Molecular and cellular mechanisms during adherence and cell signaling of pathogenic Neisseria to host cells

Sammanfattning: The pathogenic members of the genus Neisseria include N. gonorrhoeae and N. meningitidis. Both organisms are obligate human pathogens that colonize mucosal surfaces. Pili, long hair-like structures that extend from the bacterial surface, mediate and/or induce a stable anchorage of the bacteria to the cells, which in turn enables the bacteria to remain cell-bound at high numbers. We have identified CD46 (membrane cofactor protein, MCP) as a pilus-receptor for pathogenic Neisseria. CD46 is a C3b/C4b binding cell surface glycoprotein, which inhibits complement activation on host cells. Adherence to epithelial cells of pathogenic Neisseria is blocked by antibodies against CD46 or by recombinant CD46-protein. Further, purified pili bind to recombinant CD46. We also show that piliated, but not non-piliated, N. gonorrhoeae attach to COS-7 cells transfected with human CD46-cDNA. The adherence is favored to the expression of the BC1 isoform of CD46. By transfecting COS-7 cells with a number of different CD46 deletion constructs, we show that complement control protein-3 repeat, glycosylation of complement control protein-4 repeat, the serine/theronine/proline rich domain, and the cytoplasmic tail are important for adherence of the bacteria to host cells. The primary interaction between a pilus and its host cell receptor CD46 results in transient release of Ca2+ from intracellular stores. The Ca2+ -flux and the bacterial adherence was inhibited by chelators of intracellular calcium. In addition, antibodies against CD46 blocked the Ca2+ signal. The pilus consists of a major pilus subunit protein, PilE, and a minor pilus-associated protein, PiIC. PilC is required for pilus assembly and for pilus-mediated adherence. We show that PilC is expressed in all clinical isolates of N. gonorrhoeae tested, and that the protein contains both variable and conserved regions. Since PilC is needed for adherence and has been shown to be exposed on the pilus, it is possible that PilC interacts with the CD46 receptor. The pilus-mediated adherence is followed by a tight interaction that is independent of pili. The interaction between non-piliated (P-) Neisseria and host cells most likely plays a role in colonization and asymptomatic carriage of the pathogens. We show that the adherence of P- N. gonorrhoeae is blocked by GDP-ß-S, a non-hydrolyzable GTP analogue, and by C3 exotoxin, an inhibitor of the GTPase Rho. Cholera toxin, that activates GS, and fluoroaluminate, a general G-protein activator, induced bacterial adherence. Further, a shift of the extracellular free Ca2+ concentration dramatically stimulated adherence of P- Neisseria. The nasopharynx and the urogenital tract are natural entry sites of the pathogenic Neisseria species, and at both sites the epithelial cells can be subjected to wide fluctuations in Ca2+ concentration.