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

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

Mapping of the Neisseria meningitidis NadA Cell-Binding Site: Relevance of Predicted α-Helices in the NH2-Terminal and Dimeric Coiled-Coil Regions▿

NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH2-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH2-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH2 globular head domain and the NH2 dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen

Anti-hsp90 antibodies increase NadA_Δ351–405 monocyte stimulation in a polymixin B insensitive-way.

<p>A) Cells were treated with NadA_Δ351–405 in the absence (black circles) or in the presence (open circles) of purified rabbit polyclonal antibodies directed to the COOH terminal domain of hsp90. Open squares refers to cells incubated with NadA_Δ351–405 in the presence of purified rabbit polyclonal antibodies from non immunized animals. The indicated cytokines/chemokines were analyzed in the extracellular medium by BioPlex suspension arrays. Data are the mean from a representative experiment out of four run in triplicate. Bars are +/− SE. Asterisk indicates signals significantly different (p<0.05) from control (with or without an unrelated antibody). B) Monocytes were stimulated or not with NadA_Δ351–405 (1.5 µM) as indicated, in the presence of polymixin B (black bars), antibodies to the COOH terminal of hsp90 (hatched bats) and in the presence of both polymixin B and anti-hsp90 antibodies (light gray bars). After 1 hour incubation, the indicated cytokines/chemokines were analyzed in the extracellular medium as indicated above. Data are the mean of two experiments run in triplicate. Bars represent +/− SE.</p

Peptides identified by the MS/MS tandem analysis.

<p>Peptides identified by the MS/MS analysis and recognized by Mascot. In the columns are indicated, from left to right: the relative sequence position; the experimental m/z value (m/z Obs); the theoretical molecular weight, in Dalton, obtained from the Swiss-Prot and ExPASy databases (Mr_exp); the relative molecular mass calculated from the matched peptide (Mr_calc); the difference between the experimental and calculated masses (Delta); the Mascot score for the identified protein (Score) and the sequence of the peptide. The peptide with the highest score is highlighted in bold and used as example of MS/MS scan analysis in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025089#pone-0025089-g002" target="_blank">Fig. 2</a>. All shown peptides belong to human hsp90 β.</p

NadA_Δ351–405 binds to human hsp90 in an overlay-assay.

<p>A) Samples of total monocytes extracts obtained with 1% TX-100 were separated by SDS-PAGE and challenged or not as indicated with 1 µM NadA_Δ351–405 after blotting on nitrocellulose. After extensive washings, the polypeptide bands able to retain NadA were detected with antibodies to the whole adhesin or to NadA 52–70, as indicated. The picture is from a representative experiment out of four and arrows point to the NadA-interacting ∼90 KDa protein. B) Monocytes detergent extracts prepared as in A) were separated by SDS-PAGE, blotted on nitrocellulose and subjected to western blot with anti- grp94 and hsp90 antibodies or to an overlay assay with NadA/anti-NadA antibodies. The mobility of the NadA interacting protein and of hsp90 or grp94 was compared. C) Purified human hsp90 were subjected to overlay assay with (1 µM) or without NadA_Δ351–405 and HP-NAP as indicated. D) Whole TX-100 extracts from human monocytes were incubated with protein A sepharose-bound anti-NadA IgG in the presence or not of NadA_Δ351–405. After washings, beads were treated with LSB containing 4%SDS and 10% βme and analyzed by western blot after SDS-PAGE using anti-hsp90 antibodies and alkaline phosphatase-conjugated anti-IgG secondary antibodies. Arrow points to hsp90, H and L indicate antibody heavy and light chains respectively.</p

Polymixin B inhibits the induction of cytokine/chemokine by NadA_Δ351–405 in monocytes.

<p>Human monocytes were stimulated for 24 hours with the indicated concentrations of NadA_Δ351–405 in the absence (open circles) or in the presence of 10 µg/ml polymixin B in DMEM plus 10% FCS. Cytokines/chemokines were subsequently quantified in the cells extracellular medium using a BioPlex assay. Data are from a representative experiment of three, run in duplicate and bars are ranges.</p

Model of the role of NadA_Δ351–405/hsp90 interaction in monocyte activation (discussed in the text).

<p>Model of the role of NadA_Δ351–405/hsp90 interaction in monocyte activation (discussed in the text).</p