Testing the serum bactericidal activity of anti-rHpuA antisera.

<p>Meningococcal cell suspensions were prepared following growth under iron-restricted conditions. An aliquot of 10⁴ cfu was mixed with pooled human complement at a final concentration of 5% and dilutions (as indicated on the x-axis) of either a polyclonal anti-rHpuA antiserum (A) or anti-PorA monoclonal antibody P1.2 (B). Assays were incubated at 37^°C for 60 minutes and then assessed for the numbers of surviving cells (cfu/ml) by plating 10μl of aliquots on supplmented BHI agar. Inoc, cell count after 0 minutes; No Ab, cell count after 60 minutes incubation in serum alone. Graphs show mean values for two independent experiments. Red line, 8047; blue line, 8047ΔhpuAB.</p

Investigation into the Antigenic Properties and Contributions to Growth in Blood of the Meningococcal Haemoglobin Receptors, HpuAB and HmbR.

Acquisition of iron from host complexes is mediated by four surface-located receptors of Neisseria meningitidis. The HmbR protein and heterodimeric HpuAB complex bind to haemoglobin whilst TbpBA and LbpBA bind iron-loaded transferrin and lactoferrin complexes, respectively. The haemoglobin receptors are unevenly distributed; disease-causing meningococcal isolates encode HmbR or both receptors while strains with only HpuAB are rarely-associated with disease. Both these receptors are subject to phase variation and 70-90% of disease isolates have one or both of these receptors in an ON expression state. The surface-expression, ubiquity and association with disease indicate that these receptors could be potential virulence factors and vaccine targets. To test for a requirement during disease, an hmbR deletion mutant was constructed in a strain (MC58) lacking HpuAB and in both a wild-type and TbpBA deletion background. The hmbR mutant exhibited an identical growth pattern to wild-type in whole blood from healthy human donors whereas growth of the tbpBA mutant was impaired. These results suggest that transferrin is the major source of iron for N. meningitidis during replication in healthy human blood. To examine immune responses, polyclonal antisera were raised against His-tagged purified-recombinant variants of HmbR, HpuA and HpuB in mice using monolipopolysaccharide as an adjuvant. Additionally, monoclonal antibodies were raised against outer membrane loops of HmbR presented on the surface of EspA, an E. coli fimbrial protein. All antisera exhibited specific reactivity in Western blots but HmbR and HpuA polyclonal sera were reactive against intact meningococcal cells. None of the sera exhibited bactericidal activity against iron-induced wild-type meningococci. These findings suggest that the HmbR protein is not required during the early stages of disease and that immune responses against these receptors may not be protective

Growth of Hb and Tf receptor mutants of <i>N</i>. <i>meningitidis</i> in whole human blood.

<p>Bacterial strains (8047, MC58 and mutants thereof) were grown to mid-log (OD600 = ~ 0.6) in MHB supplemented with 20 μM CMP-NANA and then added to freshly-collected human blood. Cultures were incubated at 37°C, 5% CO₂ for 4 hours. Samples, collected from the inoculum and at 0, 60, 120, 180 and 240 minutes from blood cultures, were plated in duplicate onto supplemented BHI agar. The Fig shows a representative experiment. Each strain and mutant was tested in triplicate and intra-assay variation was measured by calculating the standard error of the mean of the CFU counts obtained at each timepoint. Blood samples were collected from two healthy MBL-deficient volunteers (V1 and V2) and an uncharacterised volunteer (V3). (A) strain 8047 (wt) in blood from V2 and V3, which was treated with either 5 mM or 10 mM EDTA; (B) wild-type and mutants of strain MC58 in untreated blood from V1; (C) wild-type and mutants of strain MC58 in untreated blood from V2.</p

Reactivity of anti-HmbR monoclonal and polyclonal antisera with HmbR in meningococcal lysates.

<p>Meningococcal lysates were prepared from cells grown in iron-limited (induced) or iron-replete (uninduced) conditions as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133855#pone.0133855.g003" target="_blank">Fig 3</a>. Equal amounts of cells were analysed by probing Western blots with a 1:500 dilution of a primary antiserum followed by a 1:2000 dilution of an anti-Mouse IgG HRP conjugate. Upper panel, anti-r8047-HmbR; middle panel, anti-rMC58-HmbR; lower panel, pooled mAbs raised against two surface exposed epitopes of the MC58 HmbR protein. Lysates were:- induced strain MC58ΔhmbR (lane 1); uninduced wild-type MC58 (lane 2); induced wild-type MC58 (lane 3); uninduced wild-type 8047 (lane 4); induced wild-type 8047 (lane 5); uninduced wildtype H44/76 (lane 6); induced wildtype H44/76 (lane 7). Arrows, HmbR; asterisk, non-specific reactive protein.</p

List of <i>N</i>. <i>meningitidis</i> strains.

<p>List of <i>N</i>. <i>meningitidis</i> strains.</p

Reactivity of anti-rHpuA and anti-rHpuB antisera with HpuAB in meningococcal lysates.

<p>Meningococcal cells of strain 8047, an isogenic Δhpu mutant and two phase variants of a carriage strain (N88.1, hpu-OFF; and N272.1, hpu-ON) were grown to mid-log (OD600 = ~0.5) before 30 μM of desferal was added to produce iron-limited conditions. Cultures were also grown concurrently in iron-replete conditions. All cultures were incubated for two hours before heat-inactivation at 56°C overnight. Lysates were prepared using an equal number of OD units and subject to SDS-PAGE electrophoresis and Western blotting. Blots were then probed with 1:500 dilutions of mouse polyclonal sera followed by a 1:2000 dilution of an anti-mouse IgG HRP-conjugate:- upper panel, anti-rN88-HpuA; middle panel, anti-r8047-HpuA; lower panel anti-r8047-HpuB. Lysates were from:- strain N88.1, induced (lane 1); 8047ΔhpuAB, induced (lane 2); wild-type 8047, uninduced (lane 3); wild-type 8047, induced (lane 4); N272.1 uninduced (lane 5); and N272.1 induced (lane 6).</p

Evaluation of HpuA and HpuB surface expression by flow cytometry and immunoblotting.

<p>Cultures of meningococcal strains 8047ΔhpuAB, 8047 wild-type, N88.1 and N27.12 were subjected to growth in iron-limited conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133855#pone.0133855.g003" target="_blank">Fig 3</a>). Formalin-fixed cells were assessed for surface expression of HpuA or HpuB in a FACS assay (panels A-C) by incubation with a 1:20 dilution of a polyclonal mice antiserum followed by a 1:100 dilution of a secondary antibody (Alexa Fluor anti-Mouse IgG) and detection of fluorescence in a flow cytometer. A total of 25,000 events were analysed for each assay. Primary antisera were:- (A) anti-r8047-HpuA; (B) anti-rN88-HpuA; (C) anti-r8047-HpuB. Strain are as indicated in the key. Immunoblotting (panels D-E) was performed using meningococcal cells grown on supplemented BHI agar containing 65 μM desferal and incubated at 37°C, 5% CO₂. Cells were transferred to nitrocellulose filters and probed with a 1:250 dilution of either an anti-r8047-HpuA (D) or an anti-rN88-HpuA (E) followed by a 1:2000 dilution of an anti-Mouse IgG AP-conjugate.</p

Oligonucleotide primers for cloning.

<p>Oligonucleotide primers for cloning.</p