A. Western blot analysis of OM prepared from F62 wild-type (lane 1) and F62ΔpIII strains (lane 2) using mouse anti-PIII serum. B. Expression

of the main component of the gonococcal OM prepared from F62 wild-type (lane 1) and F62ΔpIII strains (lane 2); specific antibodies against each protein were used. C. 2-DE of OM prepared from F62 wild-type (upper panel) and F62ΔpIII strains (lower panel). The PIII protein Selleck Epoxomicin and the protein encoded by the gene ng1873 are shown in circled spots. D. Western blot analysis of total lysates (TL), outer membranes (OM) and inner membranes (IM) from F62 wild-type (lane 1) and F62ΔpIII strains (lane 2) using mouse anti-NG1873 serum. To explore in more detail the composition of the outer membrane, OM deriving from the wild-type and the ΔpIII strains were analyzed by 2D electrophoresis

(Figure 3C). By comparative analysis of MK2206 the 2D electrophoresis maps, only two proteins appeared to be differentially expressed in the OM deriving from the wild-type (upper panel) and absent in the OM deriving from the ΔpIII strain (lower panel). The two spots (circled in Figure 3C) were identified by mass spectrometry and shown to be the protein PIII and the protein encoded by the ng1873 gene. Western blot analysis with mouse anti-NG1873 polyclonal antibodies showed that while the level of expression of NG1873 in total cell lysates from the wild-type and the ΔpIII mutant strains was comparable, the protein was

not detected in the OM from the ΔpIII mutant strain Carnitine dehydrogenase (Figure 3D). Interestingly, the amount of NG1873 was significantly higher in the inner membranes deriving from the ΔpIII mutant strain (Figure 3D) suggesting that the lack of the PIII protein causes a defective outer-membrane localization of NG1873 protein and its accumulation in the inner membrane. Purified PIII is able to bind to human immortalized cervical and urethral cell The C-terminal domain of PIII shows significant homology to OmpA proteins described in other microorganisms and known to mediate adhesion to eukaryotic cells, with identities and similarities ranging from 35 to 45% and from 50 to 60%, respectively. To verify whether the sequence similarity to OmpA was representative also of a functional homology, we tested the ability of PIII to bind epithelial cells. To this aim, the recombinant PIII protein (devoid of the Doramapimod cost signal peptide) was expressed in E. coli, purified from the cytoplasm in its soluble form and tested in the adhesion assay. As cell models we used three immortalized human epithelial cell lines derived from primary ectocervical, endocervical and urethral cells which maintained all main features of primary cells [22, 23]. Cells were incubated with increasing amount of the purified PIII protein and binding measured by FACS analysis. The PIII protein binds all the cell lines tested.