Characterization of staphyloferrin A biosynthetic and transport mutants in Staphylococcus aureus

Abstract

SummaryIron is critical for virtually all forms of life. The production of high-affinity iron chelators, siderophores, and the subsequent uptake of iron-siderophore complexes are a common strategy employed by microorganisms to acquire iron. Staphylococcus aureus produces siderophores but genetic information underlying their synthesis and transport is limited. Previous work implicated the sbn operon in siderophore synthesis and the sirABC operon in uptake. Here we characterize a second siderophore biosynthetic locus in S. aureus; the locus consists of four genes (in strain Newman these open reading frames are designated NWMN_2079-2082) which, together, are responsible for the synthesis and export of staphyloferrin A, a polycarboxylate siderophore. While deletion of the NWMN_2079-2082 locus did not affect iron-restricted growth of S. aureus, strains bearing combined sbn and NWMN_2079-2082 locus deletions produced no detectable siderophore and demonstrated severely attenuated iron-restricted growth. Adjacent to NWMN_2079-2082 resides the htsABC operon, encoding an ABC transporter previously implicated in haem acquisition. We provide evidence here that HtsABC, along with the FhuC ATPase, is required for the uptake of staphyloferrin A. The crystal structure of apo-HtsA was determined and identified a large positively charged region in the substrate-binding pocket, in agreement with a role in binding of anionic staphyloferrin A.

Staphylococcus aureus Transporters Hts, Sir, and Sst Capture Iron Liberated from Human Transferrin by Staphyloferrin A, Staphyloferrin B, and Catecholamine Stress Hormones, Respectively, and Contribute to Virulence

“…Catecholamine hormones have previously been shown to form 2:1 and 3:1 complexes with iron(III) (30). Bacteria equipped with catechol siderophore uptake systems could feasibly import Fe(III)-catecholamine 3 complexes as "pseudosiderophores" for growth under iron-restricted conditions, as recently demonstrated for Bacillus subtilis and Escherichia coli (40).…”

“…The molecular genetics and biochemistry for the synthesis of two S. aureus siderophores, staphyloferrin A (SA) and staphyloferrin B (SB), were recently characterized (7,9). Following capture of extracellular iron, the staphyloferrins are recognized by the highly specific receptors HtsA (SA) and SirA (SB), and at least iron, if not the iron-siderophore complex, is actively imported into the cytosol through permeases (3,7,24,25). Furthermore, it has been shown that genomic inactivation of the staphyloferrin biosynthesis loci sfa (SA) and sbn (SB) eliminates siderophore output and severely curtails S. aureus growth in animal serum (3), prompting the question of whether the target of staphylococcal siderophores in serum is transferrin.…”

“…Following capture of extracellular iron, the staphyloferrins are recognized by the highly specific receptors HtsA (SA) and SirA (SB), and at least iron, if not the iron-siderophore complex, is actively imported into the cytosol through permeases (3,7,24,25). Furthermore, it has been shown that genomic inactivation of the staphyloferrin biosynthesis loci sfa (SA) and sbn (SB) eliminates siderophore output and severely curtails S. aureus growth in animal serum (3), prompting the question of whether the target of staphylococcal siderophores in serum is transferrin. In addition to the SA and SB siderophore import systems, S. aureus also carries the ironregulated FhuBGC 2 -D2/D1 transporter, with specificity for Fe(III)-hydroxamates (56,57).…”

Accurate Mass MS/MS/MS Analysis of Siderophores Ferrioxamine B and E1 by Collision-Induced Dissociation Electrospray Mass Spectrometry

“…Chelation occurs through oxygen coordination with characteristic subunits (e.g., trihydroxamate; Figure 1). In addition to common cellular processes that require iron as a cofactor (e.g., TCA cycle, DNA replication), siderophoremediated iron acquisition aids in the virulence of organisms such as Staphylococcus aureus and Pseudomonas aeruginosa [3,4]. To date, >500 siderophores have been discovered and despite their similar function, the structures vary widely, playing a major role in recognition by membrane-bound proteins and diffusion away from cellular membranes [5,6].…”

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

“…Consequently, S. aureus has evolved multiple strategies for iron acquisition (7). S. aureus produces two siderophores, staphyloferrin A (8,9) and staphyloferrin B (10), and has a transport system that can co-opt hydroxamate-type siderophores produced by other bacteria (11). S. aureus can also obtain heme from host heme-containing proteins hemoglobin and haptoglobin, transport it across the bacterial cell envelope, cleave the porphyrin ring, and release iron for use by the cell with the well characterized ironregulated surface determinant (Isd) 3 system (12).…”