M. Pintor

Analysis of TbpA and TbpB functionality in defective mutants of Neisseria meningitidis

Iron uptake analysis suggested that the Neisseria meningitidis transferrin (Tf) binding proteins, TbpA and TbpB, form only one type of receptor complex. Mutants defective in the synthesis of either TbpA or TbpB, but not defective in both proteins, can bind Tf, suggesting that both proteins are surface exposed and function in Tf binding. Also, iron uptake from Tf into the meningococci did not require the presence of both Tbps. The TbpB-defective mutant incorporated c. 37% of the iron taken up by the wild-type strain, but this was insufficient for bacterial growth. The TbpA-defective mutant incorporated c. 50% of the iron taken up by the wild-type strain and was able to grow with Tf as the only iron source. Mouse antibodies specific for TbpA were able to block c. 70% of the iron uptake from Tf in the wild-type strain, whereas they blocked only 22% of iron uptake in the TbpB-defective mutant and did not block uptake in the TbpA-defective strain. These results emphasise that TbpA should be considered in future vaccine trials in which iron-restricted proteins are to be included in the vaccine formulation.

Antigenicity, cross-reactivity and surface exposure of the Neisseria meningitidis 37 kDa protein (Fbp)

The 37 kDa iron-repressible protein, Fbp, was purified from two Neisseria meningitidis strains by metal-affinity chromatography and used to obtain mouse monospecific polyclonal immune sera. Dot-blot, immunoblotting and whole cell ELISA results demonstrate that the Fbp is present in all 16 N. meningitidis and four commensal Neisseria species tested, is highly antigenic in mouse when injected in pure form, and shows intra- and inter-species antigenic homogeneity, anti-Fbp antibodies being fully cross-reactive using the techniques mentioned. We also found that Fbp molecules (or parts of them) are surface exposed, in disagreement with the proposed exclusively periplasmic localization, although anti-Fbp antibodies seem unable to block iron uptake or to induce complement-mediated killing of the meningococci. Taken along with the high immunogenicity of the purified protein and the complete cross-reactivity of the antibodies elicited, this suggests that the protective effect of the purified Fbp must be further studied to evaluate its inclusion in future vaccine trials.

Analysis of the expression of outer-membrane proteins in Neisseria meningitidis in iron-replete and iron-deficient media

A total of 103 Neisseria strains, including 42 carrier and 40 invasive N. meningitidis and 21 commensal species were studied. Outer-membrane proteins from carrier and invasive N. meningitidis showed similar molecular mass distributions (except in the range from 78 to 82 kDa in which 90% of the invasive but only 47.6% of the carrier strains expressed proteins), many strains showing neither class II (41 kDa) nor class III (38 kDa) proteins. When grown in iron-restricted conditions proteins were induced mainly in the ranges from 62 to 92 kDa with no significant differences between groups. Commensal species, both in normal and in iron-restricted conditions, showed outer-membrane protein patterns different from those of N. meningitidis in several molecular mass ranges. Cluster analysis based on expression of principal outer-membrane proteins allowed the differentiation of commensal species and N. meningitidis, although not that of the invasive and carrier groups within the latter.

Blocking of iron uptake by monoclonal antibodies specific for the Neisseria meningitidis transferrin- binding protein 2

The existence of epitopes common to different strains in the Neisseria meningitidis transferrin (Tf)-binding protein 2 (TBP2), combined with the ability of polyclonal anti-TBP2 antibodies to inhibit Tf binding and block iron uptake in this species, led to this study on the effect of anti-TBP1+2 monoclonal antibodies (MAbs) to determine the presence of epitopes inside the Tf-binding region. All MAbs used reacted exclusively with the homologous strain when tested by dot-blots of outer membrane vesicles, with the reaction being specific for TBP2 after SDS-PAGE and electroblotting. In contrast, ELISA and iron-uptake blocking assays were also positive with heterologous strains belonging to Rokbis group II (high mol.wt TBP2). The results confirmed the two group classification proposed by Rokbi and, in contrast to other studies, indicated the existence of epitopes in the Tf-binding region that are common only to strains of Rokbis group II. These epitopes may become denatured after drying for dot-blot assays or after SDS-PAGE and electroblotting.

Expression levels of human transferrin receptors in Neisseria species

Abstract All pathogenic Neisseria strains synthesize transferrin receptors. To determine if transferrin receptor expression is quantitatively different in Neisseria meningitidis strains from two separate origins (carrier and invasive) and in commensal Neisseria , we applied an enzyme-linked receptor assay (ELRA) and a radiolabelled receptor assay (RLRA) to evaluate the receptor expression levels after iron-limited growth. There were significant differences in receptor expression in commensal Neisseria and Neisseria meningitidis as determined by both methods and between the two origins (invasive and carrier) when the ELRA method was used, but in the latter case, the biological significance is questionable and the amount of transferrin receptor expressed may not be a useful indicator of the potential pathogenicity of strains. The RLRA method was more suitable for measuring transferrin receptor expression since it provided a lower background in strains lacking transferrin receptor and, consequently, made interpretation of the results easier.

One-step metal-affinity method for the purification of the iron-binding protein Fbp of Neisseria meningitidis

A one-step method for the purification of the iron-binding protein, Fbp, from Neisseria meningitidis has been developed using affinity chromatography on iron-Sepharose. This method allowed the purification of the protein in non-denaturing conditions and in a similar way to those used previously. Our results demonstrate the homogeneity of the purified protein as tested by SDS-PAGE and IEF.