Multiprotein Heme Shuttle Pathway in <i>Staphylococcus aureus</i>: Iron-Regulated Surface Determinant Cog-Wheel Kinetics

Iron is a critically important nutrient for all species. Bacteria have evolved specialist survival systems to chelate and transport iron across the wall and membrane into the cytoplasm. One such system in the human pathogenic bacteria <i>Staphylococcus aureus</i> involves extracting heme from hemoglobin and then transporting the intact heme across the wall and membrane. The iron-regulated surface determinant (Isd) proteins act in concert to carry out the heme scavenging and subsequent transport. While details of the static heme-binding reaction are currently quite well known, little mechanistic data are available. In this paper, we describe detailed time-resolved mass spectral and magnetic circular dichroism spectral data recorded as heme is transferred unidirectionally from holo-IsdA to apo-IsdE via IsdC. The electrospray mass spectral data simultaneously monitor the concentrations of six protein species involved in the trans-wall transport of the extracted heme and show for the first time the mechanistic details of heme transfer that is key to the <i>Staphylococcus aureus</i> Isd heme-scavenging system. Bimolecular kinetic analysis of the ESI-mass spectral data shows that heme transfer from IsdA to IsdC is very fast, whereas the subsequent heme transfer from IsdC to IsdE is slower. Under limiting IsdC conditions, the IsdC intermediary cycles between heme-free and heme-containing forms until either all heme has been transferred from holo-IsdA or no further apo-IsdE is available. The data show that a unique role for IsdC is acting as the central cog-wheel that facilitates heme transfer from IsdA to IsdE

Influence of fatty acids on growth of USA300.

<p>Each point represents the mean of OD₆₀₀ (A, C, D-G) or cfu/ml determination (B, H) from triplicate flasks of USA300 grown in TSB supplemented with the indicated amount of fatty acid; (○), TSB only; (▵), 25 µM; (□), 50 µM; (•), 100 µM; (⋄), 200 µM; (♦), 250 µM. Lauric acid (C12∶0) was provided in the form of triacylglycerol-monolaurate. Y-axes, OD₆₀₀ or cfu/ml; X-axis, growth time (h).</p

SDS-PAGE of secreted proteins (A, C) and Western blot for detection of SspA and Hla (B), in culture supernatant of USA300 after growth for 18–24 h in the presence of C16 (A) or C18 (C) fatty acids.

<p>Cultures were grown with the indicated amounts of C16∶1▵6 (sapienic acid), C16∶1▵9 (palmitoleic acid), C16∶0 (palmitic acid), C18∶2 (linoleic acid), C18∶1 (oleic acid), C18∶3 (linolenic acid) or C18∶0 (stearic acid) fatty acids. Proteins in the cell-free culture supernatant were precipitated in ice-cold TCA, and after solubilization in SDS-PAGE reducing buffer, protein equivalent to 2.0 OD₆₀₀ units of culture supernatant was loaded in each lane (A and C). For Western blot (C), 0.02 OD₆₀₀ units of cell free culture supernatant were subjected directly to SDS-PAGE, prior to detection with specific antisera (see Materials and Methods).</p

SDS-PAGE and Western blot analyses of secreted proteins produced by USA300 and isogenic variants after 8 h of growth in TSB, or TSB supplemented with 25 µM linoleic acid (A), and assay of total protease activity in culture supernatant (B).

<p>For (A), protein loading was 2.0 OD₆₀₀ units for Coomassie staining, and 0.02 OD₆₀₀ units for Western blots, which were developed with primary antibody specific for Aur, and SspA as indicated. Arrows on the Coomassie stained gel indicate the selective induction of secreted proteases in response to linoleic acid. The arrow on the right margin indicates the position of proGeh. In (B), total protease activity in 8 h culture supernatant of USA300 and isogenic variants was determined with FITC-casein substrate. Cultures were grown with 25 µM linoleic acid as indicated, and assay buffer was supplemented with 10 mM EDTA where indicated, to inhibit metalloprotease. Data are reported as fluorescence emission at 535 nm (ε₅₃₅), measured in arbitrary fluorescence units.</p

SDS-PAGE and Coomassie staining (A and C), or Western blot for detection of SspA (B, D and E), in cultures of <i>S. aureus</i> grown in TSB containing 0 or 25 µM linoleic acid (LA) as indicated.

<p>Protein loading was 2.0 OD₆₀₀ units for SDS-PAGE, and 0.02 OD₆₀₀ units for Western blot. The <i>S. aureus</i> strains are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045952#pone-0045952-t001" target="_blank">Table 1</a>. Arrows and labels on the right margins of panels A and C indicate the location of 72 kDa glycerol ester hydrolase precursor (proGeh) and mature lipase (Geh), while arrows on the protein gels point to SspA protein that is induced in response to 25 µM LA. SspA exhibits some expected variation in size, being comprised of 327 amino acids in USA400 (MW_0932), 336 amino acids in USA300 (SAUSA300_0951), and 357 amino acids in MRSA252 (SAR_1022) and other CC30 strains, due to variation in a C-terminal disordered segment comprised of tripeptide repeats. Different isomers produced by the same strain as shown on Western blot (5E), and explained in the text, are attributed to varying degrees of processing of the N-terminal propeptide of the SspA precursor, proSspA.</p

β-galactosidase reporter gene assay in cell lysate of USA300<i>aur</i> after growth for 5–8 h in TSB, or TSB supplemented with 25 µM palmitic (C16∶0) or palmitoleic (C16∶1) acid.

<p>β-galactosidase reporter gene assay in cell lysate of USA300<i>aur</i> after growth for 5–8 h in TSB, or TSB supplemented with 25 µM palmitic (C16∶0) or palmitoleic (C16∶1) acid.</p

Crystal and Solution Structure Analysis of FhuD2 from <i>Staphylococcus aureus</i> in Multiple Unliganded Conformations and Bound to Ferrioxamine‑B

Iron acquisition is a central process for virtually all organisms. In <i>Staphylococcus aureus</i>, FhuD2 is a lipoprotein that is a high-affinity receptor for iron-bound hydroxamate siderophores. In this study, FhuD2 was crystallized bound to ferrioxamine-B (FXB), and also in its ligand-free state; the latter structures are the first for hydroxamate-binding receptors within this protein family. The structure of the FhuD2–FXB conformation shows that residues W197 and R199 from the C-terminal domain donate hydrogen bonds to the hydroxamate oxygens, and a ring of aromatic residues cradles the aliphatic arms connecting the hydroxamate moieties of the siderophore. The available ligand-bound structures of FhuD from <i>Escherichia coli</i> and YfiY from <i>Bacillus cereus</i> show that, despite a high degree of structural conservation, three protein families have evolved with critical siderophore binding residues on either the C-terminal domain (<i>S. aureus</i>), the N-terminal domain (<i>E. coli</i>), or both (<i>B. cereus</i>). Unliganded FhuD2 was crystallized in five conformations related by rigid body movements of the N- and C-terminal domains. Small-angle X-ray scattering (SAXS) indicates that the solution conformation of unliganded FhuD2 is more compact than the conformations observed in crystals. The ligand-induced conformational changes for FhuD2 in solution are relatively modest and depend on the identity of the siderophore. The crystallographic and SAXS results are used to discuss roles for the liganded and unliganded forms of FhuD2 in the siderophore transport mechanism

Identification of a Positively Charged Platform in <i>Staphylococcus aureus</i> HtsA That Is Essential for Ferric Staphyloferrin A Transport

In response to iron starvation, <i>Staphylococcus aureus</i> secretes both staphyloferrin A and staphyloferrin B, which are high-affinity iron-chelating molecules. The structures of both HtsA and SirA, the ferric-staphyloferrin A [Fe­(III)-SA] and ferric-staphyloferrin B [Fe­(III)-SB] receptors, respectively, have recently been determined. The structure of HtsA identifies a novel form of ligand entrapment composed of many positively charged residues. Through ionic interactions, the binding pocket appears highly adapted for the binding of the highly anionic siderophore SA. However, biological validation of the importance of the nine SA-interacting residues (six arginines, one tyrosine, one histidine, and one lysine) has not been previously performed. Here, we mutated each of the Fe­(III)-SA-interacting residues in HtsA and found that substitutions R104A, R126A, H209A, R306A, and R306K resulted in a reduction of binding affinity of HtsA for Fe­(III)-SA. While mutation of almost all proposed ligand-interacting residues decreased the ability of <i>S. aureus</i> cells to transport ⁵⁵Fe­(III)-SA, <i>S. aureus</i> expressing HtsA R104A, R126A, R306A, and R306K showed the greatest transport defects and were incapable of growth in iron-restricted growth media in a SA-dependent manner. These three residues cluster together and, relative to other residues in the binding pocket, move very little between the apo and closed holo structures. Their essentiality for receptor function, together with structural information, suggests that they form a positively charged platform that is required for initial contact with the terminal carboxyl groups of the two citrates in the Fe­(III)-SA complex. This is a likely mechanism by which HtsA discerns iron-bound SA from iron-free SA

Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis

<i>Staphylococcus aureus</i> assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from <i>O</i>-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5′-phosphate (PLP)-dependent enzyme with homology to <i>O</i>-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of <i>O</i>-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress

Repression of branched-chain amino acid synthesis in <i>Staphylococcus aureus</i> is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide

<div><p><i>Staphylococcus aureus</i> requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for <i>S</i>. <i>aureus</i> physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed <i>S</i>. <i>aureus</i> uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that <i>S</i>. <i>aureus</i> can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a <i>trans-</i>acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a <i>cis</i>-acting leucine-responsive attenuator, uncovering how <i>S</i>. <i>aureus</i> regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in <i>S</i>. <i>aureus</i>, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on <i>S</i>. <i>aureus</i> environmental adaptation and virulence via CodY.</p></div