The Soluble Recombinant Neisseria meningitidis Adhesin NadAΔ351–405 Stimulates Human Monocytes by Binding to Extracellular Hsp90

Abstract

The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadAΔ351–405, devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadAΔ351–405 cellular effects in monocytes. We show that NadAΔ351–405 (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadAΔ351–405 cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadAΔ351–405 /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadAΔ351–405 and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadAΔ351–405 determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadAΔ351–405 alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2 antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4 transducing signal complex is necessary for the immune-stimulating activity of NadAΔ351–405 anti-MenB vaccine candidate.

Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages

“…2C and D). Our observation is consistent with reported expression of HSP90 on the surface of human monocytes determined with an antibody specific for N-terminal domain (N17) and with the proposed existence of multiple complexes comprising HSP90 and other receptors, involved in activation of immune cells by microbial patterns [6,36,37]. The immunofluorescence results mentioned above were confirmed in the next part of our study, in which we used one of the remarkably specific inhibitors of HSP90 -geldanamycin coupled to biotin or FITC molecule through a long, hydrophobic spacer.…”

Transcriptional Regulation of the nadA Gene in Neisseria meningitidis Impacts the Prediction of Coverage of a Multicomponent Meningococcal Serogroup B Vaccine

“…Its adhesive role is important in the course of colonization of the human upper respiratory tract (10,35). Following the passage across the epithelium, meningococcus invades tissues and blood, where NadA can interact with human blood leukocytes and lead to enhanced immune stimulation (36)(37)(38)(39). For these reasons, NadA is to be considered an important meningococcal virulence factor, involved in progressively invasive steps of infection.…”

Extracellular Heat Shock Proteins

“…The mitochondria Hsp60 has been reported on the surface of human T cell-lines (54), endothelial cells (55, 56), as well as in the liver and spleen of mice (57). Hsp90 was initially detected on the cell surface of tumor cells (58) and subsequently in multipotential mesenchymal precursor cells (59), human neuroblastoma cells (60, 61), human monocytes, (62). Grp94 was observed on the plasma membrane of sarcoma cells (63).…”