Iron acquisition systems in the pathogenic Neisseria.

Pathogenic 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, haemoglobin-haptoglobin 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.

Discrimination between apo and iron-loaded forms of transferrin by transferrin binding protein B and its N-terminal subfragment.

Many pathogens of the Pasteurellaceae and Neisseriaceae possess a surface receptor that binds transferrin (Tf) as an initial step in an iron acquisition process. This receptor is comprised of two proteins, transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB). Since the ability to recognize the iron-loaded form of Tf preferentially would be a useful attribute of these receptors, we examined this property in a number of bacterial species. In solid-phase binding assays with isolated membranes, only the receptor from Moraxella catarrhalis was capable of preferentially binding iron-loaded Tf. In a competitive affinity isolation assay which enabled us to resolve TbpA and TbpB, TbpA from all tested species was shown to bind both apo and iron-loaded Tf. Under these assay conditions TbpB from M. catarrhalis, Haemophilus somnus and Pasteurella haemolytica discriminated between apo and holo Tf, whereas TbpB from Neisseria meningitidis showed no discrimination. The ability of TbpB from N. meningitidis to bind iron-saturated hTf preferentially became evident in a TbpA- background or by using recombinant TbpB. In binding assays with recombinant fusion proteins, both intact TbpB and the N-terminal half of TbpB from all the tested species preferentially bound Fe-loaded Tf, indicating that this may be a conserved mechanism by which these organisms optimize their ability to acquire iron.

The Pasteurella haemolytica 35 kDa iron-regulated protein is an FbpA homologue.

In a previous investigation, a 35 kDa iron-regulated protein was identified from total cellular proteins of Pasteurella haemolytica grown under iron-depleted conditions. This study reports identification of the gene (fbpA) encoding the 35 kDa protein based on complementation of an entA Escherichia coli strain transformed with a plasmid derived from a P. haemolytica lambda ZAP II library. Cross-reactivity was demonstrated between an anti-35 kDa mAb and a 35 kDa protein expressed in this strain. Furthermore, a translated ORF identified on the recombinant plasmid corresponded with the N-terminal amino acid sequence of the intact and a CNBr-cleaved fragment of the 35 kDa iron-regulated protein. Nucleotide sequence analysis of the gene encoding the 35 kDa protein demonstrated homology with the cluster 1 group of extracellular solute-binding proteins, especially to the iron-binding proteins of this family. Complete sequence analysis of the recombinant plasmid insert identified three other predominant ORFs, two of which appeared to be in an operonic organization with fbpA. These latter components (fbpB and fbpC) showed homology to the transmembrane and ATPase components of ATP-binding cassette (ABC)-type uptake systems, respectively. Based on amino acid/DNA sequencing, citrate competition assay of iron affinity and visible wavelength spectra, it was concluded that the P. haemolytica 35 kDa protein functions as an FbpA homologue (referred to as PFbpA) and that the gene encoding this protein is part of an operon comprising a member of the FbpABC family of iron uptake systems. Primary sequence analysis revealed rather surprisingly that PFbpA is more closely related to the intracellular Mn/Fe-binding protein IdiA found in cyanobacteria than to any of the homologous FbpA proteins currently known in commensal or pathogenic members of the Pasteurellaceae or Neisseriaceae.

Biochemical characterization of a Haemophilus influenzae periplasmic iron transport operon.

Bacterial iron transport is critical for growth of pathogens in the host environment, where iron is limited as a form of nonspecific immunity. For Gram-negative bacteria such as Haemophilus influenzae, iron first must be transported across the outer membrane and into the periplasmic space, then from the periplasm to the cytosol. H. influenzae express a periplasmic iron-binding protein encoded by the hitA gene. This gene is organized as the first of a three-gene operon purported to encode a classic high affinity iron acquisition system that includes hitA, a cytoplasmic permease (hitB), and a nucleotide binding protein (hitC). In this study we describe the cloning, overexpression, and purification of the H. influenzae hitA gene product. The function of this protein is unambiguously assigned by demonstrating its ability to compete for iron bound to the chemical iron chelator 2,2'-dipyridyl, both in vitro and within the periplasmic space of a siderophore-deficient strain of Escherichia coli. Finally, the importance of a functional hitABC operon for iron acquisition is demonstrated by complementation of this siderophore-deficient E. coli to growth on dipyridyl-containing medium. These studies represent a detailed genetic, biochemical, and physiologic description of an active transport system that has evolved to efficiently transport iron and consequently is widely distributed among Gram-negative pathogenic bacteria.

Cloning and sequence analysis of the fur gene encoding an iron-regulatory protein of Neisseria meningitidis.

The fur gene, encoding an iron-regulatory protein in Neisseria meningitidis strain B16B6, has been cloned and sequenced. Its product showed a high degree of homology to known Fur sequences.

Regions located in both the N-lobe and C-lobe of human lactoferrin participate in the binding interaction with bacterial lactoferrin receptors.

As a first step in localizing the regions of human lactoferrin involved in binding to bacterial lactoferrin receptors, N-lobe and C-lobe fragments were assessed for binding to receptors on Neisseria meningitidis, Neisseria gonorrhoeae and Moraxella (Branhamella) catarrhalis. Preparations of N-lobe and C-lobe were obtained by tryptic digestion of iron-loaded human lactoferrin followed by separation of the two lobes by gel exclusion chromatography in 10% acetic acid. Solid phase binding studies demonstrated that the isolated C- and N-lobe preparations were capable of binding to membranes from iron-deficient N. meningitidis, N. gonorrhoeae and M. catarrhalis. The binding of the individual C- and N-lobes was confirmed by an analytical SDS-PAGE binding method in which the membrane-associated polypeptides were identified by prior biotinylation and subsequent binding of labelled streptavidin. This contrasts with bacterial transferrin receptors, which only bind to C-lobe fragment of human transferrin, indicating that the bacterial lactoferrin and transferrin receptors differ in their interaction with their respective glycoprotein ligands and may differ in the mechanism of iron removal.

Correlation between the ability of Haemophilus paragallinarum to acquire ovotransferrin-bound iron and the expression of ovotransferrin-specific receptors.

To investigate the mechanisms of iron acquisition in avian haemophili, strains of Haemophilus paragallinarum and H. avium were tested for siderophore production and utilization of transferrin iron for growth. No evidence of siderophore production was detected in either of these species using a functional screening assay. H. paragallinarum, but not strains of H. avium, was able to acquire iron from 30% saturated chicken and turkey transferrins but not from human, porcine, or bovine transferrins. In response to iron limitation, H. paragallinarum expressed four iron-regulated outer-membrane proteins of 53, 62, 66, and 94 kilodaltons (kDa). Only the 53- and 94-kDa proteins were detected in the H. avium strains. Using affinity methods, the 94- and 53-kDa proteins were isolated specifically by chicken or turkey transferrin, indicating that they may be equivalent to transferrin binding proteins (TBP1 and TBP2, respectively) isolated from other bacterial species. The isolation of the 62- and 66-kDa proteins in association with TBP1 and TBP2 under less stringent washing conditions only in H. paragallinarum implicates these proteins in the iron acquisition process.

Iron acquisition in Haemophilus influenzae: receptors for human transferrin.

As an adaptation to the iron-limited environment of the host, Haemophilus influenzae has a transferrin receptor-mediated mechanism of iron acquisition such that it can acquire iron directly from human transferrin. The absence of detectable siderophore production and the presence of transferrin binding in a collection of type b and nontypeable H. influenzae strains indicate that the mechanism is widespread in this species. Growth and binding studies have consistently shown that the receptor is specific for human transferrin, which correlates with the host range of this pathogen. Inhibitor experiments indicate that iron regulation of receptor activity is mediated at the gene level. Affinity isolation experiments indicate that, as observed with other bacterial pathogens, the receptor is composed of two iron-repressible outer membrane proteins, transferrin binding proteins 1 and 2.

Evidence for non-siderophore-mediated acquisition of transferrin-bound iron by Pasteurella multocida.

Two clinical isolates of Pasteurella multocida associated with bovine pneumonia were examined for iron acquisition. Both isolates were capable of obtaining iron for growth from bovine but not from human, avian, equine or porcine transferrin. This correlated with specific binding of bovine transferrin by iron-limited cells or isolated membranes. No siderophore was detected in the strains by a general screening assay. In response to iron-limited conditions, a number of high molecular mass iron-regulated outer membrane proteins were produced including an 82 kDa receptor protein which was affinity isolated with biotinylated transferrin. In contrast, avian strains of P. multocida could not use transferrin-bound for growth and did not express either transferrin binding activity or the 82 kDa receptor protein.

Response of Haemophilus somnus to iron limitation: expression and identification of a bovine-specific transferrin receptor.

Nine clinical isolates of Haemophilus somnus were screened for their ability to use different transferrins as a source of iron growth. All nine strains were capable of using bovine but not porcine, human or chicken transferrin. A screening assay for siderophore production did not show any evidence of siderophore production by these strains. When iron-deficient cells from these strains were screened for their ability to bind peroxidase-conjugated transferrin, binding was detected with conjugated bovine, but not human or porcine transferrin. Competition binding studies demonstrated that the binding of peroxidase-conjugated bovine transferrin was competitively inhibited by unconjugated bovine transferrin but not transferrin from other species. The induction of receptor expression by low iron conditions was inhibited by chloramphenicol and rifampicin but not ampicillin indicating that new protein and mRNA synthesis was required for expression of receptor activity. Affinity isolation of receptor proteins with biotinylated bovine transferrin, but not human or porcine transferrin, yielded three proteins from H. somnus strain H74. Two of the proteins were identified as 105 kDa and 73 kDa iron-regulated outer membrane proteins. A third protein of 85 kDa that was isolated did not co-migrate with any iron-regulated outer membrane protein. Affinity isolation of receptor proteins from other strains of H. somnus yielded a 73 kDa protein from all strains and a 105 kDa and 85 kDa protein in four of the six strains analysed.

Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure.

Comparison of the abilities of different protein sources of iron to enhance Neisseria meningitidis infection in mice.

This study was done primarily to determine whether the previously observed specificity of the meningococcal transferrin and lactoferrin receptors for human proteins was maintained in vivo during meningococcal infection in mice. Preliminary experiments evaluating the choice of host strain, the age and sex of mice, and the growth conditions of the meningococci indicated that 45-day-old female Swiss Webster mice challenged with meningococci grown on low-pH, low-iron Mueller-Hinton agar plates were appropriate for this study. The comparison of transferrins and lactoferrins from different species demonstrated that only the human forms of these proteins were utilized by meningococci; there was significantly greater mortality among mice treated with iron-saturated human transferrin or lactoferrin (93 and 100%, respectively) than among those not treated or treated with iron-saturated bovine transferrin or bovine lactoferrin (0%). Provision of exogenous hemoglobin also resulted in increased mortality, although not as great as that observed with amounts of transferrin with equivalent iron content, which parallels the more effective utilization of transferrin and lactoferrin in in vitro growth experiments. In addition, unlike with transferrin and lactoferrin, there was no difference in utilization of human and bovine hemoglobin in vitro or in vivo.

Mechanism of Pseudomonas aeruginosa persistence during treatment with broad-spectrum cephalosporins of lung infections in patients with cystic fibrosis.

Beta-lactam resistance in Pseudomonas aeruginosa detected only during ceftazidime therapy of cystic fibrosis patients was studied. Evaluation of resistant and susceptible isolates from one patient and resistant laboratory derivatives indicated that elevated beta-lactamase levels were the primary determinant of resistance. Susceptible isolates outgrew resistant isolates on antibiotic-free medium.

Cloning and expression of genes responsible for altered penicillin-binding proteins 3a and 3b in Haemophilus influenzae.

A Haemophilus influenzae strain (T-1,3) possessing clinical beta-lactam resistance due to altered penicillin-binding protein 3 was used to construct a recombinant cosmid gene bank in Escherichia coli. Three of the recombinant cosmids were capable of transforming a susceptible H. influenzae strain (Rdnov) simultaneously to moxalactam resistance and altered the binding of penicillin-binding proteins 3a and 3b to [35S]penicillin G. Restriction endonuclease mapping of one of the recombinant cosmids, pLB100, was performed to facilitate subsequent subcloning of the gene(s) responsible for the altered penicillin-binding protein 3 (a and b) binding phenotype. Subcloning of individual fragments derived from pLB100 indicated that two adjacent fragments of DNA were both capable of transforming a susceptible Haemophilus strain to moxalactam resistance and altered penicillin-binding protein 3 binding. Expression of plasmid-coded proteins in minicells indicated that one fragment coded for a major 55,000-molecular-weight polypeptide and that the second contained a C-terminal coding region that expressed a 28,000-molecular-weight polypeptide when fused to the N-terminal region of the tetracycline resistance gene. Initial attempts at labeling the plasmid-coded proteins expressed in minicells with [35S]penicillin G were unsuccessful.

Antigenic relationships among penicillin-binding proteins 1 from members of the families Pasteurellaceae and Enterobacteriaceae.

Penicillin-binding proteins (PBPs) from Haemophilus influenzae RD purified by a combination of affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electroelution were used to immunize rabbits to obtain specific antisera. Antisera directed against PBP 1 (1b) of H. influenzae cross-reacted with representative organisms of the family Pasteurellaceae and with many members of the family Enterobacteriaceae but not with other gram-negative organisms. Immunization with purified PBP 3 of H. influenzae produced antisera that reacted with PBP 1 (1b) of H. influenzae and showed the same cross-reactive pattern with other species as the anti-PBP 1 antiserum. A 24,000-molecular-weight polypeptide of H. influenzae, not radiolabeled by [35S]penicillin, reacted with antisera against purified PBPs 1 (1a, 1b), 2, and 3. The results suggest that antigenic epitopes are shared among similar PBPs from related species and even among different PBPs within the same species.