SummaryNhhA, Neisseria hia/hsf homologue, or GNA0992, is an oligomeric outer membrane protein of Neisseria meningitidis, recently included in the family of trimeric autotransporter adhesins. In this study we present the structural and functional characterization of this protein. By expressing in Escherichia coli the full-length gene, deletion mutants and chimeric proteins of NhhA, we demonstrated that the last 72 C-terminal residues are able to allow trimerization and localization of the N-terminal protein domain to the bacterial surface. In addition, we investigated on the possible role of NhhA in bacterial-host interaction events. We assessed in vitro the ability of recombinant purified NhhA to bind human epithelial cells as well as laminin and heparan sulphate. Furthermore, we shown that E. coli strain expressing NhhA was able to adhere to epithelial cells, and observed a reduced adherence in a meningococcal isogenic MC58DNhhA mutant. We concluded that this protein is a multifunctional adhesin, able to promote the bacterial adhesion to host cells and extracellular matrix components. Collectively, our results underline a putative role of NhhA in meningococcal pathogenesis and ascertain its structural and functional belonging to the emerging group of bacterial autotransporter adhesins with trimeric architecture.

Aims/hypothesis: Post-prandial glucose may be a risk factor for cardiovascular disease and chronic diabetic complications. We tested the hypothesis that post-prandial hyperglycaemia is common in type 2 diabetes, even among patients in apparently good glycaemic control, and that simple clinical characteristics identify subsets of diabetic patients with frequent post-prandial hyperglycaemia. Subjects and methods: Three self-assessed daily blood glucose profiles over a 1-week period, including 18 glucose readings before and 2 h after meals, were obtained from 3,284 unselected outpatients (men 51%; age 63±10 years) with non-insulin-treated type 2 diabetes mellitus attending 500 different diabetes clinics operating throughout Italy. Results: A post-prandial blood glucose value >8.89 mmol/l (160 mg/dl) was recorded at least once in 84% of patients, and 81% of patients had at least one Δglucose ≥2.22 mmol/l (40 mg/dl). Among patients with apparently good metabolic control, 38% had >40% of post-prandial blood glucose readings >8.89 mmol/l (≥4 of 9 meals in total), and 36% had >40% Δglucose ≥2.22 mmol/l. In multivariate analysis adjusted for pre-prandial glucose levels, older age, longer duration of diabetes, absence of obesity, hyperlipidaemia and hypertension, as well as treatment with sulfonylureas, were significantly associated with greater glucose excursions after meals. Conclusions/interpretation: These results indicate that post-prandial hyperglycaemia is a very frequent phenomenon in patients with type 2 diabetes mellitus on active treatment; can occur even when metabolic control is apparently good; and can be predicted by simple clinical features.

Pathophysiology of graft failure (GF) occurring after allogeneic hematopoietic stem cell transplantation (HSCT) still remains elusive. We measured serum levels of several different cytokines/chemokines in 15 children experiencing GF, comparing their values with those of 15 controls who had sustained donor cell engraftment. Already at day +3 after transplantation, patients developing GF had serum levels of interferon (IFN)-γ and CXCL9 (a chemokine specifically induced by IFNγ) significantly higher than those of controls (8859±7502 vs. 0 pg/mL, P=0.03, and 1514.0±773 vs. 233.6±50.1 pg/mlL, P=0.0006, respectively). The role played by IFNγ in HSCT-related GF was further supported by the observation that a rat anti-mouse IFNγ-neutralizing monoclonal antibody promotes donor cell engraftment in Ifngr1−/−mice receiving an allograft. In comparison to controls, analysis of bone marrow-infiltrating T lymphocytes in patients experiencing GF documented a predominance of effector memory CD8+ cells, which showed markers of activation (overexpression of CD95 and downregulation of CD127) and exhaustion (CD57, CD279, CD223 and CD366). Finally, we obtained successful donor engraftment in 2 out of 3 children with primary hemophagocytic lymphohistiocytosis who, after experiencing GF, were re-transplanted from the same HLA-haploidentical donor under the compassionate use coverage of emapalumab, an anti-IFNγ monoclonal antibody recently approved by the US Food and Drug Administration for treatment of patients with primary hemophagocytic lymphohistiocytosis. Altogether, these results suggest that the IFNγ pathway plays a major role in GF occurring after HSCT. Increased serum levels of IFNγ and CXCL9 represent potential biomarkers useful for early diagnosis of GF and provide the rationale for exploring the therapeutic/preventive role of targeted neutralization of IFNγ.

A novel vaccine against serogroup B meningococcal disease - containing a combination of protein antigens identified by reverse vaccinology: fHBP fused to GNA2091, GNA2132 fused to GNA1030, and NadA - is currently in Phase III clinical trials. In order to determine the role of these antigens in the growth, survival and fitness of the meningococcus, we generated a mutant lacking the expression of all five protein antigens (5KO), a mutant lacking the three main antigens (fHBP, GNA2132 and NadA; 3KO), as well as strains lacking the single antigens. Our results show that abrogation of expression of these antigens in Neisseria meningitidis results in reduced growth in vitro, increased sensitivity of the bacterium to stresses it may encounter in the host, as well as reduced fitness in ex vivo models of infection and in an in vivo infant rat competitive index assay. These results support a multivalent vaccine approach, which was undertaken to strengthen the protective activity of the vaccine antigens, increase the breadth of MenB strains targeted by the vaccine, and limit the potential for selection of vaccine escape mutants.

SummaryNadA (Neisseria meningitidis adhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/ invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.

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 NH 2 -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 NH 2 -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 NH 2 globular head domain and the NH 2 dimeric intrachain coiled-coil ␣-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.

In glucocorticoid-remediable aldosteronism (GRA), there is a large interfamily variation of phenotype. We report three subjects with GRA in a single family (parents, two brothers and two sisters), of whom only one (proband) displayed classical features of the mineralocorticoid excess. The proband was a man found to be hypertensive and hypokalaemic at the age of 24 years. Plasma renin activity was suppressed and plasma aldosterone was repeatedly elevated. Blood pressure and aldosterone levels normalized within 5 days of dexamethasone therapy. The presence of a chimaeric CYP11B1/CYP11B2 gene was demonstrated by long-PCR and Southern blotting (crossover site at the end of intron 3) in the proband, in the younger sister (sibling 1) and in the father. In these patients, sequencing of the chimaeric portion of CYP11B1 did not reveal any mutation, while sequencing of the chimaeric portion of CYP11B2 showed a V386A polymorphism in exon 7, known to cause only a minimal impairment of enzymatic activity. Sibling 1 was normotensive, normokalaemic and had normal PRA and aldosterone. The father had normal blood pressure and potassium, low-normal PRA and normal aldosterone. All three subjects had elevated levels of urinary 18-hydroxycortisol and 18-oxocortisol. Baseline 11-deoxycorticosterone (DOC), corticosterone (B) and aldosterone were high in the proband and normal in the father and sibling 1; 11-deoxycortisol (S) and cortisol (F) were normal. ACTH induced a normal increase of B, DOC, S and F, and an excessive aldosterone increase in all three patients. Abnormalities in the chimaeric portions of CYB11B1 or CYP11B2 genes did not account for the phenotypic disparity of the different members in a single GRA family. Altered regulation of the chimaeric gene may be responsible for differences in its activity.

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.