SummaryPathogenic neisseriae have a repertoire of high-affinity iron uptake systems to facilitate acquisition of this essential element in the human host. They possess surface receptor proteins that directly bind the extracellular host iron-binding proteins transferrin and lactoferrin. Alternatively, they have siderophore receptors capable of scavenging iron when exogenous siderophores are present. Released intracellular haem iron present in the form of haemoglobin, haemoglobinhaptoglobin or free haem can be used directly as a source of iron for growth through direct binding by specific surface receptors. Although these receptors may vary in complexity and composition, the key protein involved in the transport of iron (as iron, haem or iron-siderophore) across the outer membrane is a TonB-dependent receptor with an overall structure presumably similar to that determined recently for Escherichia coli FhuA or FepA. The receptors are potentially ideal vaccine targets in view of their critical role in survival in the host. Preliminary pilot studies indicate that transferrin receptor-based vaccines may be protective in humans. Availability of iron in the human hostIron is an essential element, as it serves as a cofactor in key metabolic processes, such as nucleotide biosynthesis and energy production. In iron-containing proteins, the iron is bound directly by amino acids or may be incorporated into haem, a complex of iron with porphyrin (Bridges and Seligman, 1995). Excess iron within cells is stored in the protein ferritin and, in spite of the redox status inside cells, the iron is stored as ferric and not ferrous ions. In the human host, iron bound to haemoglobin within erythrocytes plays a critical role in the transport and exchange of oxygen with tissues throughout the body. The tetrameric haemoglobin molecule consists of four homologous 16 kDa subunits that each bind a single haem moiety.Iron is transported throughout the body by the serum glycoprotein transferrin (Tf) (Bridges and Seligman, 1995). Human Tf is an 80 kDa bilobed monomeric glycoprotein with a ferric ion and bicarbonate anion binding site present in each lobe. Iron-loaded Tf is bound by transferrin receptors on the surface of cells requiring iron, and the iron is removed through a cyclical process involving receptormediated endocytosis. Tf is present in substantial levels (25-44 M) in serum, but is only a minor component (0.2-1.3 M) of mucosal secretions. In serum, the iron saturation of Tf is usually 33%, with iron primarily occupying the N-lobe, and the level of iron saturation can be reduced substantially during infection.Lactoferrin (Lf ) is a related iron-binding glycoprotein that is present in mucosal secretions and is released by leucocytes at sites of inflammation. Lf is structurally very similar to Tf but differs in that it is capable of retaining iron under acidic conditions (pH < 6). In contrast to Tf, Lf is involved in a variety of physiological functions other than iron sequestration (Spik et al., 1998). Lf is present at very low level...

The upper respiratory tract (URT) is a crucial site for host defense, as it is home to bacterial communities that both modulate host immune defense and serve as a reservoir of potential pathogens. Young children are at high risk of respiratory illness, yet the composition of their URT microbiota is not well understood. Microbial profiling of the respiratory tract has traditionally focused on culturing common respiratory pathogens, whereas recent culture-independent microbiome profiling can only report the relative abundance of bacterial populations. In the current study, we used both molecular profiling of the bacterial 16S rRNA gene and laboratory culture to examine the bacterial diversity from the oropharynx and nasopharynx of 51 healthy children with a median age of 1.1 years (range 1-4.5 years) along with 19 accompanying parents. The resulting profiles suggest that in young children the nasopharyngeal microbiota, much like the gastrointestinal tract microbiome, changes from an immature state, where it is colonized by a few dominant taxa, to a more diverse state as it matures to resemble the adult microbiota. Importantly, this difference in bacterial diversity between adults and children accompanies a change in bacterial load of three orders of magnitude. This indicates that the bacterial communities in the nasopharynx of young children have a fundamentally different structure from those in adults and suggests that maturation of this community occurs sometime during the first few years of life, a period that includes ages at which children are at the highest risk for respiratory disease.

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Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.

Pathogenic bacteria from the Neisseriaceae and Pasteurellacea families acquire iron directly from the host iron-binding glycoprotein, transferrin (Tf), in a process mediated by surface receptor proteins that directly bind host Tf, extract the iron, and transport it across the outer membrane. The bacterial Tf receptor is comprised of a surface exposed lipoprotein, Tf-binding protein B (TbpB), and an integral outer-membrane protein, Tf-binding protein A (TbpA), both of which are essential for survival in the host. In this study, we report the 1.98 A resolution structure of TbpB from the porcine pathogen Actinobacillus pleuropneumoniae, providing insights into the mechanism of Tf binding and the role of TbpB. A model for the complex of TbpB bound to Tf is proposed. Mutation of a single surface-exposed Phe residue on TbpB within the predicted interface completely abolishes binding to Tf, suggesting that the TbpB N lobe comprises the sole high-affinity binding region for Tf.