Molecular characterization of hpuAB, the haemoglobin–haptoglobin‐utilization operon of Neisseria meningitidis

Abstract

SummaryWe previously identified HpuB, an 85 kDa Fe-repressible protein required for utilization of Fe from, and binding to, haemoglobin and the haemoglobin-haptoglobin complex. The gene for hpuB was cloned from Neisseria meningitidis strain DNM2 and the predicted amino acid sequence indicates that HpuB is an outer membrane receptor belonging to the TonB family of high-affinity transport proteins. A second open reading frame, predicted to encode a 34.8 kDa lipoprotein, was discovered 5Ј to hpuB, and was designated hpuA. HpuA was identified in a total-membrane-protein preparation by construction of a mutant lacking HpuA. Acylation of HpuA was confirmed by [ 3 H]-palmitic acid labelling of meningococci. Consensus promoter sequences were not apparent 5Ј to hpuB. The hpuA insertion mutation exerted a polar effect, abolishing expression of hpuB, suggesting that hpuA and hpuB are co-transcribed. The 3.5 kb polycistronic hpuAB mRNA was identified and shown to be transcriptionally repressed by iron. The transcriptional start site was identified 33 nucleotides 5Ј to the hpuA translational start site, appropriately positioned around consensus promoter and ferric uptake regulator (Fur)-box sequences. The structure of this operon suggests that HpuA-HpuB is a two-component receptor analogous to the bipartite transferrin receptor TbpB-TbpA.

Characterization and Functional Analysis of the Siderophore-Iron Transporter CaArn1p in Candida albicans

“…However, microbial pathogens have collectively evolved a diverse repertoire of highly effective iron acquisition mechanisms, some of which appear to be specifically "designed" to defeat the ironwithholding system or to target iron-rich niches of the host (3)(4)(5)(6). For example, pathogenic Neisseria species have surface receptors specific for the host extracellular iron-binding proteins transferrin and lactoferrin, turning the two major components of the host iron-withholding system into their iron sources (7,8). Candida albicans possesses mechanisms for attracting and lysing erythrocytes and utilizing the iron in hemoglobin (9,10).…”

Analysis of Haptoglobin and Hemoglobin-Haptoglobin Interactions with theNeisseria meningitidisTonB-Dependent Receptor HpuAB by Flow Cytometry

“…No known homologues of HpuA exist in public databases, and its role in HpuAB function remains to be determined. We have reported data suggesting that HpuA and HpuB physically interact to form a functional receptor complex (51,69). Characterization of isogenic single mutants of HpuA and HpuB suggested that both receptor components are absolutely required for the utilization of Hb or HbHp (48,69).…”

“…This model was based on the highly conserved three-dimensional X-ray crystallographic structures of three E. coli TonB-dependent siderophore receptors: FepA, FhuA, and FecA (6,(23)(24)(25)52). The 35-kDa HpuA accessory lipoprotein is peripherally associated with the OM via an N-terminally linked fatty acid tether (49,69). No known homologues of HpuA exist in public databases, and its role in HpuAB function remains to be determined.…”

“…This study was focused on the meningococcal two-component receptor HpuAB, previously shown to bind Hb, apo-Hp, and HbHp (47,49). Based on its homology to the TonBdependent receptor family, we proposed that the 85-kDa HpuB interacts with TonB and forms a beta-barrel transport channel across the OM that is gated by an N-terminal periplasmic "plug" domain (49,68).…”

“…Based on its homology to the TonBdependent receptor family, we proposed that the 85-kDa HpuB interacts with TonB and forms a beta-barrel transport channel across the OM that is gated by an N-terminal periplasmic "plug" domain (49,68). This model was based on the highly conserved three-dimensional X-ray crystallographic structures of three E. coli TonB-dependent siderophore receptors: FepA, FhuA, and FecA (6,(23)(24)(25)52).…”