Molecular Cloning and Analysis of a Putative Siderophore ABC Transporter from Staphylococcus aureus

Abstract

From a mass-excised Staphylococcus aureus ZapII expression library, we cloned an operon encoding a novel ABC transporter with significant homology to bacterial siderophore transporter systems. The operon encodes four genes designated sstA, -B, -C, and -D encoding two putative cytoplasmic membrane proteins (sstA and sstB), an ATPase (sstC), and a membrane-bound 38-kDa lipoprotein (sstD). The sst operon is preceded by two putative Fur boxes, which indicated that expression of the sst operon was likely to be iron dependent. SstD was overexpressed in Escherichia coli, purified by Triton X-114 phase partitioning, and used to generate monospecific antisera in rats. Immunoblotting studies located SstD in the membrane fraction of S. aureus and showed that expression of the lipoprotein was reduced under iron-rich growth conditions. Triton X-114 partitioning studies on isolated membranes provided additional biochemical evidence that SstD in S. aureus is a lipoprotein. Immunoreactive polypeptides of approximately 38 kDa were detected in a wide range of staphylococcal species, but no antigenic homolog was detected in Bacillus subtilis. Expression of SstD in vivo was confirmed by immunoblotting studies with S. aureus recovered from a rat intraperitoneal chamber implant model. To further define the contribution of SstD in promoting growth of S. aureus in vitro and in vivo, we used antisense RNA technology to modulate expression of SstD. Expression of antisense sstD RNA in S. aureus resulted in a decrease in SstD expression under both iron-rich and iron-restricted growth conditions. However, this reduction in SstD levels did not affect the growth of S. aureus in vitro in an iron-limited growth medium or when grown in an intraperitoneal rat chamber implant model in vivo.Acquisition of nutrients, such as iron, for growth in the host environment is essential for bacterial pathogens to establish an infection. An effective mechanism for scavenging iron involves the production and secretion of low-molecular-weight ferriciron chelators, siderophores, which scavenge iron from the host and transport it into the cell via specific ABC transporters (27). In comparison to the wealth of information available concerning gram-negative siderophore transport (11, 26), very little is known about ferric-siderophore uptake in staphylococci. It has been demonstrated that Staphylococcus aureus produces at least three siderophores, staphyloferrins A (22) and B (8, 12) and aureochelin (7), and it also utilizes a range of exogenous siderophores, such as the enterobacterial siderophore enterobactin (21). However, to date there are no published reports on staphylococcal genes coding for siderophore biosynthesis and very little is known about siderophore uptake. Three iron-regulated staphylococcal ABC transporters have been identified, but these have only been partially characterized, and in each case the transported solute has not been identified. In S. aureus, the sirABC operon has homology to gram-negative siderophore transporters, in particular, the c...

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

“…Previously, inactivation of sst was shown to make no significant contribution to bacterial survival in a rat intraperitoneal cage model of infection (44). In the current study, sst inactivation on its own also failed to have an impact on virulence within typically characterized murine organs (e.g., kidneys and liver) but did significantly decrease virulence in the absence of siderophore transport.…”

“…1), characterizing the contribution of catecholamines to iron uptake from serum or transferrin was made possible only after constructing and characterizing two whole-locus siderophore biosynthesis knockout strains. Catecholamine iron uptake was subsequently shown to be mediated by a distinct transporter, SstABCD, previously implicated in the S. aureus adaptive response to low-iron environments (44).…”

“…The S. aureus sst operon (NWMN_0702_0705) is iron regulated in vitro and in vivo and encodes a membrane-tethered lipoprotein, SstD (44). SstD is a member of the class III substrate binding protein family, a family which includes the S. aureus Fe(III)-staphyloferrin receptors HtsA (25) and SirA (24) and the heme-binding IsdE protein (26).…”

“…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). One additional iron-regulated transporter, SstABCD, was described as a putative siderophore transporter based on sequence similarities to catechol transport systems in Gram-negative and Gram-positive bacteria (44), but it has not yet been characterized for substrate specificity.…”

At the Crossroads of Bacterial Metabolism and Virulence Factor Synthesis in Staphylococci

“…S. aureus cells synthesize and secrete high-affinity iron chelators called siderophores (i.e., staphyloferrins A and B, staphylobactin, and aureochelin) that, when complexed with iron, are transported into the bacterial cytoplasm, and the iron is extracted. Transport of the ironsiderophore complexes is mediated by the ATP binding cassette (ABC) transporters encoded within sirABC, sitABC, fhuCBG, and sstABCD (38,43,158,200). In addition, S. aureus has an elaborate system to transport and extract iron from heme encoded within the iron-regulated surface determinant (isd) locus and the hrtAB and htsABC loci (151,209,223).…”