These cellular systems allowed to overcome the problems of limited life span and limited number of primary cells deriving from surgical tissues; moreover, it is a better model respect to the cancer-derived cell lines which can strongly differ from in vivo tissues. In our studies we show that the pIII-deficient strain has an impaired ability to associate to cervical cells and, to a lesser extent, to urethral cells. These observations, together with the evidence that the purified PIII protein is able to specifically bind to all the three cell lines, support the hypothesis that PIII could have a role in PXD101 mouse gonococcal colonization

of the genital tract. The impaired adhesive phenotype was not a secondary effect of the outer membrane reorganization since we demonstrated that deletion of the pIII gene has no major effects on the NVP-HSP990 datasheet expression of the main outer membrane proteins. We previously described an OmpA-like protein in gonococcus, denoted as

Ng-OmpA [25] which plays a significant role in the adhesion and invasion processes into human cervical and AZD9291 cost endometrial cells. These results suggest that the OmpA domain has a redundant function in gonococcus and that it could have a role at different stages of infection; however, additional studies will be needed to explore the respective role of these two proteins in gonococcal pathogenesis. Conclusions In conclusion, we demonstrated that PIII protein of N. gonorrhoeae does not influence the outer membrane integrity as well as bacterial shape, morphology and strain sensitivity to detergents. However, the loss of expression of PIII protein causes a defective membrane localization of NG1873,

a protein having a LysM domain with a putative peptidoglycan binding function. Ureohydrolase Our study also demonstrated that PIII has a role in the interaction with human cervical and urethral cells, suggesting an involvement in the gonococcal adhesion process. Methods Bacterial strains and growth conditions Neisseria gonorrhoeae F62 strain was grown overnight in gonococcus medium (GC) agar (Difco) or in liquid GC broth supplemented with 1% isovitalex (BBL) at 37°C in 5% CO2. Cloning and construction of isogenic mutants The pIII and ng1873 genes devoid of the sequence for the predicted leader peptide (sequences coding for amino acids 1–22) and the stop codon were amplified using the primers FOR-pIII-5′-cgcggatcccatatg GGCGAGGCGTCCGTT-3′ (NdeI site), REV-pIII-5′-cccgctcgagGTGTTGGTGATGATTGCG-3′ (XhoI site), FOR-ng1873-5′-cgcggatcccatatgGCAAATCTGGAGGTGCGCC-3′ (NdeI site), REV-ng1873-5′-cccgctcgagTTGGAAAGGGTCGGAATCG-3′ (XhoI site). The PCR products were inserted into the NdeI/XhoI sites of the pET21b expression vector in order to obtain the pET-pIII-His and pET-ng1873-His constructs. Knockout mutants in F62 strain, in which the pIII and the ng1873 genes were truncated and replaced with an antibiotic cassette, were prepared as described in [25].