Extracellular iron reductases: identification of a new class of enzymes by siderophore-producing microorganisms.

This study identifies extracellular iron reductases in culture supernatant fluids of the siderophore-producing microorganisms Escherichia coli and Pseudomonas aeruginosa. These enzymes were constitutively produced and reduced and released iron from a variety of ferric chelators. Dialyzable cofactors, necessary for the transfer of electrons in the enzymatic reduction of iron, were identified. The reductases were sensitive to treatment with proteinase K and guanidine-HCl, were not associated with siderophore activity, and were apparently released from the cell as extracellular enzymes. The acquisition of 59Fe2+ by cell suspensions of E. coli and P. aeruginosa was saturable, suggesting that the ferrous iron generated by these reductases can be bound and transported. Salmonella typhimurium mutants feoB, tonB, entB, and entBfeoB, deficient in numerous known iron uptake pathways, were found to exhibit substantial extracellular iron-reducing activities over that of the parent. A hypothesis is proposed in which the extracellular iron reductases excreted by siderophore-producing microorganisms may be responsible for the mobilization of iron during conditions of iron repletion when siderophores are repressed and may also function in concert with siderophores during periods of iron starvation.

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients.

We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR+ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3-, CD19-, CD20-, CD14-, CD11b-, CD16-, CD56-). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean +/- SE: 0.36 +/- 0.05%, 0.14 +/- 0.06%, and 0.75 +/- 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system.

Activated lymphocytes reduce adherence of Aspergillus fumigatus.

Lymphocytes comprise up to 30% of the cells present in human bronchoalveolar lavage fluid and thus could participate in host response to infectious Aspergillus fumigatus conidia. We have examined the possibility that lymphocytes might play a role during early infection by either damaging the fungus or interfering with adherence. When incubated with A. fumigatus conidia for 20 h, highly purified 5-day-old lymphocytes activated with either IL-2 or phytohaemagglutinin, but not untreated lymphocytes, were consistently able to reduce residual fungal biomass as estimated by a metabolic assay. T lymphocytes, but not NK cells, appeared to be responsible for this activity. Lymphocytes bound both A. fumigatus conidia and hyphae, and the antifungal activity of the lymphocytes required direct lymphocyte fungus contact. In a separate set of experiments using release of 51Cr from 51Cr-loaded fungi as an estimate of fungal damage, lymphocyte-induced loss of fungal biomass was found to be due to loss of fungal adherence rather than to direct fungal damage. The detached hyphae were also found to be metabolically intact and to have normal morphology by electron microscopy. These data demonstrate that IL-2- and phytohaemagglutinin-activated lymphocytes exhibit a contact-dependent ability to reduce adherence of germinating conidia of A. fumigatus to a surface.

Aspergillus fumigatus conidia induce a Th1-type cytokine response.

The response of human peripheral blood mononuclear cells (MNC) to Aspergillus fumigatus in vitro was evaluated. In studies of the proliferative response of MNC from 18 healthy donors to heat-killed A. fumigatus conidia, 15 displayed a significant response, with a stimulation index (SI) between 4 and 193. In contrast, all donors displayed a positive response to Candida albicans blastoconidia (SI ranged from 10 to 224). Despite the variability in reactivity to A. fumigatus conidia, the response of a particular individual was stable when retested over periods of 1-2 weeks. Supernatant from cocultures of A. fumigatus conidia with MNC contained increased levels of interferon-gamma, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and interleukin (IL)-2, compared with unstimulated cells, but not IL-10 or IL-4. In addition, A. fumigatus induced lymphocyte surface expression of adhesion/activation-associated molecules. These results suggest that lymphocytes may contribute to host defense against Aspergillus by generating a Th1-type response.

Extracellular iron reductase activity produced by Listeria monocytogenes.

Little is known about how pathogenic microorganisms that do not produce low-molecular-weight iron-chelating agents, termed siderophores, acquire iron from their environment. We have identified an extracellular enzyme produced by Listeria monocytogenes that can mobilize iron from a variety of iron-chelate complexes via reduction of the metal. The iron reductase requires Mg2+, flavin mononucleotide (FMN), and reduced nicotinamide adenine dinucleotide (NADH) for activity. Saturation kinetics were found when initial velocity studies of iron reduction were carried out as a function of variable FMN concentrations in the presence of 100 microM NADH and 10 mM Mg2+. Hyperbolic kinetics were also found when these studies were repeated as a function of variable NADH concentrations along with 20 microM FMN and 10 mM Mg2+. This process of extracellular reduction, in all likelihood, could be involved in the mobilization of iron from soils and aqueous environments and from host tissues in pathogenic processes. This is the first report of the extracellular enzymic reduction of iron by microorganisms.

Iron acquisition by Cryptococcus neoformans.

Iron is an essential element for the growth and metabolism of microbial cells. Most pathogenic microbes elaborate powerful iron chelating agents (siderophores) to mobilize iron from ferric ligands. The pathogenic yeast, Cryptococcus neoformans has not been found to produce siderophores and its mechanism of iron acquisition is unknown. This investigation explored an alternative pathway for iron acquisition by examining the interactions of iron with the cell surface. Iron uptake experiments were conducted utilizing radiolabelled ferrous iron and ferric iron chelates, with evidence for the presence of iron(II) receptors and the generation of ferrous iron by surface reduction. Hyperbolic kinetics were found when 59FeII was presented to the organism and uptake was blocked with bathophenanthroline sulphonate, an Fe2+ chelator. The yeast also acquired iron as [59Fe3+]-citrate and [59Fe3+]-pyrophosphate while bathophenanthroline sulphonate reduced the acquisition of these ferric ligands by 48% and 52% respectively. Pre-incubation with either ferric ligand also reduced iron acquisition by 50%. KCN inhibited uptake of iron(II) by 90% and uptake of [59Fe3+]-pyrophosphate and [59Fe3+]-citrate by 46% and 56% respectively; dinitrophenol had no effect on these processes. The data suggest that C. neoformans can (i) generate ferrous iron at the cell surface via a reduction of ferric chelates, with the subsequent acquisition of the ferrous iron, and (ii) acquire iron through the interaction of ferric chelates with a surface component.

A comparison of bathophenanthrolinedisulfonic acid and ferrozine as chelators of iron(II) in reduction reactions.

Bathophenanthrolinedisulfonic acid (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid [BPS]) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine), chelators of ferrous iron, are often used to determine iron(II) concentrations in various samples and for identifying or measuring iron reduction in biological systems. In this study, the efficacy of ferrozine and BPS to chelate iron(II) reduced from Fe(3+)-ligands by selected reducing agents was determined. Our results indicate that (i) BPS and ferrozine are not equivalent as kinetic indicators of iron reducing activity; (ii) apparent initial rates of reduction of Fe(3+)-ligands by dithiothreitol, as indicated by formation of complexes of iron(II) with either BPS or ferrozine, differed by a factor of 50; and (iii) nonspecific reduction of some Fe(3+)-ligands by both BPS and ferrozine occurred. Under identical conditions, rates of formation of Fe(2+)-ferrozine generally were slower than rates of formation of Fe(2+)-BPS. These data suggest careful consideration should be given in the design of any experiments where kinetics of iron reduction are monitored with BPS or ferrozine.

Regulation of cryptococcal capsular polysaccharide by iron.

Iron is tightly controlled in mammalian tissues and regulates virulence factors in various pathogenic organisms. The influence of Fe availability upon production of cryptococcal capsular polysaccharide was studied. Polysaccharide, measured as cell-bound glucuronyl residues, increased more than threefold as available Fe in the culture medium was varied from repletion to tight sequestration and depletion in five incremental steps. Since physiologic CO2 concentration may serve as stimulus for cryptococcal polysaccharide synthesis, the combined effect of Fe availability and CO2 on encapsulation was studied. Addition of dissolved, loosely chelated Fe moderated the effect of CO2. Tight chelation of dissolved Fe potentiated the CO2 effect. Tissue from infected mice showed heavily encapsulated organisms, consistent with results with physiologic CO2 concentration and Fe deprivation. In conclusion, cryptococcal polysaccharide synthesis is increased by limitation of ferric iron availability to the cell and by dissolved CO2, and the two effects are additive.

Iron acquisition systems of Listeria monocytogenes.

The uptake of iron by Listeria monocytogenes was studied. The microorganism was found to bind both 59Fe(II) and [59Fe3+]citrate. In contrast, L. monocytogenes was unable to acquire iron from [59Fe3+]ferroxamine or [59Fe3+]EDTA or as 59FeCl3. The data suggest that iron is acquired principally as iron(II) and that a citrate-inducible iron uptake system is also operative.

Adherence of a virulent strain of Listeria monocytogenes to the surface of a hepatocarcinoma cell line via lectin-substrate interaction.

Listeria monocytogenes was examined for the presence of surface carbohydrates to ascertain whether surface sugars, if present, would interact with eucaryotic surface carbohydrate receptors. We found that a virulent, but not two avirulent strains had a surface alpha-D-galactose residue as determined by agglutination with Griffonia simplicifolia (GS-I) and other lectins. The virulent strain bound to a human hepatocarcinoma cell line (HepG2), which has a well characterized receptor for alpha-D-galactose. This interaction could be blocked by pretreatment of the HepG2 cells with either alpha-D-galactose or neuraminidase, the latter of which will render the galactose receptor functionally inactive. We propose that the attachment of the virulent Listeria to eucaryotic cells occurs as a result of the interaction of the microbial alpha-D-galactose with that of the eucaryotic galactose receptor. This surface carbohydrate may provide an explanation for the mechanism of attachment and penetration of virulent Listeria into host cells during infection. As such, this may allow for amplification of pathogenesis through intracellular multiplication in nonprofessional phagocytes prior to macrophage involvement.

The exchange of Fe3+ between pyrophosphate and transferrin. Probing the nature of an intermediate complex with stopped flow kinetics, rapid multimixing, and electron paramagnetic resonance spectroscopy.

A detailed study of the exchange of Fe3+ between pyrophosphate and human serum transferrin was undertaken to test the hypothesis of a generalized reaction route for exchange of Fe3+ between transferrin and chelators. The initial rate of Fe3+ transfer from pyrophosphate to apotransferrin-CO2-3 is highly sensitive to the pyrophosphate to iron ratio with a maximal rate being observed at a ratio of 3:1, consistent with the presence of slowly reactive polymeric species at ratios less than 3:1 as revealed by EPR and kinetic measurements. At a ratio of 4:1 the reaction is distinctly biphasic. The rapid first phase results in the formation of an intermediate postulated as a mixedligand complex of the type PPi-Fe3+-transferrin-CO2-3. The intermediate has a distinct EPR spectrum and an absorption spectrum similar to that of Fe3+-transferrin-CO2-3, but with a spectral maximum at 450 nm rather than 465 nm. The second phase principally arises from the slow reaction of polymeric iron-pyrophosphate with the apoprotein and has contributions from the breakdown of the intermediate formed in the first phase. The rate of formation of the intermediate shows a hyperbolic dependence on NaHCO3 and apotransferrin concentrations, the latter suggesting a rate-limiting labilization of Fe3+(PPi)3, perhaps to form species of the type Fe3+(PPi)2, prior to attack by apotransferrin-CO2-3. Multimixing stopped flow spectrophotometry was employed to test the chemical reactivity of the Fe3+ to reduction at various times during the first phase. Surprisingly, a diminution of reactivity of 1000-fold was noted after only 2% of the first phase was completed, indicating a fast initial reaction which is not observed by normal rapid flow spectrophotometry. This initial reaction may involve the binding of iron-pyrophosphate to allosteric sites on the protein. The kinetics of iron removal from Fe3+-transferrin-CO2-3 by PPi are consistent with a rate-limiting conformational change in the protein as proposed earlier.

Differential effects of iron on the growth of Listeria monocytogenes: minimum requirements and mechanism of acquisition.

The differential effects of iron on the growth of virulent and avirulent Listeria monocytogenes were examined. We found that virulent strains exhibited faster rates of growth as a function of iron than did the avirulent strains. We also noted that serum was microbiostatic, but this microbiostasis was overcome either by saturating the serum transferrin with iron or by increasing the number of organisms initially inoculated into the serum. We were unable to identify any component of a high-affinity iron transport system. We did find, however, that this microorganism removes iron from Fe -transferrin-CO3-- by a reductive pathway, and we propose that this pathway is a nonspecific mechanism of iron acquisition.

The exchange of Fe3+ between acetohydroxamic acid and transferrin. Spectrophotometric evidence for a mixed ligand complex.

Transferrin, the serum iron transport protein, provides an excellent model for studying biological metal ion exchange reactions. A curious problem is that while a mixed ligand species of chelate-Fe3+-protein is anticipated from theoretical considerations and supported by kinetic results, no clear spectrophotometric evidence for such an intermediate has heretofore been obtained. In this study of the exchange of Fe3+ between acetohydroxamic acid and transferrin such evidence has been found. The reaction of Fe2+-acetohydroxamic acid with apotransferrin-CO3(2-) is distinctly biphasic when examined by stopped flow spectrophotometry. The first phase is complete within approximately 4 s and results in the formation of a transient species with a distinct spectral maximum at 432 nm. The second phase requires approximately 2 min and results in the formation of Fe3+-transferrin-CO3(2-). We suggest that the transient species is a mixed ligand complex. The reaction rate-concentration relationship for the formation of the intermediate is linear for Fe3+-acetohydroxamic acid and hyperbolic for apotransferrin-CO3(2-). This suggests a rate-limiting labilization of Fe3+-(acetohydroxamic acid)3 preceding attack by the apotransferrin-CO3(2-). The reverse reaction, the removal of Fe3+ from the Fe3+-transferrin-CO3(2-) by acetohydroxamic acid, does not provide spectral evidence for the intermediate. The velocity-concentration relationship shows a hyperbolic dependence on acetohydroxamic acid concentration and a linear dependence of Fe3+-transferrin-CO3(2-), suggesting a rate-limiting labilization of the Fe3+ of Fe3+-transferrin-CO3(2-) resulting from a conformational change.