Vaccine potential of Adhesin Complex Protein (ACP) from <i>Neisseria gonorrhoeae</i>

Sexual transmitted disease Gonorrhoea is caused by the organism Neisseria gonorrhoeae, infecting ~106 million cases annually. Currently, a lack of an effective vaccine against this pathogen and treatments using last generation of antibiotics are misleading due to emerging antibiotic-resistant superbugs. Recently has been described a small protein in a closed related bacteria, N. meningitidis (Nm), termed as an Adhesin Complex Protein (ACP). Nm-ACP is an outer-membrane protein which is a conserved protein in commensal and pathogenic bacteria. Besides Nm-ACP is capable of induce cross-protective bactericidal antibodies. A homologue gene, ng-acp (NGO1981) from Neisseria gonorrhoeae strain P9-17 is highly conserved among gonococcal isolates reported until the date. The ng-acp gene product was cloned into pRSET-A and pET-22b cloning vector systems and expressed as a recombinant protein in E. coli BL21pLysS to be used in immunization trials in murine model using a range of adjuvants and delivery formulations. Raised mice serum demonstrated a great reactivity against recombinant rNg-ACP by ELISA and displayed cross-strain reactivity in gonococcal outer-membrane (OMV) and lysate preparations from N. gonorrhoeae strains P9-17 and FA1090 by western-blot. Antisera r-Ng-ACP showed high bactericidal properties against homologous and heterologous wild type strains compared to the knockout strains. Furthermore, Ng-ACP plays a role of association on different epithelial cells showing a reduction ̴75-50% by comparison the wild-type and knockout. Three–dimensional structure of rNgACP the overall fold resemble of the lysozyme inhibitors from Salmonella typhimurium (PliC family) and recently described Neisseria meningitidis.Taken all together, suggest that Ng-ACP from N. gonorrhoeae is a potential candidate to develop an anti-gonococcal vaccine

Basic methods for examining <i>Neisseria gonorrhoeae</i> interactions with host cells in vitro

The obligate human pathogen Neisseria gonorrhoeae colonizes primarily the mucosal columnar epithelium of the male urethra and the female endocervix. In addition, gonococci can infect the anorectal, pharyngeal, and gingival mucosae and epithelial cells of the conjunctiva. More rarely, the organism can disseminate through the bloodstream, which can involve interactions with other host cell types, including blood vessel endothelial cells and innate immune cells such as dendritic cells, macrophages, and neutrophils. “Disseminated gonococcal infection” is a serious condition with various manifestations resulting from the seeding of organs and tissues with the pathogen. The host response to gonococcal infection is inflammatory. Knowledge of the biology of gonococcal interactions has been served well through the use of a wide variety of ex vivo models using host tissues and eukaryotic cell monocultures. These models have helped identify bacterial surface adhesins and invasins and the corresponding cell surface receptors that play roles in gonococcal pathogenesis. Furthermore, they have been useful for understanding virulence mechanisms as well as innate and adaptive immune responses. In this chapter, readers are provided with protocols for examining the basic interactions between gonococci and a representative human cell line.</p

Structure of the recombinant Neisseria gonorrhoeae adhesin complex protein (rNg-ACP) and generation of murine antibodies with bactericidal activity against gonococci

Neisseria gonorrhoeae (gonococcus [Ng]) is the causative organism of the sexually transmitted disease gonorrhoea, and no effective vaccine exists currently. In this study, the structure, biological properties, and vaccine potential of the Ng-adhesin complex protein (Ng-ACP) are presented. The crystal structure of recombinant Ng-ACP (rNg-ACP) protein was solved at 1.65 Å. Diversity and conservation of Ng-ACP were examined in different Neisseria species and gonococcal isolates (https://pubmlst.org/neisseria/ database) in silico, and protein expression among 50 gonococcal strains in the Centers for Disease Control and Prevention/Food and Drug Administration (CDCP/FDA) AR Isolate Bank was examined by Western blotting. Murine antisera were raised to allele 10 (strain P9-17)-encoded rNg-ACP protein with different adjuvants and examined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and a human serum bactericidal assay. Rabbit antiserum to rNg-ACP was tested for its ability to prevent Ng-ACP from inhibiting human lysozyme activity in vitro. Ng-ACP is structurally homologous to Neisseria meningitidis ACP and MliC/PliC lysozyme inhibitors. Gonococci expressed predominantly allele 10- and allele 6-encoded Ng-ACP (81% and 15% of isolates, respectively). Murine antisera were bactericidal (titers of 64 to 512, P &lt; 0.05) for the homologous P9-17 strain and heterologous (allele 6) FA1090 strain. Rabbit anti-rNg-ACP serum prevented Ng-ACP from inhibiting human lysozyme with ∼100% efficiency. Ng-ACP protein was expressed by all 50 gonococcal isolates examined with minor differences in the relative levels of expression. rNg-ACP is a potential vaccine candidate that induces antibodies that (i) are bactericidal and (ii) prevent the gonococcus from inhibiting the lytic activity of an innate defense molecule