Expression of Factor H Binding Protein of Meningococcus Responds to Oxygen Limitation through a Dedicated FNR-Regulated Promoter

Factor H binding protein (fHBP) is a surface-exposed lipoprotein in Neisseria meningitidis, which is a component of several investigational vaccines against serogroup B meningococcus (MenB) currently in development. fHBP enables the bacterium to evade complement-mediated killing by binding factor H, a key downregulator of the complement alternative pathway, and, in addition, fHBP is important for meningococcal survival in the presence of the antimicrobial peptide LL-37. In this study, we investigate the molecular mechanisms involved in transcription and regulation of the fHBP-encoding gene, fhbp. We show that the fHBP protein is expressed from two independent transcripts: one bicistronic transcript that includes the upstream gene and a second shorter monocistronic transcript from its own dedicated promoter, P_fhbp. Transcription from the promoter P_fhbp responds to oxygen limitation in an FNR-dependent manner, and, accordingly, the FNR protein binds to a P_fhbp probe in vitro. Furthermore, expression in meningococci of a constitutively active FNR mutant results in the overexpression of the fHBP protein. Finally, the analysis of fHBP regulation was extended to a panel of strains expressing different fHBP allelic variants at different levels, and we demonstrate that FNR is involved in the regulation of this antigen in all but one of the strains tested. Our data suggest that oxygen limitation may play an important role in inducing the expression of fHBP from a dedicated FNR-regulated promoter. This implies a role for this protein in microenvironments lacking oxygen, for instance in the submucosa or intracellularly, in addition to its demonstrated role in serum resistance in the blood.Neisseria meningitidis is a Gram-negative bacterial pathogen that is carried asymptomatically in the upper respiratory tracts of approximately 5 to 10% of the human population. Occasionally, the bacterium spreads from the nasopharynx into the bloodstream, causing bacteremia with possible progression to sepsis, meningitis, and death (41). The outcome of infection depends on the capacity of the bacterium to avoid the host immune defense and survive within the circulation (44). Therefore, prevention through vaccination can bring about a major advance in preventing meningococcal disease, which remains a major worldwide cause of morbidity and mortality. However, no vaccine is currently available to provide universal protection against all serogroups of N. meningitidis. Capsular polysaccharide-based vaccines have been developed to protect against the main disease-associated serogroups A, C, Y, and W135; tailor-made vaccines, based on outer membrane vesicles (OMV) of serogroup B epidemic strains, have been used in Norway (17), Cuba (47), and New Zealand (21) to control epidemics.Serogroup B capsular polysaccharide is poorly immunogenic due to its similarity with glycoproteins in human neural tissues (15). Therefore, the strategy to develop a vaccine against serogroup B of N. meningitidis (MenB) has instead focused on meningococcal noncapsular surface antigens (38). One of the proteins, which is currently being investigated as a meningococcal vaccine candidate, is factor H binding protein (fHBP), previously referred to as GNA1870 or LP2086 (16, 31). This antigen was identified by genome analysis of the MC58 strain of N. meningitidis and is currently included together with other recombinant protein antigens in an investigational MenB vaccine, which has entered phase III clinical trials. Recombinant fHBP (rLP2086) is the principal antigen of another MenB vaccine containing recombinant proteins from two different variant subfamilies (16, 56). More recent studies also support the strategy to prepare native OMV vaccines from mutant strains engineered to express different fHBP alleles that would confer broad protection against meningococcal strains expressing fHBP from each of the antigenic variant groups (22, 25).In recent years, many studies have supported the importance of this antigen as a MenB vaccine candidate and also as an important virulence factor. fHBP is able to elicit antibodies that have high bactericidal activity (31, 53) that have also been shown to confer passive protection in the infant rat model (31). It has been demonstrated that the antigen enables N. meningitidis to evade complement-mediated killing by binding factor H, a key downregulator of the complement alternative pathway (AP), allowing meningococcal survival in human blood (29, 43). As reported for Neisseria gonorrhoeae (35), the specificity of this binding is restricted to human binding factor H (19), and this important feature could explain the reason why N. meningitidis is exclusively a human pathogen. Furthermore, anti-fHBP antibodies can block binding of fH, increasing the susceptibility to killing by the complement AP (6, 29). It has been demonstrated that the antigen also confers protection against killing by the antimicrobial peptide LL-37 (46), a compound produced by human cells that may be involved in the innate host immune response against meningococcal disease.Through many studies using monoclonal antibodies (4, 5, 53), the protective epitopes (18) and the residues involved in the binding to factor H have been identified, and the structure of the full-length fHBP has been recently solved (10, 30, 45). fHBP is a surface-exposed lipoprotein expressed in a global panel of representative meningococcal strains, albeit at different levels in different strains which can be classified as high, intermediate, and low expressers (31). In a more recent study, 98% of a panel of 1,263 invasive clinical isolates showed surface expression of fHBP at various levels, and accessibility of the protein to bactericidal antibodies was independent of capsule expression (33). Unlike the well-known role of the protein, the mechanisms that regulate and influence its expression have remained largely unexplored.In the present study, we have focused our analysis on the mechanism of regulation of the gene encoding the fHBP protein by mimicking some environmental conditions encountered by the bacterium during infection. Transcription of the fhbp gene was studied under oxygen-restricted conditions and in response to the FNR regulatory protein. Furthermore, we broaden our analysis to a representative panel of strains expressing different variant alleles of fHBP from different clonal complexes and found that regulation of transcription is widely conserved.