33 research outputs found
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 OD<sub>600</sub> (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; (ā), TSB only; (āµ), 25 ĀµM; (ā”), 50 ĀµM; (ā¢), 100 ĀµM; (ā), 200 ĀµM; (ā¦), 250 ĀµM. Lauric acid (C12ā¶0) was provided in the form of triacylglycerol-monolaurate. Y-axes, OD<sub>600</sub> 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 18ā24 h in the presence of C16 (A) or C18 (C) fatty acids.
<p>Cultures were grown with the indicated amounts of C16ā¶1āµ6 (sapienic acid), C16ā¶1āµ9 (palmitoleic acid), C16ā¶0 (palmitic acid), C18ā¶2 (linoleic acid), C18ā¶1 (oleic acid), C18ā¶3 (linolenic acid) or C18ā¶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 OD<sub>600</sub> units of culture supernatant was loaded in each lane (A and C). For Western blot (C), 0.02 OD<sub>600</sub> 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 ĀµM linoleic acid (A), and assay of total protease activity in culture supernatant (B).
<p>For (A), protein loading was 2.0 OD<sub>600</sub> units for Coomassie staining, and 0.02 OD<sub>600</sub> 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 ĀµM 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 (Īµ<sub>535</sub>), 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 ĀµM linoleic acid (LA) as indicated.
<p>Protein loading was 2.0 OD<sub>600</sub> units for SDS-PAGE, and 0.02 OD<sub>600</sub> 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 ĀµM 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
Ī²-galactosidase reporter gene assay in cell lysate of USA300<i>aur</i> after growth for 5ā8 h in TSB, or TSB supplemented with 25 ĀµM palmitic (C16ā¶0) or palmitoleic (C16ā¶1) acid.
<p>Ī²-galactosidase reporter gene assay in cell lysate of USA300<i>aur</i> after growth for 5ā8 h in TSB, or TSB supplemented with 25 ĀµM palmitic (C16ā¶0) or palmitoleic (C16ā¶1) acid.</p
Crystal and Solution Structure Analysis of FhuD2 from <i>Staphylococcus aureus</i> in Multiple Unliganded Conformations and Bound to FerrioxamineāB
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 FhuD2āFXB 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 [FeĀ(III)-SA] and ferric-staphyloferrin B [FeĀ(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 FeĀ(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 FeĀ(III)-SA. While mutation
of almost all proposed ligand-interacting residues decreased the ability
of <i>S. aureus</i> cells to transport <sup>55</sup>FeĀ(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 FeĀ(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), Ī±-ketoglutarate, and citrate. Recently, SbnA and SbnB
were shown to produce l-Dap and Ī±-ketoglutarate from <i>O</i>-phospho-l-serine (OPS) and l-glutamate.
SbnA is a pyridoxal 5ā²-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-Ī±-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 Ī±-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