Chemical Synthesis of Staphyloferrin B Affords Insight into the Molecular Structure, Iron Chelation, and Biological Activity of a Polycarboxylate Siderophore Deployed by the Human Pathogen Staphylococcus aureus

Abstract

Staphyloferrin B (SB) is a citrate-based polycarboxylate siderophore produced and utilized by the human pathogen Staphylococcus aureus for acquiring iron when colonizing the vertebrate host. The first chemical synthesis of SB is reported, which enables further molecular and biological characterization and provides access to structural analogues of the siderophore. SB was prepared in twelve steps from commercially available starting materials with full control of the stereochemistry at the citric acid moiety achieved by using a citric acid synthon containing an alkene moiety. NMR spectroscopic studies confirmed that SB adopts a hemiaminal structure.Addition of synthetic SB to bacterial growth medium recovered the growth of the antibiotic resistant community isolate S. aureus USA300 JE2 cultured under conditions of iron limitation.Further, SB-dependent growth recovery required expression of the SB receptor SirA. Two structural analogues of SB, epiSB and SBimide, were also synthesized and employed to investigate how epimerization of the citric acid moiety or imide formation influence its function as a siderophore. Epimerization of the citric acid stereocenter perturbed the iron-binding properties and siderophore function of SB as evidenced by experimental and computational modeling studies. Although epiSB provided growth recovery to S. aureus USA300 JE2 cultured in irondeficient medium, the effect was attenuated relative to that of SB. Moreover, SB more effectively sequestered the Fe(III) bound to human holo-transferrin, an iron source of S. aureus, than epiSB. SBimide is an imide analogous to the imide forms of other citric acid siderophores that are often observed when these molecules are isolated from natural sources. Here, SBimide is shown to be unstable, converting to native SB at physiological pH. SB is considered to be a virulence factor of S. aureus, a pathogen that poses a particular threat to public health because of the number of drug-resistant strains emerging in hospital and community settings. Iron acquisition by S. aureus is important for its ability to colonize the human host and cause disease, and new chemical insights into the structure and function of SB will inform the search for new therapeutic strategies for combating S. aureus infections.3

Structural and functional delineation of aerobactin biosynthesis in hypervirulent Klebsiella pneumoniae

“…Experiments presented herein confirmed the structural predictions by functionally demonstrating the stereoselective mechanisms of IucA and IucC. Although the final symmetry of aerobactin renders the citratederived C3 position achiral, stereochemistry at the C3 position has been shown to play a critical role in influencing siderophore potency and iron affinity in the NIS staphyloferrin B (43).…”

“…This implies that all the necessary substrates can be harvested from the cellular metabolic pool, not requiring any additional enzymatic activity to supply the pathway. It is interesting to note that the operon of the closely related, and virulence-promoting, NIS staphyloferrin B contains specialized enzymes dedicated to citrate and amino acid precursor production (43). The relatively low burden of the 5-gene, 8-kb aerobactin operon likely contributes to its relatively wide distribu-tion in pathogenic members of the Enterobacteriaceae family, including Escherichia, Klebsiella, Salmonella, Yersinia, Enterobacter, Shigella, Serratia, and Citrobacter, as well as marine Vibrio (44) species.…”

Human calprotectin affects the redox speciation of iron

“…6). We supplemented the medium with 3 μM of desferrioxamine B (DFO), 53 enterobactin (Ent), 54 or staphyloferrin B (SB) 55 (Fig. S7).…”

“…Desferrioxamine B (DFOB) was purchased from Sigma, and a 100-mM stock solution was prepared in water. Enterobactin (Ent) and staphyloferrin B (SB) were synthesized as previously described, 55,66 and stock solutions (≈20 mM) were prepared in anhydrous dimethyl sulfoxide (DMSO, Sigma). For metal-depletion samples containing pyocyanin (PYO), 10 μM PYO (≥98%, Sigma, 10 mM stock solution in DMSO) was added to Tris:TSB −BME prior to the CP-treatment protocol described above.…”

A facile label-free electrochemiluminescent biosensor for specific detection of Staphylococcus aureus utilizing the binding between immunoglobulin G and protein A

“…It exists in most kinds of environments and disseminates rapidly by such media as water, air, food and biological contact, afflicting people worldwide and mostly in developing and underdeveloped countries [1][2][3][4]. Therefore, a reliable, rapid, facile, and low-cost detection technique for S. aureus is urgently demanded for control of infectious diseases.…”