Neisseria meningitidis is an encapsulated, Gram-negative bacterium that colonizes the upper respiratory tract of ~10% of humans. With a frequency of one to three cases per 100.000 of the population, the bacterium enters the bloodstream, where it multiplies to high density and cause sepsis. From the bloodstream the bacterium can cross the blood-brain barrier and cause meningitis. The invasive infection is very dramatic, affecting mostly infants, children, and adolescents who do not have bactericidal antibodies against the infecting strains. Immunity against the disease can be acquired naturally or induced by vaccination and correlates with the presence of antibodies able to kill the bacterium in the presence of complement. There are no effective vaccines currently licensed in the Unit States or Europe for prevention of the disease caused by serogroup B meningococcus. The genome sequencing of Neisseria meningitidis, serogroup B, allowed the identification of unknown surface proteins termed GNA (Genome derived Neisseria Antigens) among which NadA (Neisseria Adhesin A). NadA is a highly conserved protein among disease-associated strains and capable of eliciting bactericidal antibodies in mice. Structure prediction and homology comparison with know virulence-associated factors suggest that NadA belongs to the group of OCA (Oligomeric Coiled-coil Adhesin) nonfimbrial adhesins.
Recently, NadA has been characterized as a new meningococcic factor, involved in the colonization and the invasion of host cells. Probably there is a unique receptor expressed in different cellular lines. In this thesis, we have focused on unravelling the identity of this possible receptor for NadA in these cells.
To identify the specific receptor for NadA, experiments of co-immunoprecipitation and overlay were performed in Chang total cell lysates. Our data suggest as possible receptor a protein of 90 kDa, that was present in the co-immunoprecipitation samples incubated with NadA and absent in controls.
Subsequently, this protein obtained by co-immunoprecipitation of NadA, was enriched, separated by 12% SDS-PAGE, then excised from the gel and subjected to tryptic proteolysis; resulting peptides were analyzed by liquid chromatography/MS and data were analyzed with the Mascot software. In this way, we identified the human Heat shock protein 90, as the recognized peptides provide a coverage of 25% of the total protein sequence .
To confirm the MS data, co-immunoprecipitation samples and membrane proteins from Chang cells were incubated with antibody anti-Hsp90, in order to demonstrate a membrane localization of Hsp90, which is generally known as a cytoplasmic protein. However, as described in the literature, Hsp90 can be expressed on the surface of various cell types, such as tumor and apoptotic cells, but also on HeLa cells, monocytes, macrophages and dendritic cells. Our results confirm that Hsp90 is also found in the cell membrane of Chang cells.
Moreover, we found that in co-immunoprecipitation experiments addition of polymyxin B, a cationic antibiotic similar to antimicrobic peptides produced by monocytes that binds both to NadA and Hsp90, is able to interfere in the interaction of NadA with Hsp90.
In order to investigate the effect of the association between NadA and Hsp90 in cells, we quantified the superficial expression of Hsp90 on Chang cells and on human monocytes, isolates from Buffy coat , which were found to be 2% and 5% of total cell surface protein, respectively
Since the mechanism of transport to the plasma membrane remains enigmatic, we quantified the superficial expression of Hsp90 in Chang cells after the incubation with rHsp90, but we didn’t see an increase of protein expression.
We also performed experiments on human monocytes, which are the main agonist of the innate immune system, in order to study the receptorial function of this association, and also the involvement of the immune system.
Monocytes were incubated with antibodies anti-Hsp90, with or without Polymyxin B, and then they were analyzed by FACS, to quantify the binding of NadA-alexa. These experiments showed that the NadA binding to the cells is not influenced by the presence of the anti-Hsp90 antibody.
We also investigated whether the superficial expression of Hsp90 in monocytes changed after pre-incubation with NadA. By FACS analysis, we quantified the fluorescence of a PE conjugated secondary antibody after the incubation with anti-Hsp90 antibodies. The results showed that pre-incubation with NadA interferes with the recognition of the superficial Hsp90 by its specific antibodies, showing a decrease of 40%, which could be explained by the competive association between NadA and Hsp90 on the plasmatic membrane. Moreover, when monocytes were incubated with NadA for 3h at 37°C in presence of Polymyxin B, we did not observe decrease on the signal.
Taken together, these data indicate that NadA-Hsp90 association is not a receptorial one, indicating that, under physiological conditions, these proteins bind closely and strongly each others, probably producing clusters on the plasma membrane.
Finally, to analyze the effect of these complexes on the activation of the immune system, we analyzed the pattern of cytokines produced by human monocytes stimulated for 24h with NadA, antibodies anti-Hsp90 and antibodies anti-TLR2 and 4, and with or without Polymyxin B.
These results show a synergic effect of NadA and antibody anti-Hsp90 on cytokines production, mainly IL-6, TNF? and MCP-1; on the contrary, Polymyxin B and antibody anti-TLR4 inhibited cytokines production. The cytokine pattern secreted demonstrated that NadA and Hsp90 induce a macrophage-like phenotype and that these two agonists promote a Th2 response.