Transferrin-mediated iron acquisition by pathogenic Neisseria

The pathogenic Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and transferrin. In the meningococcus this uptake system is dependent on two surface-exposed transferrin-binding proteins. This short account will review our current knowledge of the transferrin-mediated iron-acquisition system of pathogenic Neisseria.     

Iron transport systems in Neisseria meningitidis.

Acquisition of iron and iron complexes has long been recognized as a major determinant in the pathogenesis of Neisseria meningitidis. In this review, high-affinity iron uptake systems, which allow meningococci to utilize the human host proteins transferrin, lactoferrin, hemoglobin, and haptoglobin-hemoglobin as sources of essential iron, are described. Classic features of bacterial iron transport systems, such as regulation by the iron-responsive repressor Fur and TonB-dependent transport activity, are discussed, as well as more specific features of meningococcal iron transport. Our current understanding of how N. meningitidis acquires iron from the human host and the vaccine potentials of various components of these iron transport systems are also reviewed.     

Sulphur acquisition by Neisseria meningitidis

Group B Neisseria meningitidis (SD1C) was grown on defined medium supplemented with each of a variety of sulphur compounds as the sole source of sulphur. The organism grew on sulphate, sulphite, bisulphite, thiosulphate, dithionite, hydrosulphide, thiocyanate, L-cysteine, L-cystine, reduced glutathione, methionine, mercaptosuccinate, and lanthionine, but not on dithionate unless previously sulphur starved. Good growth was seen on concentrations of sulphate or thiosulphate as low as 10 microM. When pregrown on and subsequently starved for sulphate, the meningococcus showed enhanced transport capacity for this ion. Optimal conditions for assessing sulphur transport by active sulphur-limited cells were determined. The maximal sulphate uptake velocity was 9.3 nmol sulphate X mg protein-1 X min-1, and the apparent Km was 1.4 microM, far below human nasopharyngeal or serum sulphate levels.     

Iron Transport Systems in Neisseria meningitidis

Acquisition of iron and iron complexes has long been recognized as a major determinant in the pathogenesis of Neisseria meningitidis. In this review, high-affinity iron uptake systems, which allow meningococci to utilize the human host proteins transferrin, lactoferrin, hemoglobin, and haptoglobin-hemoglobin as sources of essential iron, are described. Classic features of bacterial iron transport systems, such as regulation by the iron-responsive repressor Fur and TonB-dependent transport activity, are discussed, as well as more specific features of meningococcal iron transport. Our current understanding of how N. meningitidis acquires iron from the human host and the vaccine potentials of various components of these iron transport systems are also reviewed.     

Iron acquisition in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, has an absolute requirement for iron and must obtain this element in the human host as well as in its varied environmental niches. It has multiple systems for iron acquisition, including the TonB-dependent transport of heme, the endogenous siderophore vibriobactin and several siderophores that are produced by other microorganisms. There is also a Feo system for the transport of ferrous iron and an ABC transporter, Fbp, which transports ferric iron. There appears to be at least one additional high affinity iron transport system that has not yet been identified. In iron replete conditions, iron acquisition genes are repressed by Fur. Fur also represses the synthesis of a small, regulatory RNA, RyhB, which negatively regulates genes for iron-containing proteins involved in the tricarboxylic acid cycle and respiration as well as genes for motility and chemotaxis. The redundancy in iron transport systems has made it more difficult to determine the role of individual systems in vivo and in vitro, but it may reflect the overall importance of iron in the growth and survival of V. cholerae.     

Iron acquisition systems for ferric hydroxamates, haemin and haemoglobin in Listeria monocytogenes

Listeria monocytogenes is a Gram-positive bacterium that causes severe opportunistic infections in humans and animals. We biochemically characterized, for the first time, the iron uptake processes of this facultative intracellular pathogen, and identified the genetic loci encoding two of its membrane iron transporters. Strain EGD-e used iron complexes of hydroxamates (ferrichrome and ferrichrome A, ferrioxamine B), catecholates (ferric enterobactin, ferric corynebactin) and eukaryotic binding proteins (transferrin, lactoferrin, ferritin, haemoglobin). Quantitative determinations showed 10-100-fold lower affinity for ferric siderophores (Km approximately 1-10 nM) than Gram-negative bacteria, and generally lower uptake rates. Vmax for [59Fe]-enterobactin (0.15 pMol per 10(9) cells per minute) was 400-fold lower than that of Escherichia coli. For [59Fe]-corynebactin, Vmax was also low (1.2 pMol per 10(9) cells per minute), but EGD-e transported [59Fe]-apoferrichrome similarly to E. coli (Vmax=24 pMol per 10(9) cells per minute). L. monocytogenes encodes potential Fur-regulated iron transporters at 2.031 Mb (the fur-fhu region), 2.184 Mb (the feo region), 2.27 Mb (the srtB region) and 2.499 Mb (designated hupDGC region). Chromosomal deletions in the fur-fhu and hupDGC regions diminished iron uptake from ferric hydroxamates and haemin/haemoglobin respectively. In the former locus, deletion of fhuD (lmo1959) or fhuC (lmo1960) strongly reduced [59Fe]-apoferrichrome uptake. Deletion of hupC (lmo2429) eliminated the uptake of haemin and haemoglobin, and decreased the virulence of L. monocytogenes 50-fold in mice. Elimination of srtB region genes (Deltalmo2185, Deltalmo2186, Deltalmo2183), both sortase structural genes (DeltasrtB, DeltasrtA, DeltasrtAB), fur and feoB did not impair iron transport. However, deletion of bacterioferritin (Deltafri, lmo943; 0.97 Mb) decreased growth and altered iron uptake: Vmax of [59Fe]-corynebactin transport tripled in this strain, whereas that of [59Fe]-apoferrichrome decreased 20-fold.     

The ferric iron-binding protein of pathogenic Neisseria spp. functions as a periplasmic transport protein in iron acquisition from human transferrin

The ferric iron-binding protein (Fbp) expressed by pathogenic Neisseria spp. has been proposed to play a central role in the high-affinity acquisition of iron from human transferrin. The results of this investigation provide evidence that Fbp participates in this process as a functional analogue of a Gram-negative periplasmic-binding protein component, which operates as a part of a general active transport process for the receptor-mediated, high-affinity transport of iron from human transferrin. Known properties of Fbp are correlated with those of other well-characterized periplasmic-binding proteins, including structural features and the reversible binding of ligand. Predictive of a periplasmic-binding protein, which functions in the high-affinity acquisition of iron, is that Fbp is a transient participant in the process of iron acquisition from human transferrin. Evidence for this is demonstrated by results of pulse–chase experiments. Taken together, the data described here and elsewhere suggest that pathogenic Neisseria spp. use a periplasmic-binding protein-mediated active transport mechanism for the acquisition of iron from human transferrin.     

Population coverage analysis of T-Cell epitopes of Neisseria meningitidis serogroup B from Iron acquisition proteins for vaccine design

Although the concept of Reverse Vaccinology was first pioneered for sepsis and meningococcal meningitidis causing bacterium, Neisseria meningitides, no broadly effective vaccine against serogroup B meningococcal disease is yet available. In the present investigation, HLA distribution analysis was undertaken to select three most promiscuous T-cell epitopes out of ten computationally validated epitopes of Iron acquisition proteins from Neisseria MC58 by using the population coverage tool of Immune Epitope Database (IEDB). These epitopes have been determined on the basis of their binding ability with maximum number of HLA alleles along with highest population coverage rate values for all the geographical areas studied. The comparative population coverage analysis of moderately immunogenic and high immunogenic peptides suggests that the former may activate T-cell response in a fairly large proportion of people in most geographical areas, thus indicating their potential for development of epitope-based vaccine.     

Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.     

Differences in the ultrastructure of colored variants of colonies of pathogenic Neisseria

Electron-transparent zones (vacuoles) of different size and localization have been detected in the cytoplasm of meningococci forming orange colonies. These vacuoles have been detected in cells from meningococcal cultures of different age, grown both in liquid and solid culture media. This phenomenon requires further deeper study. The presence of a relationship between newly detected vacuoles and the pathogenicity of meningococci is suggested.     

Iron acquisition through the bacterial transferrin receptor

Transferrin is one of the sources of iron that is most readily available to colonizing and invading pathogens. In this review, we look at iron uptake by the bacterial transferrin receptor that is found in the families Neisseriaceae, Pasteurellaceae and Moraxellaceae. This bipartite receptor consists of the TonB-dependent transporter, TbpA, and the surface lipoprotein, TbpB. In the past three decades, major advancements have been made in our understanding of the mechanism through which the Tbps take up iron. We summarize these findings and discuss how they relate to the diversity and specificity of the transferrin receptor. We also outline several of the remaining unanswered questions about iron uptake via the bacterial transferrin receptor and suggest directions for future research.     

Antimutator role of DNA glycosylase MutY in pathogenic Neisseria species

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2'-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC-->AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.     

Iron modulates ecto-phosphohydrolase activities in pathogenic trichomonads

The presence of iron in the extracellular medium is essential for both in vivo and in vitro survival of pathogenic microorganisms, including Trichomonas vaginalis and Tritrichomonas foetus. In these parasites, iron is directly involved in the proliferation, protein expression and activation of critical enzymes. The purpose of this study was to investigate the role of iron in ecto-ATPase, ecto-phophatase and secreted phosphatase activities of these trichomonads. We observed that trichomonads grown in iron-depleted medium exhibited a remarkable decrease in both ecto-ATPase and ecto-phosphatase activities, when compared to those cultivated under control conditions (iron-rich medium). Furthermore, parasites grown in iron-depleted medium restored their enzyme activities when they were re-inoculated into fresh iron-rich medium. We demonstrated that modulation of ecto-phosphohydrolase activities is due neither to enzyme–iron nor to substrate–iron complex formation, since iron addition directly to the medium where the enzymatic reactions occurred did not alter their activities. Previously, we had reported that a fresh clinical isolate of T. vaginalis was much more cytotoxic to epithelial cell monolayers than a long-term cultured one. In this study we witnessed that the fresh isolate of T. vaginalis presented higher activities to all herein investigated enzymes than the long-term cultured one. Altogether, our data clearly point out that iron has a pivotal role in the expression of phosphohydrolases in both trichomonads.     

Iron acquisition by plants.

In nongraminaceous plants, the FeII-transporter gene and ferric-chelate reductase gene have been cloned from Arabidopsis thaliana, whereas FeIII-reductase has not. In graminaceous monocots, the genes for mugineic acids (MAs) synthesis, nas (nicotianamine synthase) and naat (nicotianamine aminotransferase), have been cloned from barley, whereas the FeIII-MAs transporter gene is yet to be cloned. Transferrin absorption in Dunaliella has been reported, suggesting a phagocytotic (endocytotic) Fe-acquisition mechanism. Work to develop transgenic cultivars tolerant to Fe-deficiency in calcareous soils is now in progress.     

Iron acquisition by teleost fish

Iron is a vital micronutrient for teleost fish, being an integral component of proteins involved in cellular respiration and oxygen transfer. However, in excess iron is toxic, and fish need to balance uptake to prevent deficiency vs. potential toxicity. This review assesses the current physiological and molecular knowledge of the mechanisms of iron acquisition in the teleost fish. It focuses on freshwater teleost fish when assessing the gill as a possible site for iron acquisition, and includes a summary of geochemical processes that govern aquatic iron bioavailability. It focuses on marine teleost fish for assessing the mechanism of intestinal iron uptake. Physiological evidence indicates that iron preferentially crosses the apical membrane of both the gills and intestine in the ferrous (Fe2+) state. Molecular evidence supports this, demonstrating the presence of homologues in fish to the large Slc 11a family of evolutionary conserved proteins linked to Fe2+ transport. This symporter is probably linked to a reductase, which reduces either ferric (Fe3+) or organic complexed iron to Fe2+ prior to uptake.     

Iron acquisition by parasitic protozoa

The efficient uptake of iron by microorganisms is essential for their survival. Mammalian hosts possess elaborate means of sequestering their iron stores to protect themselves against invading pathogens. In this review, Mary Wilson and Bradley Britigan summarize mechanisms by which bacteria and protozoa effectively scavenge iron from their hosts during infection, as well as the potential and proven effects of these mechanisms on microbial virulence.