The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence.

GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that nmb2091 is part of an operon and is cotranscribed with the nmb2089, nmb2090, and nmb2092 adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the nmb2091 gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as znuD. Finally, the ∆2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival in vivo, and a model for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.-Seib, K. L., Haag, A. F., Oriente, F., Fantappiè, L., Borghi, S., Semchenko, E. A., Schulz, B. L., Ferlicca, F., Taddei, A. R., Giuliani, M. M., Pizza, M., Delany, I. The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence.

A novel Hfq-dependent sRNA that is under FNR control and is synthesized in oxygen limitation in Neisseria meningitidis.

Small non-coding RNAs (sRNA) are emerging as key elements of post-transcriptional gene regulation in bacteria. The conserved Hfq protein is thought to function as an RNA chaperone and facilitate base-pairing between sRNAs and mRNA targets. In this study we identify a novel sRNA of Neisseria meningitidis through global gene expression studies of regulated transcripts in the Hfq mutant. The synthesis of this sRNA, named AniS, is anaerobically induced through activation of its promoter by the FNR global regulator. Whole-genome expression analyses led to the identification of putative mRNA targets, two of which are predicted to base pair with AniS. We show that Hfq binds the AniS transcript in vitro and is necessary for the downregulation of the identified target mRNAs in vivo. Contrary to many Hfq-dependent sRNA of the Enterobacteriaceae, Hfq promotes decay of AniS in N. meningitidis. Our analysis shows that the AniS regulator is part of the FNR regulon and may be responsible for the downregulation of FNR-repressed genes. Furthermore the presence of similar conserved regulatory sequences in all Neisseria spp. to date suggests that an analogous FNR-regulated sRNA, with a variable 5' sequence, may be ubiquitous to all commensals and pathogens of the Genus.

Influence of serogroup B meningococcal vaccine antigens on growth and survival of the meningococcus in vitro and in ex vivo and in vivo models of infection.

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.

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.

The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA- OMVs, without further stimulating their proinflammatory activity on circulating monocytes.

Hypervirulent MenB causing fatal human infections frequently display the oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10-kDa COOH terminal membrane domain (NadA(Delta351-405)) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA(+) OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA(-) OMVs. To do this we investigated the activity of purified free NadA(Delta351-405) and of OMVs from MenB and Escherichia coli strains, expressing or not full-length NadA. NadA(Delta351-405) stimulated monocytes and macrophages to secrete cytokines (IL-1beta, TNF-alpha, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8, MIP-1alpha, MCP-1, RANTES), and full-length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadA(Delta351-405) induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA(+) OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than NadA(-) OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadA(Delta351-405) ones, were much less IFN-gamma-sensitive. The activity of NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations approximately 10(6) times lower than those required to stimulate cells with free NadA(Delta351-405).

The RNA chaperone Hfq is involved in stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression.

The well-conserved protein Hfq has emerged as the key modulator of riboregulation in bacteria. This protein is thought to function as an RNA chaperone and to facilitate base pairing between small regulatory RNA (sRNA) and mRNA targets, and many sRNAs are dependent on the Hfq protein for their regulatory functions. To address the possible role of Hfq in riboregulated circuits in Neisseria meningitidis, we generated an Hfq mutant of the MC58 strain, and the knockout mutant has pleiotropic phenotypes; it has a general growth phenotype in vitro in culture media, and it is sensitive to a wide range of stresses, including those that it may encounter in the host. Furthermore, the expression profile of a vast number of proteins is clearly altered in the mutant, and we have identified 27 proteins by proteomics. All of the phenotypes tested to date are also restored by complementation of Hfq expression in the mutant strain. Importantly, in ex vivo and in vivo models of infection the Hfq mutant is attenuated. These data indicate that Hfq plays a key role in stress response and virulence, and we propose a major role for Hfq in regulation of gene expression. Moreover, this study suggests that in meningococcus there is a large Hfq-mediated sRNA network which so far is largely unexplored.