Structural requirements for hemoglobin to induce fibronectin receptor expression in Candida albicans

Hemoglobin (Hb) is a host factor that induces expression of a promiscuous receptor on Candida albicans for fibronectin (FN) and several other extracellular matrix proteins. FN receptor expression was induced by ferric (Hb(+)Met and Hb(+)CN), ferrous (HbCO and HbO(2)), and cobalt-protoporphyrin derivatives of Hb, whereas globin was inactive. The Hb derivatives all exhibited saturable, dose-dependent kinetics of FN receptor induction, suggesting that Hb may be acting as a receptor ligand. Soluble Hb bound saturably to a low-affinity binding site [K(d) = (1.1+/-0.2) x 10(-6) M] on C. albicans blastospores. However, uptake of (55)FeHb revealed that heme or iron transport into the cell is not required for induction, since internalization of (55)Fe from Hb did not occur until after induction of FN binding. The serum Hb-binding protein, haptoglobin, specifically abrogated this response, indicating that protein structure rather than the heme ligand or iron is necessary for induction of this signaling pathway. C. albicans also adhered to immobilized Hb, which was sufficient to induce FN receptor expression, and to Hb polymers that formed in defined Hb liquid media in the presence of cells. Formation of Hb polymers in solution required metabolic energy, since the aggregation process was halted with azide addition. Collectively, these data demonstrate that C. albicans recognizes polymerized Hb through multivalent low-affinity interactions, and this may be a host environmental cue that triggers extracellular matrix receptor expression at a septic site.     

Role of clathrin‐mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life‐threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non‐iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin‐mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin‐containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.     

Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin.

Corynebacterium diphtheriae was examined for the ability to utilize various host compounds as iron sources. C. diphtheriae C7(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin but was unaffected in acquisition of iron from heme and hemoglobin, which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and Corynebacterium ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated following chemical mutagenesis and streptonigrin enrichment. A recombinant clone, pCD293, obtained from a C7(-) genomic plasmid library complemented several of the C. ulcerans mutants and three of the C. diphtheriae mutants. The nucleotide sequence of the gene (hmuO) required for complementation was determined and shown to encode a protein with a predicted mass of 24,123 Da. Sequence analysis revealed that HmuO has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. Heme oxygenases, which have been well characterized in eukaryotes but have not been identified in prokaryotes, are involved in the oxidation of heme and subsequent release of iron from the heme moiety. It is proposed that the HmuO protein is essential for the utilization of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene whose product has homology to heme oxygenases.     

Candida albicans iron acquisition within the host

As a commensal and opportunistic pathogen, Candida albicans possesses a range of determinants that contribute to survival, persistence and virulence. Among this repertoire of fitness and virulence attributes are iron acquisition factors and pathways, which allow fungal cells to gain this essential mineral in the iron-poor environment of the host. The aim of this review is to present the strategies used by C. albicans to exploit host iron reservoirs and their impact on C. albicans pathogenicity. Because iron in the human host is mostly linked to host proteins, pathogens such as C. albicans must possess mechanisms to gain iron from these proteins. Here, we introduce the most important groups of human proteins, including haemoglobin, transferrin, lactoferrin and ferritin, which contain iron and that are potential iron sources for invading microorganisms. We then summarize and discuss the known and proposed strategies by which C. albicans exploits or may exploit iron from host proteins and compare these with strategies from other pathogenic microorganisms.     

Heme catabolism in the causative agent of anthrax

A challenge common to all bacterial pathogens is to acquire nutrients from hostile host environments. Iron is an important cofactor required for essential cellular processes such as DNA repair, energy production and redox balance. Within a mammalian host, most iron is sequestered within heme, which in turn is predominantly bound by hemoglobin. While little is understood about the mechanisms by which bacterial hemophores attain heme from host‐hemoglobin, even less is known about intracellular heme processing. Bacillus anthracis, the causative agent of anthrax, displays a remarkable ability to grow in mammalian hosts. Hypothesizing this pathogen harbors robust ways to catabolize heme, we characterize two new intracellular heme‐binding proteins that are distinct from the previously described IsdG heme monooxygenase. The first of these, HmoA, binds and degrades heme, is necessary for heme detoxification and facilitates growth on heme iron sources. The second protein, HmoB, binds and degrades heme too, but is not necessary for heme utilization or virulence. The loss of both HmoA and IsdG renders B. anthracis incapable of causing anthrax disease. The additional loss of HmoB in this background increases clearance of bacilli in lungs, which is consistent with this protein being important for survival in alveolar macrophages.     

A screen for genes of heme uptake identifies the FLC family required for import of FAD into the endoplasmic reticulum

Although Candida albicans and Saccharomyces cerevisiae express very similar systems of iron uptake, these species differ in their capacity to use heme as a nutritional iron source. Whereas C. albicans efficiently takes up heme, S. cerevisiae grows poorly on media containing heme as the sole source of iron. We identified a gene from C. albicans that would enhance heme uptake when expressed in S. cerevisiae. Overexpression of CaFLC1 (for flavin carrier 1) stimulated the growth of S. cerevisiae on media containing heme iron. In C. albicans, deletion of both alleles of CaFLC1 resulted in a decrease in heme uptake activity, whereas overexpression of CaFLC1 resulted in an increase in heme uptake. The S. cerevisiae genome contains three genes with homology to CaFLC1, and two of these, termed FLC1 and FLC2, also stimulated growth on heme when overexpressed in S. cerevisiae. The S. cerevisiae Flc proteins were detected in the endoplasmic reticulum and the FLC genes encoded an essential function, as strains deleted for either FLC1 or FLC2 were viable, but deletion of both FLC1 and FLC2 was synthetically lethal. FLC gene deletion resulted in pleiotropic phenotypes related to defects in cell wall integrity. High copy suppressors of this synthetic lethality included three mannosyltransferases, VAN1, KTR4, and HOC1. FLC deletion strains exhibited loss of cell wall mannose phosphates, defects in cell wall assembly, and delayed maturation of carboxypeptidase Y. Permeabilized cells lacking FLC proteins exhibited dramatic loss of FAD import activity. We propose that the FLC genes are required for import of FAD into the lumen of the endoplasmic reticulum, where it is required for disulfide bond formation.     

Heme Iron Uptake by Caco-2 Cells is a Saturable,Temperature Sensitive and Modulated by Extracellular pH and Potassium

It is known that heme iron and inorganic iron are absorbed differently. Heme iron is found in the diet mainly in the form of hemoglobin and myoglobin. The mechanism of iron absorption remains uncertain. This study focused on the heme iron uptake by Caco-2 cells from a hemoglobin digest and its response to different iron concentrations. We studied the intracellular Fe concentration and the effect of time, K⁺ depletion, and cytosol acidification on apical uptake and transepithelial transport in cells incubated with different heme Fe concentrations. Cells incubated with hemoglobin-digest showed a lower intracellular Fe concentration than cells grown with inorganic Fe. However, uptake and transepithelial transport of Fe was higher in cells incubated with heme Fe. Heme Fe uptake had a low V _max and K _m as compared to inorganic Fe uptake and did not compete with non-heme Fe uptake. Heme Fe uptake was inhibited in cells exposed to K⁺ depletion or cytosol acidification. Heme oxygenase 1 expression increased and DMT1 expression decreased with higher heme Fe concentrations in the media. The uptake of heme iron is a saturable and temperature-dependent process and, therefore, could occur through a mechanism involving both a receptor and the endocytic pathway.     

Identification of Candida albicans genes that induce Saccharomyces cerevisiae cell adhesion and morphogenesis

Morphogenesis and adhesion to host tissues and medical devices contribute to the virulence of Candida albicans, the most common fungal pathogen isolated from humans. However, identification of molecular mechanisms of C. albicans adhesion and morphogenesis has been impaired by the lack of effective molecular and genetic tools available for this organism. Saccharomyces cerevisiae provides an attractive model system for studying C. albicans adhesion and morphogenesis because of its well-characterized genetics and gene expression systems. To gain insight into the genetic mechanisms of C. albicans adhesion and morphogenesis, we used a parallel plate flow chamber to screen and quantitatively characterize attachment to polystyrene of an adhesion-deficient nonfilamentous flo8Delta S. cerevisiae strain expressing a C. albicans genomic library. We identified six C. albicans genes that are capable of promoting cell adhesion and pseudohyphal development in S. cerevisiae. We also analyzed the ability of these adhesion-promoting genes to regulate the expression of FLO11, which encodes an endogenous S. cerevisiae adhesin. One C. albicans gene, EAP1, appears to directly mediate adhesion and morphogenesis while the remaining five (EAP2, SWI1, MSB1, AAF1, and TEC1) upregulate expression of endogenous S. cerevisiae adhesins. These results suggest that S. cerevisiae is a useful system for molecular characterization of factors that regulate C. albicans adhesion and morphogenesis and that parallel plate flow chamber-based adhesion assays can be used in conjunction with genetic screens to identify molecular mechanisms regulating fungal cell adhesion.     

Iron-binding proteins and heme compounds as iron sources forVibrio anguillarum

Utilization of several iron sources available from the host was investigated in different strains ofVibrio anguillarum. We tested the ability to use transferrins, heme, hemoglobin, and haptoglobin-hemoglobin as iron sources in strains ofV. anguillarum possessing different iron uptake systems mediated by siderophores. Only the wild-type pathogenic strains with an intact siderophore-mediated iron transport system were able to obtain iron from transferrins. None of the low-virulence derivatives lacking siderophore production could grow in the presence of transferrins. However, all strains, wild-type and iron-deficient derivatives, could utilize heme, hemoglobin, and haptoglobin-hemoglobin as iron sources when added to iron-deficient media. The ability to grow in fish serum was also evaluated. Although only wild-type strains could grow in fresh serum, derivative strains lacking siderophore production also were able to grow when serum was heat inactivated or when a utilizable siderophore was present in serum. The results indicate that besides the siderophore-mediated mechanism,V. anguillarum can also obtain iron from other sources presumably available from the host, although its importance for growth in vivo is so far unknown.     

Solution structure of the NEAT (NEAr Transporter) domain from IsdH/HarA: the human hemoglobin receptor in Staphylococcus aureus

During infections the pathogen Staphylococcus aureus procures the essential nutrient iron from its host using iron-regulated surface determinant (Isd) proteins, which scavenge heme bound iron from host hemoproteins. Four Isd proteins are displayed in the cell wall, where they function as receptors for host proteins and heme. Each of the receptors contains one or more copies of a recently discovered domain called NEAT (NEAr Transporter) that has been shown to mediate protein binding. Here we report the three-dimensional solution structure of the NEAT domain from the IsdH/HarA protein, which is the hemoglobin receptor in the Isd system. This is the first structure of a NEAT domain and reveals that they adopt a beta sandwich fold that consists of two five-stranded antiparallel beta sheets. Although unrelated at the primary sequence level, our results indicate that NEAT domains belong to the immunoglobulin superfamily. Binding studies indicate that two IsdH/HarA NEAT domains bind a single molecule of methemoglobin, while the distantly related NEAT domain from the S. aureus IsdC protein binds only heme. A comparison of their primary sequences in light of the new structure is used to predict the hemoglobin and heme binding surfaces on NEAT domains.     

The five near-iron transporter (NEAT) domain anthrax hemophore, IsdX2, scavenges heme from hemoglobin and transfers heme to the surface protein IsdC

Pathogenic bacteria require iron to replicate inside mammalian hosts. Recent studies indicate that heme acquisition in Gram-positive bacteria is mediated by proteins containing one or more near-iron transporter (NEAT) domains. Bacillus anthracis is a spore-forming, Gram-positive pathogen and the causative agent of anthrax disease. The rapid, extensive, and efficient replication of B. anthracis in host tissues makes this pathogen an excellent model organism for the study of bacterial heme acquisition. B. anthracis secretes two NEAT hemophores, IsdX1 and IsdX2. IsdX1 contains a single NEAT domain, whereas IsdX2 has five, a novel property among hemophores. To understand the functional significance of harboring multiple, non-identical NEAT domains, we purified each individual NEAT domain of IsdX2 as a GST fusion and analyzed the specific function of each domain as it relates to heme acquisition and transport. NEAT domains 1, 3, 4, and 5 all bind heme, with domain 5 having the highest affinity. All NEATs associate with hemoglobin, but only NEAT1 and -5 can extract heme from hemoglobin, seemingly by a specific and active process. NEAT1, -3, and -4 transfer heme to IsdC, a cell wall-anchored anthrax NEAT protein. These results indicate that IsdX2 has all the features required to acquire heme from the host and transport heme to the bacterial cell wall. Additionally, these results suggest that IsdX2 may accelerate iron import rates by acting as a "heme sponge" that enhances B. anthracis replication in iron-starved environments.     

Iron Acquisition in Bacillus cereus: The Roles of IlsA and Bacillibactin in Exogenous Ferritin Iron Mobilization

In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.     

Calcineurin A of Candida albicans: involvement in antifungal tolerance,cell morphogenesis and virulence

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.     

Kinetics of heme transfer by the Shr NEAT domains of Group A Streptococcus

The hemolytic Group A Streptococcus (GAS) is a notorious human pathogen. Shr protein of GAS participates in iron acquisition by obtaining heme from host hemoglobin and delivering it to the adjacent receptor on the surface, Shp. Heme is then conveyed to the SiaABC proteins for transport across the membrane. Using rapid kinetic studies, we investigated the role of the two heme binding NEAT modules of Shr. Stopped-flow analysis showed that holoNEAT1 quickly delivered heme to apoShp. HoloNEAT2 did not exhibit such activity; only little and slow transfer of heme from NEAT2 to apoShp was seen, suggesting that Shr NEAT domains have distinctive roles in heme transport. HoloNEAT1 also provided heme to apoNEAT2, by a fast and reversible process. To the best of our knowledge this is the first transfer observed between isolated NEAT domains of the same receptor. Sequence alignment revealed that Shr NEAT domains belong to two families of NEAT domains that are conserved in Shr orthologs from several species. Based on the heme transfer kinetics, we propose that Shr proteins modulate heme uptake according to heme availability by a mechanism where NEAT1 facilitates fast heme delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface.     

Effect of iron deprivation on surface composition and virulence determinants of Candida albicans

Six strains of Candida albicans were grown in defined medium which had been deferrated by ion-exchange chromatography and then supplemented with FeCl3 to give iron concentrations ranging from 0.026 microM to 0.8 microM. Growth in 0.026 microM-iron (measured as increase in biomass) was reduced by 26-59% as compared with that in excess (0.8 microM) iron. With five of the strains, adhesion to buccal epithelial cells was maximal after growth in 0.2-0.4 microM-iron, but strain GDH 2023 adhered best when grown in 0.026 microM-iron. Differences in yeast cell-wall composition were revealed by Zymolyase treatment of whole cells and by 125I-labelling of surface proteins. SDS-PAGE of iodinated proteins, followed by autoradiography, showed quantitative but no qualitative differences in protein profiles of iron-deficient and iron-replete organisms. The ability of all strains to form germ tubes in serum was near-maximal after growth in 0.2-0.4 microM-iron but was inhibited by up to 93% following growth in lower concentrations. These results indicate that expression of important virulence attributes by C. albicans is highly dependent on available iron and that expression in vivo may therefore be significantly different from that observed under conventional laboratory conditions.