Functional Activity of Antibodies against the Recombinant OpaJ Protein from Neisseria meningitidis

ABSTRACT The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.

Interactions of Neisseria gonorrhoeae with Mature Human Macrophage Opacity Proteins Influence Production of Proinflammatory Cytokines

ABSTRACT The pathological features of ascending gonococcal infection suggest that proinflammatory mediators secreted by tissue-resident macrophages are important components of the host response. Challenge of fully differentiated, mature macrophages with variants of Neisseria gonorrhoeae strain P9 or purified bacterial surface components (pili, lipooligosaccharide, and outer membrane vesicles) induced the secretion of interleukin 6 (IL-6), tumor necrosis factor alpha, growth-related protein α, macrophage inflammatory protein 1α (MIP-1α), and RANTES cytokines but had no effect on IL-8 production. No secretion of IL-1β, epithelial-derived neutrophil attractant 78, granulocyte-macrophage colony-stimulating factor, IL-10, or IL-12 cytokines was observed. Notably, the P9-Opab protein, in comparison to P9-Opaa, increased the association of gonococci with macrophages and elevated the secretion of cytokines. Thus, variation in Opa protein expression by the gonococcus may be a determining factor in the severity of pelvic inflammatory disease.

Several Carcinoembryonic Antigens (CD66) Serve as Receptors for Gonococcal Opacity Proteins

Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Although pili are required for the initial adherence, the interaction of GC with epithelial cells is also promoted by a family of outer membrane proteins, the opacity (Opa) proteins such as OpaA protein from strain MS11. Studies have demonstrated that the interaction of the OpaA GC with epithelial cells involves binding to heparan sulfate attached to syndecan receptors. However, other Opa proteins interact with CEA gene family member 1 (CGM1) or biliary glycoprotein (BGP), members of the CD66 antigen family. In this study, we demonstrate that, in addition, the 180-kD carcinoembryonic antigen (CEA) is a receptor for Opa proteins. This conclusion was based on the following observations. First, transfected HeLa cells expressing CEA (HeLaCEA) and the CEA-expressing colon cancer cell line (LS 174T) bound and subsequently engulfed the Opa+ bacteria. These interactions were inhibited by anti-CEA antibody, but could not be inhibited by addition of heparin. Furthermore, OpaI E. coli directly bound purified CEA. We also compared the adherence and invasion by Opa+ bacteria of CD66 transfected HeLa cells: HeLa-BGPa, HeLa-CGM6, HeLa-NCA, HeLa-CGM1a, HeLa-CEA, and HeLa-Neo serving as negative control. Using OpaI as the prototype, the relative ability of the transfected HeLa cell lines to support adherence was (CEA = BGPa >CGM1a >NCA >>CGM6 = Neo). The ability to mediate invasion of the transfectant cells was (CGM1a >CEA >BGPa >NCA >CGM6 = Neo). Among the Opa proteins tested, OpaC proved to be bifunctional, able to mediate adherence to both syndecan receptors and to CD66 antigens.