Tino Krell

Role of Transferrin Receptor from a Neisseria meningitidis tbpB Isotype II Strain in Human Transferrin Binding and Virulence

ABSTRACT Neisseria meningitidis acquires iron through the action of the transferrin (Tf) receptor, which is composed of the Tf-binding proteins A and B (TbpA and TbpB). Meningococci can be classified into isotype I and II strains depending on whether they harbor a type I or II form of TbpB. Both types of TbpB have been shown to differ in their genomic, biochemical, and antigenic properties. Here we present a comparative study of isogenic mutants deficient in either or both Tbps from the isotype I strain B16B6 and isotype II strain M982. We show that TbpA is essential in both strains for iron uptake and growth with iron-loaded human Tf as a sole iron source. No growth has also been observed for the TbpB− mutant of strain B16B6, as shown previously, whereas the growth of the analogous mutant in M982 was similar to that in the wild type. This indicates that TbpB in the latter strain plays a facilitating but not essential role in iron uptake, which has been observed previously in similar studies of other bacteria. These data are discussed in relation to the fact that isotype II strains represent more than 80% of serogroup B meningococcal strains. The contribution of both subunits in the bacterial virulence of strain M982 has been assessed in a murine model of bacteremia. Both the TbpB− TbpA− mutant and the TbpA− mutant are shown to be nonvirulent in mice, whereas the virulence of the TbpB− mutant is similar to that of the wild type.

Effect of the β-Propiolactone Treatment on the Adsorption and Fusion of Influenza A/Brisbane/59/2007 and A/New Caledonia/20/1999 Virus H1N1 on a Dimyristoylphosphatidylcholine/Ganglioside GM3 Mixed Phospholipids Monolayer at the Air–Water Interface

The production protocol of many whole cell/virion vaccines involves an inactivation step with β-propiolactone (BPL). Despite the widespread use of BPL, its mechanism of action is poorly understood. Earlier work demonstrated that BPL alkylates nucleotide bases, but its interaction with proteins has not been studied in depth. In the present study we use ellipsometry to analyze the influence of BPL treatment of two H1N1 influenza strains, A/Brisbane/59/2007 and A/New Caledonia/20/1999, which are used for vaccine production on an industrial scale. Analyses were conducted using a mixed lipid monolayer containing ganglioside GM3, which functions as the viral receptor. Our results show that BPL treatment of both strains reduces viral affinity for the mixed monolayer and also diminishes the capacity of viral domains to self-assemble. In another series of experiments, the pH of the subphase was reduced from 7.4 to 5 to provoke the pH-induced conformational change of hemagglutinin, which occurs following endocytosis into the endosome. In the presence of the native virus the pH decrease caused a reduction in domain size, whereas lipid layer thickness and surface pressure were increased. These observations are consistent with a fusion of the viral membrane with the lipid monolayer. Importantly, this fusion was not observed with adsorbed inactivated virus, which indicates that BPL treatment inhibits the first step of virus-membrane fusion. Our data also indicate that BPL chemically modifies hemagglutinin, which mediates the interaction with GM3.

Transferrin-Binding Protein B of Neisseria meningitidis: Sequence-Based Identification of the Transferrin-Binding Site Confirmed by Site-Directed Mutagenesis

A sequence-based prediction method was employed to identify three ligand-binding domains in transferrin-binding protein B (TbpB) of Neisseria meningitidis strain B16B6. Site-directed mutagenesis of residues located in these domains has led to the identification of two domains, amino acids 53 to 57 and 240 to 245, which are involved in binding to human transferrin (htf). These two domains are conserved in an alignment of different TbpB sequences from N. meningitidis and Neisseria gonorrhoeae, indicating a general functional role of the domains. Western blot analysis and BIAcore and isothermal titration calorimetry experiments demonstrated that site-directed mutations in both binding domains led to a decrease or abolition of htf binding. Analysis of mutated proteins by circular dichroism did not provide any evidence for structural alterations due to the amino acid replacements. The TbpB mutant R243N was devoid of any htf-binding activity, and antibodies elicited by the mutant showed strong bactericidal activity against the homologous strain, as well as against several heterologous tbpB isotype I strains.

The Use of Microcalorimetric Techniques to Study the Structure and Function of the Transferrin Receptor from Neisseria meningitidis

Meningococcal disease continues to be a worldwide health problem and can lead to death within several hours if untreated (Begg et al. 1999). There is currently no vaccine to prevent serogroup B meningococcal disease. The proteins which form the transferrin receptor of Neisseria meningitidis are promising candidates for inclusion in such a vaccine (Gorringe et al. 1995). The receptor consists of two types of subunits, transferrin-binding protein A and B (TbpA and B), which both have the capacity to independently bind their ligand, human transferrin (htf) (Cornelissen and Sparling 1996). TbpA (100 kDa) is thought to be a porin-like integral membrane protein, which is proposed to serve as a channel for the transport of iron across the outer membrane. TbpB (65–85 kDa) is considered to be an outer membrane protein, which is anchored to the membrane via the lipidated N-terminal part of the protein and an interaction between TbpA and TbpB has been demonstrated (Fuller et al. 1998).