Structure of the hemoglobin-IsdH complex reveals the molecular basis of iron capture by Staphylococcus aureus.

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and ß Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/ß globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.

Staphylococcus aureus uses a novel multidomain receptor to break apart human hemoglobin and steal its heme.

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S. aureus using two closely related receptors, IsdH and IsdB. Here we demonstrate that each receptor captures heme using two conserved near iron transporter (NEAT) domains that function synergistically. NMR studies of the 39-kDa conserved unit from IsdH (IsdH(N2N3), Ala(326)-Asp(660)) reveals that it adopts an elongated dumbbell-shaped structure in which its NEAT domains are properly positioned by a helical linker domain, whose three-dimensional structure is determined here in detail. Electrospray ionization mass spectrometry and heme transfer measurements indicate that IsdH(N2N3) extracts heme from Hb via an ordered process in which the receptor promotes heme release by inducing steric strain that dissociates the Hb tetramer. Other clinically significant Gram-positive pathogens capture Hb using receptors that contain multiple NEAT domains, suggesting that they use a conserved mechanism.

Direct evidence for self-propagation of different amyloid-ß fibril conformations.

BACKGROUND: Amyloid fibrils formed by amyloid-ß (Aß) peptides are associated with Alzheimer's disease and can occur in a range of distinct morphologies that are not uniquely determined by the Aß sequence. Whether distinct conformations of Aß fibrils can be stably propagated over multiple cycles of seeding and fibril growth has not been established experimentally. OBJECTIVE: The ability of the 40-residue peptide Aß1-40 to assemble into fibrils with the conformation of the mutant Aß1-40 peptide containing the 'Osaka' mutation E22Δ was investigated. METHODS: Fibril formation of highly pure, recombinant Aß1-40 in the presence of distinct, preformed seeds in vitro was recorded with thioflavin T fluorescence, and distinct fibrillar structures were identified and distinguished by fluorescence spectroscopy and electron microscopy. RESULTS: We propagated the specific quaternary structure of Aß1-40 E22Δ fibrils with wild-type Aß1-40 over up to seven cycles of seeding and fibril elongation. As a result of a 10(7)-fold dilution of the initially present Aß1-40 E22Δ seeds, the vast majority of fibrils formed after the seventh propagation cycle with Aß1-40 did not contain a single molecule of Aß1-40 E22Δ, but still retained the conformation of the initial Aß1-40 E22Δ seeds. Increased critical concentrations of Aß1-40 fibrils formed in the presence of Aß1-40 E22Δ nuclei suggest that these fibrils are less stable than homologously seeded Aß1-40 fibrils, consistent with a kinetically controlled mechanism of fibril formation. CONCLUSION: The propagation of a distinct Aß fibril conformation over multiple cycles of seeded fibril growth demonstrates the basic ability of the Aß peptide to form amyloid strains that in turn may cause phenotypes in Alzheimer's disease.

Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH.

Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.

Sortase enzymes in Gram-positive bacteria.

In Gram-positive bacteria proteins are displayed on the cell surface using sortase enzymes. These cysteine transpeptidases join proteins bearing an appropriate sorting signal to strategically positioned amino groups on the cell surface. Working alone, or in concert with other enzymes, sortases either attach proteins to the cross-bridge peptide of the cell wall or they link proteins together to form pili. Because surface proteins play a fundamental role in microbial physiology and are frequently virulence factors, sortase enzymes have been intensely studied since their discovery a little more than a decade ago. Based on their primary sequences and functions sortases can be partitioned into distinct families called class A to F enzymes. Most bacteria elaborate their surfaces using more than one type of sortase that function non-redundantly by recognizing unique sorting signals within their protein substrates. Here we review what is known about the functions of these enzymes and the molecular basis of catalysis. Particular emphasis is placed on 'pilin' specific class C sortases that construct structurally complex pili. Exciting new data have revealed that these enzymes are amazingly promiscuous in the substrates that they can employ and that there is a startling degree of diversity in their mechanism of action. We also review recent data that suggest that sortases are targeted to specific sites on the cell surface where they work with other sortases and accessory factors to properly function.

Transient weak protein-protein complexes transfer heme across the cell wall of Staphylococcus aureus.

Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus . Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins. A central step in this process is the transfer of heme between the cell wall associated IsdA and IsdC hemoproteins. Biochemical evidence indicates that heme is transferred via an activated IsdA:heme:IsdC heme complex. Transfer is rapid and occurs up to 70,000 times faster than indirect mechanisms in which heme is released into the solvent. To gain insight into the mechanism of transfer, we modeled the structure of the complex using NMR paramagnetic relaxation enhancement (PRE) methods. Our results indicate that IsdA and IsdC transfer heme via an ultraweak affinity "handclasp" complex that juxtaposes their respective 3(10) helices and ß7/ß8 loops. Interestingly, PRE also identified a set of transient complexes that could represent high-energy pre-equilibrium encounter species that form prior to the stereospecific handclasp complex. Targeted amino acid mutagenesis and stopped-flow measurements substantiate the functional relevance of a PRE-derived model, as mutation of interfacial side chains significantly slows the rate of transfer. IsdA and IsdC bind heme using NEAr Transporter (NEAT) domains that are conserved in many species of pathogenic Gram-positive bacteria. Heme transfer in these microbes may also occur through structurally similar transient stereospecific complexes.

The autoinducer synthase LqsA and putative sensor kinase LqsS regulate phagocyte interactions, extracellular filaments and a genomic island of Legionella pneumophila.

The amoebae-resistant opportunistic pathogen Legionella pneumophila employs a biphasic life cycle to replicate in host cells and spread to new niches. Upon entering the stationary growth phase, the bacteria switch to a transmissive (virulent) state, which involves a complex regulatory network including the lqs gene cluster (lqsA-lqsR-hdeD-lqsS). LqsR is a putative response regulator that promotes host-pathogen interactions and represses replication. The autoinducer synthase LqsA catalyses the production of the diffusible signalling molecule 3-hydroxypentadecan-4-one (LAI-1) that is presumably recognized by the sensor kinase LqsS. Here, we analysed L. pneumophila strains lacking lqsA or lqsS. Compared with wild-type L. pneumophila, the DeltalqsS strain was more salt-resistant and impaired for the Icm/Dot type IV secretion system-dependent uptake by phagocytes. Legionella pneumophila strains lacking lqsS, lqsR or the alternative sigma factor rpoS sedimented more slowly and produced extracellular filaments. Deletion of lqsA moderately reduced the uptake of L. pneumophila by phagocytes, and the defect was complemented by expressing lqsA in trans. Unexpectedly, the overexpression of lqsA also restored the virulence defect and reduced filament production of L. pneumophila mutant strains lacking lqsS or lqsR, but not the phenotypes of strains lacking rpoS or icmT. These results suggest that LqsA products also signal through sensors not encoded by the lqs gene cluster. A transcriptome analysis of the DeltalqsA and DeltalqsS mutant strains revealed that under the conditions tested, lqsA regulated only few genes, whereas lqsS upregulated the expression of 93 genes at least twofold. These include 52 genes clustered in a 133 kb high plasticity genomic island, which is flanked by putative DNA-mobilizing genes and encodes multiple metal ion efflux pumps. Upon overexpression of lqsA, a cluster of 19 genes in the genomic island was also upregulated, suggesting that LqsA and LqsS participate in the same regulatory circuit.

Pilus chaperones represent a new type of protein-folding catalyst.

Adhesive type 1 pili from uropathogenic Escherichia coli strains have a crucial role during infection by mediating the attachment to and potentially the invasion of host tissue. These filamentous, highly oligomeric protein complexes are assembled by the 'chaperone-usher' pathway, in which the individual pilus subunits fold in the bacterial periplasm and form stoichiometric complexes with a periplasmic chaperone molecule that is essential for pilus assembly. The chaperone subsequently delivers the subunits to an assembly platform (usher) in the outer membrane, which mediates subunit assembly and translocation to the cell surface. Here we show that the periplasmic type 1 pilus chaperone FimC binds non-native pilus subunits and accelerates folding of the subunit FimG by 100-fold. Moreover, we find that the FimC-FimG complex is formed quantitatively and very rapidly when folding of FimG is initiated in the presence of both FimC and the assembly-competent subunit FimF, even though the FimC-FimG complex is thermodynamically less stable than the FimF-FimG complex. FimC thus represents a previously unknown type of protein-folding catalyst, and simultaneously acts as a kinetic trap preventing spontaneous subunit assembly in the periplasm.

Synergistic contribution of the Legionella pneumophila lqs genes to pathogen-host interactions.

The causative agent of Legionnaires' disease, Legionella pneumophila, is a natural parasite of environmental protozoa and employs a biphasic life style to switch between a replicative and a transmissive (virulent) phase. L. pneumophila harbors the lqs (Legionella quorum sensing) cluster, which includes genes encoding the autoinducer synthase LqsA, the sensor kinase LqsS, the response regulator LqsR, and a homologue of HdeD, which is involved in acid resistance in Escherichia coli. LqsR promotes host-cell interactions as an element of the stationary-phase virulence regulatory network. Here, we characterize L. pneumophila mutant strains lacking all four genes of the lqs cluster or only the hdeD gene. While an hdeD mutant strain did not have overt physiological or virulence phenotypes, an lqs mutant showed an aberrant morphology in stationary growth phase and was defective for intracellular growth, efficient phagocytosis, and cytotoxicity against host cells. Cytotoxicity was restored upon reintroduction of the lqs genes into the chromosome of an lqs mutant strain. The deletion of the lqs cluster caused more-severe phenotypes than deletion of only lqsR, suggesting a synergistic effect of the other lqs genes. A transcriptome analysis indicated that in the stationary phase more than 380 genes were differentially regulated in the lqs mutant and wild-type L. pneumophila. Genes involved in protein production, metabolism, and bioenergetics were upregulated in the lqs mutant, whereas genes encoding virulence factors, such as effectors secreted by the Icm/Dot type IV secretion system, were downregulated. A proteome analysis revealed that a set of Icm/Dot substrates is not produced in the absence of the lqs gene cluster, which confirms the findings from DNA microarray assays and mirrors the virulence phenotype of the lqs mutant strain.

The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin.

Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdH(N2N3), A326-D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.

Backbone 1H, 13C and 15N resonance assignments of the 39 kDa staphylococcal hemoglobin receptor IsdH.

During infections Stahpylococcus aureus preferentially uses heme as an iron source, which it captures from human hemoglobin using the Iron regulated surface determinant (Isd) system. On the cell surface two related staphylococcal surface receptors called IsdH and IsdB bind to hemoglobin and extract its heme. Both receptors contain multiple NEAr iron Transporter (NEAT) domains that either bind to hemoglobin, or to heme. All previous structural studies have investigated individual NEAT domains and have not explored how the domains might interact with one another to synergistically extract heme from hemoglobin. Here, we report the near complete (1)H, (13)C and (15)N backbone resonance assignments of a bi-domain unit from IsdH that contains the N2 and N3 NEAT domains, which bind to hemoglobin and heme, respectively (IsdH(N2N3), residues 326-660, 39 kDa). The assigned backbone resonances lay the foundation for future NMR studies that will explore the molecular basis of IsdH function.

Bacterial gene regulation by alpha-hydroxyketone signaling.

Bacteria produce diffusible, small signaling molecules termed autoinducers to promote cell-cell communication. Recently, a novel class of signaling molecules, the alpha-hydroxyketones (AHKs), was discovered in the facultative human pathogens Legionella pneumophila and Vibrio cholerae. In this review, we summarize and compare findings on AHK signaling in these bacteria. The L. pneumophila lqs (Legionella quorum sensing) and V. cholerae cqs (cholera quorum sensing) gene clusters synthesize and detect Legionella autoinducer 1 (3-hydroxypentadecan-4-one) or cholera autoinducer-1 (3-hydroxytridecan-4-one), respectively. In addition to the autoinducer synthase and cognate sensor kinase encoded in the cqs locus, the lqs cluster also harbors a prototypic response regulator. AHK signaling regulates pathogen-host cell interactions, bacterial virulence, formation of biofilms or extracellular filaments, and expression of a genomic island. The lqs/cqs gene cluster is present in several environmental bacteria, suggesting that AHKs are widely used for cell-cell signaling.

The Legionella autoinducer synthase LqsA produces an alpha-hydroxyketone signaling molecule.

The opportunistic pathogen Legionella pneumophila replicates in human lung macrophages and in free-living amoebae. To accommodate the transfer between host cells, L. pneumophila switches from a replicative to a transmissive phase. L. pneumophila harbors a gene cluster homologous to the Vibrio cholerae cqsAS quorum sensing system, encoding a putative autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a response regulator (lqsR). LqsR is an element of the L. pneumophila virulence regulatory network, which promotes pathogen-host cell interactions and inhibits entry into the replicative growth phase. Here, we show that lqsA functionally complements a V. cholerae cqsA autoinducer synthase deletion mutant and, upon expression in L. pneumophila or Escherichia coli, produces the diffusible signaling molecule LAI-1 (Legionella autoinducer-1). LAI-1 is distinct from CAI-1 (Cholerae autoinducer-1) and was identified as 3-hydroxypentadecan-4-one using liquid chromatography coupled to high resolution tandem mass spectrometry. The activity of both LqsA and CqsA was abolished upon mutation of a conserved lysine, and covalent binding of the cofactor pyridoxal 5'-phosphate to this lysine was confirmed by mass spectrometry. Thus, LqsA and CqsA belong to a family of pyridoxal 5'-phosphate-dependent autoinducer synthases, which produce the alpha-hydroxyketone signaling molecules LAI-1 and CAI-1.

The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA.

Legionella pneumophila is an opportunistic human pathogen that replicates within environmental amoebae including Acanthamoeba castellanii and Dictyostelium discoideum. The Icm/Dot type IV secretion system promotes phagocytosis and intracellular replication of L. pneumophila in an endoplasmic reticulum-derived 'Legionella-containing vacuole' (LCV). L. pneumophila adopts a biphasic life cycle consisting of a replicative growth phase and a transmissive (stationary) phase, the latter of which is characterized by the preferential expression of genes required for motility and virulence. A bioinformatic analysis of the L. pneumophila genome revealed a gene cluster homologous to the Vibrio cholerae cqsAS genes, encoding a putative quorum sensing autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a novel response regulator (lqsR). We report here that an L. pneumophila lqsR deletion mutant grew in broth with the same rate as wild-type bacteria, but entered the replicative growth phase earlier. Overexpression of lqsR led to an elongated morphology of the bacteria. The lqsR mutant strain was found to be more salt-resistant and impaired for intracellular growth in A. castellanii, D. discoideum and macrophages, formation of the ER-derived LCV and toxicity. Moreover, L. pneumophila lacking LqsR, as well as strains lacking the stationary sigma factor RpoS or the two-component response regulator LetA, were phagocytosed less efficiently by A. castellanii, D. discoideum or macrophages. The expression of lqsR was dependent on RpoS and, to a lesser extent, also on LetA. DNA microarray experiments revealed that lqsR regulates the expression of genes involved in virulence, motility and cell division, consistent with a role for LqsR in the transition from the replicative to the transmissive (virulent) phase. Our findings indicate that LqsR is a novel pleiotropic regulator involved in RpoS- and LetA-controlled interactions of L. pneumophila with phagocytes.

Expression of Legionella pneumophila paralogous lipid A biosynthesis genes under different growth conditions.

Legionella pneumophila is an opportunistic pathogen that in the environment colonizes biofilms and replicates within amoebae. The bacteria employ the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to grow intracellularly in a specific vacuole. Using Acanthamoeba castellanii as a host cell, we have previously identified lcsC (Legionella cytotoxic suppressor), a paralogue of the lipid A disaccharide synthase lpxB, as a cytotoxic factor of L. pneumophila. A bioinformatic analysis of the genome revealed that L. pneumophila is unique in harbouring two paralogues of lpxB and two and three paralogues of the lipid A biosynthesis acyltransferases lpxA and lpxD, respectively. LcsC (lpxB1) forms a transcriptional unit with gnnA, encoding a putative UDP-GlcNAc oxidase in the biosynthetic pathway leading to 3-aminoglucosamine analogues of lipid A. LpxB2 clusters with lpxD2, lpxA2 and lpxL paralogues, encoding secondary acyltransferases. LcsC/lpxB1 and lpxB2 were found to partially complement the growth defect of an Escherichia coli lpxB conditional mutant strain, indicating that both corresponding enzymes possess lipid A disaccharide synthase activity. The two L. pneumophila lpxB paralogues are not functionally equivalent, since expression of lcsC/lpxB1 but not lpxB2 in an L. pneumophila icmG mutant is cytotoxic for A. castellanii, and LPS purified from the two strains triggers CD14-dependent tumour necrosis factor (TNF)alpha production by macrophages with a different potency. The lpxB and lpxA paralogues are expressed under various growth conditions, including broth, biofilms and in A. castellanii. While the flagellar gene flaA is mainly expressed in late stationary phase, the lpxB and lpxA paralogues are preferentially expressed in the exponential and early stationary phases. Upon exposure to hypotonic stress and nutrient deprivation, lpxA1, and to a lesser extent lcsC/lpxB1, is upregulated. The differential regulation of lpxB or lpxA paralogues in response to changing environmental conditions might allow L. pneumophila to adapt its lipid A structure.

Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions.

Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate within biofilms in the absence of protozoa. In this study, we analyzed surface adherence of and biofilm formation by L. pneumophila in a rich medium that supported axenic replication. Biofilm formation by the virulent L. pneumophila strain JR32 and by clinical and environmental isolates was analyzed by confocal microscopy and crystal violet staining. Strain JR32 formed biofilms on glass surfaces and upright polystyrene wells, as well as on pins of "inverse" microtiter plates, indicating that biofilm formation was not simply due to sedimentation of the bacteria. Biofilm formation by an L. pneumophila fliA mutant lacking the alternative sigma factor sigma(28) was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase. L. pneumophila neither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition of Acanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming) L. pneumophila expressed beta-galactosidase activity to similar extents, and therefore, the observed lack of proliferation of surface-attached bacteria was not due to impaired protein synthesis or metabolic activity. Cocultivation of green fluorescent protein (GFP)- and DsRed-labeled L. pneumophila led to randomly interspersed cells on the substratum and in aggregates, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially adhered in a continuous-flow chamber system but detached over time; the detachment correlated with the flow rate, and there was no accumulation of biomass. Under these conditions, L. pneumophila persisted in biofilms formed by Empedobacter breve or Microbacterium sp. but not in biofilms formed by Klebsiella pneumoniae or other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.