Biomedical subjects

I C Boulton

Publications and source records attributed to I C Boulton.

9 recordsLinked to original sources

PubMed2000

Identification of discrete domains within gonococcal transferrin-binding protein A that are necessary for ligand binding and iron uptake functions.

The availability of free iron in vivo is strictly limited, in part by the iron-binding protein transferrin. The pathogenic Neisseria spp. can sequester iron from this protein, dependent upon two iron-repressible, transferrin-binding proteins (TbpA and TbpB). TbpA is a TonB-dependent, integral, outer membrane protein that may form a beta-barrel exposing multiple surface loops, some of which are likely to contain ligand-binding motifs. In this study we propose a topological model of gonococcal TbpA and then test some of the hypotheses set forth by the model by individually deleting three putative loops (designated loops 4, 5, and 8). Each mutant TbpA could be expressed without toxicity and was surface exposed as assessed by immunoblotting, transferrin binding, and protease accessibility. Deletion of loop 4 or loop 5 abolished transferrin binding to whole cells in solid- and liquid-phase assays, while deletion of loop 8 decreased the affinity of the receptor for transferrin without affecting the copy number. Strains expressing any of the three mutated TbpAs were incapable of growth on transferrin as a sole iron source. These data implicate putative loops 4 and 5 as critical determinants for receptor function and transferrin-iron uptake by gonococcal TbpA. The phenotype of the DeltaL8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.

Amino Acid Sequence↗

PubMed2000

Antigenic and sequence diversity in gonococcal transferrin-binding protein A.

Neisseria gonorrhoeae is a gram-negative pathogen that is capable of satisfying its iron requirement with human iron-binding proteins such as transferrin and lactoferrin. Transferrin-iron utilization involves specific binding of human transferrin at the cell surface to what is believed to be a complex of two iron-regulated, transferrin-binding proteins, TbpA and TbpB. The genes encoding these proteins have been cloned and sequenced from a number of pathogenic, gram-negative bacteria. In the current study, we sequenced four additional tbpA genes from other N. gonorrhoeae strains to begin to assess the sequence diversity among gonococci. We compared these sequences to those from other pathogenic bacteria to identify conserved regions that might be important for the structure and function of these receptors. We generated polyclonal mouse sera against synthetic peptides deduced from the TbpA sequence from gonococcal strain FA19. Most of these synthetic peptides were predicted to correspond to surface-exposed regions of TbpA. We found that, while most reacted with denatured TbpA in Western blots, only one antipeptide serum reacted with native TbpA in the context of intact gonococci, consistent with surface exposure of the peptide to which this serum was raised. In addition, we evaluated a panel of gonococcal strains for antigenic diversity using these antipeptide sera.

Amino Acid Sequence↗

PubMed1999

A dynamic model of the meningococcal transferrin receptor.

Iron is an essential nutrient for all organisms and consequently, the ability to bind transferrin and sequester iron from his source constitutes a distinct advantage to a blood-borne bacterial pathogen. Levels of free iron are strictly limited in human serum, largely through the action of the iron-binding protein transferrin. The acquisition of trasferrin-iron is coincident with pathogenicity among Neisseria species and a limited number of other pathogens of human and veterinary significance. In Neisseria meningitidis, transferrin binding relies on two co-expressed, outer membrane proteins distinct in aspects of both structure and function. These proteins are independently and simultaneously capable of binding human transferrin and both are required for the optimal uptake of iron from this source. It has been established that transferrin-binding proteins (designated TbpA and TbpB) form a discrete, specific complex which may be composed of a transmembrane species (composed of the TbpA dimer) associated with a single surface-exposed lipoprotein (TbpB). This more exposed protein is capable of selectively binding iron-saturated transferrin and the receptor complex has ligand-binding properties which are distinct from either of its components. Previous in vivo analyses of N. gonorrhoeae, which utilizes a closely related transferrin-iron uptake system, indicated that this receptor exists in several conformations influenced in part by the presence (or absence) of transferrin. Here we propose a dynamic model of the meningococcal transferrin receptor which is fully consistent with the current data concerning this subject. We suggest that TbpB serves as the initial binding site for iron-saturated transferrin and brings this ligand close to the associated transmembrane dimer, enabling additional binding events and orientating transferrin over the dual TbpA pores. The antagonistic association of these receptor proteins with a single ligand molecule may also induce conformational change in transferrin, thereby favouring the release of iron. As, in vivo, transferrin may have iron in one or both lobes, this dynamic molecular arrangement would enable iron uptake from either iron-binding site. In addition, the predicted molecular dimensions of the putative TbpA dimer and hTf are fully consistent with these proposals. Given the diverse data used in the formulation of this model and the consistent characteristics of transferrin binding among several significant Gram-negative pathogens, we speculate that such receptor-ligand interactions may be, at least in part, conserved between species. Consequently, this model may be applicable to bacteria other than N. meningitidis.

Blood-Borne Pathogens↗

PubMed1999

Purified meningococcal transferrin-binding protein B interacts with a secondary, strain-specific, binding site in the N-terminal lobe of human transferrin.

Neisseria meningitidis, grown in iron-limited conditions, produces two transferrin-binding proteins (TbpA and TbpB) that independently and specifically bind human serum transferrin (hTF) but not bovine serum transferrin (bTF). We have used surface plasmon resonance to characterize the interaction between individual TbpA and TbpB and a series of full-length human-bovine chimaeric transferrins (hbTFs) under conditions of variable saturation with iron. A comparative analysis of hTF and hbTF chimaera-binding data confirmed that the major features involved in Tbp binding are located in the C-terminal lobe of hTF and that isolated TbpA can recognize distinct sites present in, or conformationally influenced by, residues 598-679. Binding by TbpB was maintained at a significant but decreased level after replacement of the entire hTF C-terminal lobe by the equivalent bovine sequence. The extent of this binding difference was dependent on the meningococcal strain and on the presence of hTF residues 255-350. This indicated that TbpB from strain SD has a secondary, strain-specific, binding site located within this region, whereas TbpB from strain B16B6 does not share this recognition site. Binding of TbpA was influenced primarily by sequence substitutions in the hTF C-terminal lobe, and co-purified TbpA and TbpB (TbpA+B) was functionally distinct from either of its components. The limited divergence between hTF and bTF has been related to observed differences in binding by Tbps and has been used to delineate those regions of hTF that are important for such interactions.

Bacterial Outer Membrane Proteins↗

PubMed1998

Transferrin-binding protein B isolated from Neisseria meningitidis discriminates between apo and diferric human transferrin.

Neisseria meningitidis utilization of human serum transferrin (hTF)-bound iron is an important pathogenicity determinant. The efficiency of this system would clearly be increased through preferential binding of diferric hTF over the iron-free form. To characterize this process, functionally active meningococcal transferrin-binding protein A (TbpA) and TbpB have been purified from N. meningitidis using a novel purification procedure. The association of isolated Tbps and Tbps in the presence of hTF was investigated by gel filtration. Co-purified TbpA+B formed a complex of molecular mass 300 kDa which bound 1-2 molecules of hTF. Purified TbpA formed a complex of 200 kDa, indicating association as a dimer, whereas TbpB aggregated to form multimers of variable sizes. On recombining TbpA and TbpB, a stable complex of equivalent size to co-purified TbpA+B was formed. This complex may be composed of a single TbpA dimer and 1 molecule of TbpB. The technique of surface plasmon resonance (SPR) was used to demonstrate clearly that TbpB of either high (85 kDa) or low (68 kDa) molecular-mass preferentially bound diferric hTF in comparison with iron-free hTF. This selectivity was not observed with TbpA, but was found at low levels with co-purified TbpA+B. Individual TbpA and TbpB, recombined in a 1:1 molecular ratio, showed iron-mediated discriminatory binding at an intermediate level. SPR was also used to show that TbpA and TbpB bound to distinct regions of hTF, and that prior saturation with TbpB reduced subsequent TbpA binding. The results demonstrated that hTF bound more TbpA than TbpB, with an approximate ratio of 2:1. We have demonstrated that in vitro, TbpA+B exists as a receptor complex composed of a TbpA dimer and one molecule of TbpB, and that TbpB selectively binds diferric hTF. We propose that, in vivo, TbpA and TbpB also exist as a receptor complex, with TbpB selectively binding diferric hTF, bringing it close to TbpA, the transmembrane component, where the ferric iron can be transported to the periplasm.

Apoproteins↗

PubMed1997

Characterisation of the meningococcal transferrin binding protein complex by photon correlation spectroscopy.

Photon correlation spectroscopy demonstrated for the first time that co-purified meningococcal TbpA+B form a complex in solution. This structure bound hTf and the resultant species underwent partial dissociation after exposure to additional hTf or following prolonged incubation. Purified TbpA and TbpB had similar apparent sizes but showed distinctive size profiles suggesting that TbpA forms a largely homogeneous population while TbpB may produce more variable particle sizes under these conditions.

Bacterial Outer Membrane Proteins↗

PubMed1997

Fluoride-induced lesions in the teeth of the short-tailed field vole (Microtus agrestis): a description of the dental pathology.

The effect of fluoride on the appearance of the teeth of the short-tailed field vole, Microtus agrestis, was investigated in both wild animals collected from field sites affected by different levels of industrial fluoride contamination and laboratory-reared animals consuming experimental grass diets of known fluoride concentration or with known fluoride concentrations in drinking water. The extent and severity of lesions on the surface and structure of both incisors and molars are described as six lesion types and related to the amount of biologically available orally ingested fluoride. In the incisors of voles consuming relatively low fluoride diets, lesions are mainly confined to those resulting from disruption of enamel pigmentation expressing itself as concentric bands of pigmentation-free areas on incisor surfaces. The visible effects on molars at low fluoride levels are confined to minor alterations in surface appearance. At higher levels of available dietary fluoride, effects on enamel pigmentation are superseded by alterations in the formation, composition, and strength of both enamel and dentine. The incisors exhibit a marked to severe increase in the cutting tip erosion rates with comparable increases in the extent of abnormal surface changes (enamel hypoplasia) and the loss of enamel pigmentation. The grinding surfaces of molars from animals exposed to high levels of dietary fluoride exhibit increasingly severe erosion of outer enamel regions, combined with cavitation and staining of the exposed central dentine. The mechanisms through which fluoride elicits increasingly visible and pathological alterations to the surface and subsurfaces of rodent teeth are discussed.

Animals↗

PubMed1994

Fluoride accumulation and toxicity in wild small mammals.

Populations of two species of small mammal, the field vole (Microtus agrestis L.) and the bank vole (Clethrionomys glareolus L.), inhabiting grasslands contaminated by industrial sources of fluoride were examined for fluoride concentrations in skeletal tissue and for morphological changes in the teeth. Concentrations of fluoride in teeth and bones were higher for C. glareolus than for M. agrestis at the chemical works and smelter sites. Severe dental lesions were recorded on the incisor and molar teeth of both species at the chemical works and smelter sites, with less marked damage at the mine tailings dam. This is attributed to inter-site differences in fluoride speciation and the consequent effects on the availability of fluoride in the diet for bioassimilation.

Journal Article↗

PubMedDate not supplied

Fluoride accumulation and toxicity in laboratory populations of wild small mammals and white mice.

A laboratory experiment was conducted to compare the toxicological response and metabolism of inorganic fluoride by three species of wild mammals and laboratory white mice (Mus musculus L.). The experimental populations of the wild species--the short-tailed field vole (Microtus agrestis L.), the bank vole (Clethrionomys glareolus L.) and the the wood mouse (Apodemus sylvaticus L.)--were laboratory reared from wild stock, and all test animals were exposed to 0, 40 or 80 microgram F per ml in their drinking water for up to 84 days. The 40 and 80 microgram F per ml treatments induced premature mortalities in M. agrestis and C. glareolus only. Differential intakes, absorption and retention of fluoride were evident between M. musculus and M. agrestis, the two species subject to cage studies of fluoride metabolism budgets. Interspecific variation in accumulation of fluoride with time was also evident between all four species as regards the femur, molars and incisors. Severe dental lesions were apparent in species surviving the 80 microgram ml(-1) treatment for 84 days. Overall, however, there were few clear differences in inherent species sensitivity to fluoride, the interspecific variation in metabolism and accumulation rates being attributable mainly to variation in intake.

Animals↗