Structure of the Neisseria Adhesin Complex Protein (ACP) and its role as a novel lysozyme inhibitor.

Pathogenic and commensal Neisseria species produce an Adhesin Complex Protein, which was first characterised in Neisseria meningitidis (Nm) as a novel surface-exposed adhesin with vaccine potential. In the current study, the crystal structure of a recombinant (r)Nm-ACP Type I protein was determined to 1.4 Å resolution: the fold resembles an eight-stranded ß-barrel, stabilized by a disulphide bond between the first (Cys38) and last (Cys121) ß-strands. There are few main-chain hydrogen bonds linking ß4-ß5 and ß8-ß1, so the structure divides into two four-stranded anti-parallel ß-sheets (ß1-ß4 and ß5-ß8). The computed surface electrostatic charge distribution showed that the ß1-ß4 sheet face is predominantly basic, whereas the ß5-ß8 sheet is apolar, apart from the loop between ß4 and ß5. Concentrations of rNm-ACP and rNeisseria gonorrhoeae-ACP proteins ≥0.25 µg/ml significantly inhibited by ~80-100% (P<0.05) the in vitro activity of human lysozyme (HL) over 24 h. Specificity was demonstrated by the ability of murine anti-Neisseria ACP sera to block ACP inhibition and restore HL activity. ACP expression conferred tolerance to HL activity, as demonstrated by significant 3-9 fold reductions (P<0.05) in the growth of meningococcal and gonococcal acp gene knock-out mutants in the presence of lysozyme. In addition, wild-type Neisseria lactamica treated with purified ACP-specific rabbit IgG antibodies showed similar fold reductions in bacterial growth, compared with untreated bacteria (P<0.05). Nm-ACPI is structurally similar to the MliC/PliC protein family of lysozyme inhibitors. However, Neisseria ACP proteins show <20% primary sequence similarity with these inhibitors and do not share any conserved MliC/PliC sequence motifs associated with lysozyme recognition. These observations suggest that Neisseria ACP adopts a different mode of lysozyme inhibition and that the ability of ACP to inhibit lysozyme activity could be important for host colonization by both pathogenic and commensal Neisseria organisms. Thus, ACP represents a dual target for developing Neisseria vaccines and drugs to inhibit host-pathogen interactions.

Visualizing multistep elevator-like transitions of a nucleoside transporter.

Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii. Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion.

A Comparative Structure/Function Analysis of Two Type IV Pilin DNA Receptors Defines a Novel Mode of DNA Binding.

DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors.

Identification of an antibacterial protein by functional screening of a human oral metagenomic library.

Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375.

Pseudomonas aeruginosa minor pilins prime type IVa pilus assembly and promote surface display of the PilY1 adhesin.

Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility.

Optimization of intracellular product release from Neisseria denitrificans using microfluidizer.

Disruption of Neisseria denitrificans cells by microfluidizer was optimized using a factorial experiments design. The pH, pretreatment time, cell concentration, NaCl, ethylenediamine tetraacetic acid (EDTA) and Triton X-100 concentrations showed significant impact on disruption process and the process was optimized using central composite design and response surface methodology (RSM). Investigation revealed optimum conditions: 90 min pretreatment at pH 9.0 containing 110 g L(-1) cells (dry cell weight), 50 mM NaCl, 10 mM EDTA, and 0.2% Triton X-100. At optimized conditions, the disruption rate increased twofold, up to 5.62 ± 0.27 × 10(-3) MPa(-a); meanwhile, yield of intracellular content was increased by 26%, with 1 g of cells resulting in 113.2 ± 8.2 mg proteins, 12.1 ± 0.7 mg nucleic acids, 21.0 ± 1.2 mg polysaccharides, 0.99 ± 0.08 kU glucose-6-phosphate dehydrogenase (G6PD), and 10,100 ± 110 kU restriction endonuclease NdeI endonuclease. Particle size distribution analysis revealed nearly twofold larger cell lysate particles with diameter of 120 nm. For optimal release of intracellular content, 9200 J/g of energy was needed (95% confidence), yielding 6900 J/g energy savings. Model equations generated from RSM on cell disruption of N. denitrificans were found adequate to determine significant factors and its interaction. The results showed that optimized combination of known pretreatment and disruption methods could considerably improve cell disruption efficiency.

Distribution and diversity of the haemoglobin-haptoglobin iron-acquisition systems in pathogenic and non-pathogenic Neisseria.

A new generation of vaccines containing multiple protein components that aim to provide broad protection against serogroup B meningococci has been developed. One candidate, 4CMenB (4 Component MenB), has been approved by the European Medicines Agency, but is predicted to provide at most 70-80 % strain coverage; hence there is a need for second-generation vaccines that achieve higher levels of coverage. Prior knowledge of the diversity of potential protein vaccine components is a key step in vaccine design. A number of iron import systems have been targeted in meningococcal vaccine development, including the HmbR and HpuAB outer-membrane proteins, which mediate the utilization of haemoglobin or haemoglobin-haptoglobin complexes as iron sources. While the genetic diversity of HmbR has been described, little is known of the diversity of HpuAB. Using whole genome sequences deposited in a Bacterial Isolate Genome Sequence Database (BIGSDB), the prevalence and diversity of HpuAB among Neisseria were investigated. HpuAB was widely present in a range of Neisseria species whereas HmbR was mainly limited to the pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae. Patterns of sequence variation in sequences from HpuAB proteins were suggestive of recombination and diversifying selection consistent with strong immune selection. HpuAB was subject to repeat-mediated phase variation in pathogenic Neisseria and the closely related non-pathogenic Neisseria species Neisseria lactamica and Neisseria polysaccharea but not in the majority of other commensal Neisseria species. These findings are consistent with HpuAB being subject to frequent genetic transfer potentially limiting the efficacy of this receptor as a vaccine candidate.

Western blot analysis of neisserial lipooligosaccharide at the lower femtomole level with normal human sera.

Lipooligosaccharide (LOS) is a major immunogenic component of pathogenic Neisseria species such as Neisseria meningitidis and N. gonorrhoeae. Recent immunochemical studies have found that normal human sera (NHS) contain bactericidal anti-LOS antibodies that bind to the oligosaccharide (OS) moiety of neisserial LOS. Although affinity-purified anti-LOS antibodies can be characterized using 10-100 ng of LOS samples (up to a few tens of pmoles), a more sensitive immunoblotting assay must be established in order to analyze NHS directly and characterize anti-LOS antibodies without affinity purification. We examined analytical PAGE/blot conditions using a 15-well mini gel. For the first time, Western blot detection of LOS at the lower femtomole level was accomplished by both chromogenic and chemiluminescent detection. A model LOS, 15253 LOS, was detected in a low femtomole range (62.5-500 pg, 16-125 femtomole) even with 10 pM of a monoclonal antibody (MAb) 2C7. Furthermore, detection of similar amounts (50-250 femtomole) of neisserial LOSs and Salmonella truncated lipopolysaccharides (LPSs) was also possible with 1:50 and with 1:100 diluted NHS. The results obtained here indicate that the binding of IgG in NHS to the LOS and LPS samples is probably due to their carbohydrate moieties. The detection level accomplished in this study should help not only to further characterize anti-LOS antibodies in blood and body fluids but also to analyze carbohydrate structures that are recognized by them.

Neisserial Correia repeat-enclosed elements do not influence the transcription of pil genes in Neisseria gonorrhoeae and Neisseria meningitidis.

Two human-specific neisserial pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, require the expression of type IV pili (tfp) for initial attachment to the host during infection. However, the mechanisms controlling the assembly and functionality of tfp are poorly understood. It is known that the gonococcal pilE gene, encoding the major subunit, is positively regulated by IHF, a multifunctional DNA binding protein. A neisserial specific repetitive DNA sequence, termed the Correia repeat-enclosed element (CREE) is situated upstream of three pil loci: pilHIJKX (pilH-X), pilGD, and pilF. CREEs have been shown to contain strong promoters, and some CREE variants contain a functional IHF binding site. CREEs might therefore be involved in the regulation of tfp biogenesis in pathogenic Neisseria. Site-directed and deletion mutagenesis on promoter::cat reporter constructs demonstrated that transcription of pilH-X and pilGD is from a σ(70) promoter and is independent of the CREE. The insertion of a CREE in the pilF promoter region in N. meningitidis generated a functional σ(70) promoter. However, there is also a functional promoter at this position in N. gonorrhoeae, where there is no CREE. These results suggest CREE insertion in these three pil loci does not influence transcription and that IHF does not coordinately regulate tfp biogenesis.

Structural characterization of outer membrane components of the type IV pili system in pathogenic Neisseria.

Structures of the type IV pili secretin complexes from Neisseria gonorrhoeae and Neisseria meningitidis, embedded in outer membranes were investigated by transmission electron microscopy. Single particle averaging revealed additional domains not observed previously. Secretin complexes of N. gonorrhoeae showed a double ring structure with a 14-15-fold symmetry in the central ring, and a 14-fold symmetry of the peripheral ring with 7 spikes protruding. In secretin complexes of N. meningitidis, the spikes were absent and the peripheral ring was partly or completely lacking. When present, it had a 19-fold symmetry. The structures of the complexes in several pil mutants were determined. Structures obtained from the pilC1/C2 adhesin and the pilW minor pilin deletion strains were similar to wild-type, whereas deletion of the homologue of N. meningitidis PilW resulted in the absence of secretin structures. Remarkably, the pilE pilin subunit and pilP lipoprotein deletion mutants showed a change in the symmetry of the peripheral ring from 14 to 19 and loss of spikes. The pilF ATPase mutant also lost the spikes, but maintained 14-fold symmetry. These results show that secretin complexes contain previously unidentified large and flexible extra domains with a probable role in stabilization or assembly of type IV pili.

Neisseria wadsworthii sp. nov. and Neisseria shayeganii sp. nov., isolated from clinical specimens.

An analysis of 16S rRNA gene sequences from archived clinical reference specimens has identified two novel Neisseria species. For each species, two strains from independent sources were identified. Amongst species with validly published names, the closest species to the newly identified organisms were Neisseria canis, N. dentiae, N. zoodegmatis, N. animaloris and N. weaveri. DNA-DNA hybridization studies demonstrated that the newly identified isolates represent species that are distinct from these nearest neighbours. Analysis of partial 23S rRNA gene sequences for the newly identified strains and their nearest neighbours provided additional support for the species designation. Bayesian analysis of 16S rRNA gene sequences suggested that the newly identified isolates belong to distinct but related species of the genus Neisseria, and are members of a clade that includes N. dentiae, N. bacilliformis and N. canis. The predominant cellular fatty acids [16 : 0, summed feature 3 (16 : 1ω7c and/or iso-15 : 0 2-OH) and 18 : 1ω7c], as well as biochemical and morphological analyses further support the designation of Neisseria wadsworthii sp. nov. (type strain 9715(T) =DSM 22247(T) =CIP 109934(T)) and Neisseria shayeganii sp. nov. (type strain 871(T) =DSM 22246(T) =CIP 109933(T)).

Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction.

Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically.

Application of atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry for rapid identification of Neisseria species.

Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI MS) was applied to develop a proteomics-based method to detect and identify Neisseria species. Heat-inactivated clinical isolate cell suspensions of Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to on-probe protein/peptide extraction and tryptic digestion followed by AP-MALDI tandem MS (MS/MS)-based proteomic analysis. Amino acid sequences derived from three protonated peptides with m/z values of 1743.8, 1894.8, and 1946.8 were identified by AP-MALDI MS/MS and MASCOT proteome database search analysis as belonging to neisserial acyl carrier protein, neisserial-conserved hypothetical protein, and neisserial putative DNA binding protein, respectively. These three peptide masses can thus be potential biomarkers for neisserial species identification by AP-MALDI MS.

Towards the molecular understanding of glycogen elongation by amylosucrase.

Amylosucrase from Neisseria polysaccharea (AS) is a transglucosidase from the glycoside-hydrolase family 13 that catalyzes the synthesis of an amylose-like polymer from sucrose, without any primer. Its affinity towards glycogen is particularly noteworthy since glycogen is the best D-glucosyl unit acceptor and the most efficient activator (98-fold k(cat) increase) known for this enzyme. Glycogen-enzyme interactions were modeled starting from the crystallographic AS: maltoheptaose complex, where two key oligosaccharide binding sites, OB1 and OB2, were identified. Two maltoheptaose molecules were connected by an alpha-1,6 branch by molecular modeling to mimic a glycogen branching. Among the various docking positions obtained, four models were chosen based on geometry and energy criteria. Robotics calculations enabled us to describe a back and forth motion of a hairpin loop of the AS specific B'-domain, a movement that assists the elongation of glycogen branches. Modeling data combined with site-directed mutagenesis experiments revealed that the OB2 surface site provides an anchoring platform at the enzyme surface to capture the polymer and direct the branches towards the OB1 acceptor site for elongation. On the basis of the data obtained, a semiprocessive glycogen elongation mechanism can be proposed.

Azurin, Plasmodium falciparum malaria and HIV/AIDS: inhibition of parasitic and viral growth by Azurin.

Azurin, a member of a family of copper-containing proteins involved in electron transfer called cupredoxins, demonstrates structural features similar to the variable domains of the immunoglobulin superfamily members. An azurin-like protein called Laz with an additional N-terminal 39 amino acid peptide known as H.8 epitope is present on the surface of gonnococci and meningococci. We demonstrate that azurin, Laz and H.8-azurin can bind to the C-terminal cleavage product MSP1-19 of merozoite surface protein 1 (MSP1) of the malarial parasite Plasmodium falciparum and significantly reduce parasitemia. Azurin and Laz also bound strongly to HIV-1 gp120. Interestingly, azurin could not only bind to gp120 but also to the dendritic cell-specific adhesion receptor DC-SIGN, mimicking the functionality of the intercellular adhesion molecule ICAM-3 with which it also binds avidly. Furthermore, these three proteins significantly suppressed HIV-1 growth in peripheral blood mononuclear cells and such suppression appeared to be occurring at an entry stage in the infection process. The presence of both antimalarial and antiretroviral activity in azurin, H.8-azurin and Laz makes these proteins, or peptides derived from them, potential therapeutic agents in the treatment of malaria, HIV-1 infections or coinfections with both P. falciparum and HIV-1.

Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport.

The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process.

Analysis of outer membrane protein complexes and heat-modifiable proteins in Neisseria strains using two-dimensional diagonal electrophoresis.

Two-dimensional diagonal SDS-PAGE was used to resolve membrane complexes and identify proteins with temperature-dependent mobility in Neisseria meningitidis and N. lactamica. The main membrane complexes were composed of porins and were formed by heteromers of PorA, PorB and RmpM in N. meningitidis, and by PorB and RmpM in N. lactamica. Also, other proteins, including Opa, with temperature-dependent mobility were clearly demonstrated. The method allows improved detection of the components of membrane complexes and proteins with temperature-dependent mobility which is difficult to resolve with other analytical approaches.

Determination of methylation site of DNA-methyltransferase NlaX by a hybrid method.

Using a new method based on a combination of bisulfite reaction, the repair enzyme uracil-DNA glycosylase, and synthetic oligodeoxyribonucleotides, the methylation site of DNA-methyltransferase NlaX (M.NlaX) from Neisseria lactamica was established to be the inner cytosine in the double-stranded pentanucleotide recognition sequence 5'-CCNGG-3' (where N = any nucleoside). 5-Methylcytosine (m5C) type modification by M-N1aX was confirmed by the use of oligonucleotide substrates that contain 5-fluoro-2'-deoxycytidine.

Expression of pathogen-like Opa adhesins in commensal Neisseria: genetic and functional analysis.

Several species of commensal Neisseriae (Cn) may colonize the human nasopharynx, but little is known about their adhesion mechanisms. We have investigated structural and functional similarities between adhesins of Cn and of Neisseria meningitidis (Nm), also a frequent colonizer of the nasopharynx. In this study, we demonstrate the expression of Opa-like proteins in nine strains of Cn. Phylogenetic analysis segregated the majority of the Cn Opa in a cluster separated from the pathogenic cluster with a few exceptions. One Opa, which located within the pathogenic cluster, was strikingly similar (74%) to an Opa of a Neisseria gonorrhoeae (Ng) strain and, like Ng, it lacked the extra Y11 or the 136DKF138 triplet insert, which are conserved among many N. meningitidis Opa proteins. Most importantly, the majority of the Cn Opa proteins were able to interact with human CEACAM1 (CD66a) molecules, previously identified as receptors for pathogenic Opa proteins. By the use of CEACAM1 N-domain mutants, we demonstrate that Cn Opa target the same region of the N-domain of the receptor as that used by Nm. Furthermore, Cn strains bound to cell-expressed human CEACAM1. In competition assays, adherent Cn strain C450, exhibiting high affinity for CEACAM1, was not displaced by a Nm isolate and vice versa. But in simultaneous incubation, Nm out-competed the Cn strain. This is the first study to demonstrate the expression of adhesins in Cn that are structurally and functionally closely related to pathogenic adhesins. The studies imply that some Cn have the potential to occupy and thus compete with the pathogens for receptors on human mucosa, their common and exclusive niche.

The neisserial 37 kDa ferric binding protein (FbpA).

The ferric binding protein (FbpA) is one of the major proteins regulated by the level of environmental iron in the genus Neisseria. Its conservation in all species of pathogenic Neisseria has been demonstrated, and the possible role that it plays in the iron uptake mechanisms in these bacteria has been postulated. Similar proteins in Haemophilus influenzae (HitA) and in Serratia marcescens (SfuA) have been described, but relationships with the meningococcal FbpA could not be proven. Although supposedly periplasmic, the exact location of FbpA remains controversial because some molecules, or parts of them, have been found exposed to the bacterial outer surface. The DNA sequence downstream of the fbpA gene has been recently analysed, finding an operon composed of three open reading frames: fbpA, encoding for FbpA; fbpB, that codifies a cytoplasmic permease, and fbpC, that contains the information for a nucleotide binding protein. These proteins would form an iron transport system through the periplasmic space. FbpA is highly antigenic in mice when injected in purified form, shows intraspecies and interspecies antigenic homogenicity, and specific anti-FbpA antibodies are fully cross-reactive; nevertheless, the in vivo induction of anti-FbpA antibodies in man is still polemical. Recent studies reveal that the purified FbpA induces a fair response of bactericidal antibodies in mice.