intracellular processes of host cells to the advantage of the invading organism (including delaying apoptosis). In our study, attempts to purify GapA-1 under native conditions were unsuccessful.