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

Abstract

Staphylococcus aureus is a frequent cause of bloodstream, respiratory tract, and skin and soft tissue infections. In the bloodstream, the iron-binding glycoprotein transferrin circulates to provide iron to cells throughout the body, but its iron-binding properties make it an important component of innate immunity. It is well established that siderophores, with their high affinity for iron, in many instances can remove iron from transferrin as a means to promote proliferation of bacterial pathogens. It is also established that catecholamine hormones can interfere with the iron-binding properties of transferrin, thus allowing infectious bacteria access to this iron pool. The present study demonstrates that S. aureus can use either of two carboxylate-type siderophores, staphyloferrin A and staphyloferrin B, via the transporters Hts and Sir, respectively, to access the transferrin iron pool. Growth of staphyloferrin-producing S. aureus in serum or in the presence of holotransferrin was not enhanced in the presence of catecholamines. However, catecholamines significantly enhanced the growth of staphyloferrin-deficient S. aureus in human serum or in the presence of human holotransferrin. It was further demonstrated that the Sst transporter was essential for this activity as well as for the utilization of bacterial catechol siderophores. The substrate binding protein SstD was shown to interact with ferrated catecholamines and catechol siderophores, with low to submicromolar affinities. Experiments involving mice challenged intravenously with wild-type S. aureus and isogenic mutants demonstrated that the combination of Hts, Sir, and Sst transport systems was required for full virulence of S. aureus.

Fig. 1.

Growth of S. aureus Newman and derivatives in Chelex-treated Tris-minimal succinate medium containing either 20% human serum (A) or 10 μM human holotransferrin (holo-hTf) (B). The iron-restricted growth observed was dependent on high-affinity iron acquisition, since supplementation of either medium with 100 μM FeCl₃ (inset) obviated any growth differences between strains. All data points represent average values for at least three independent biological replicates, and error bars indicate the corresponding standard deviations from the means.

Fig. 2.

In the presence of human serum or transferrin, catecholamines stimulate the growth of staphyloferrin-deficient S. aureus. Growth of S. aureus Newman and derivatives was measured in TMS medium containing either 20% human serum (A and C) or 10 μM human holotransferrin (B). Additions to the media were as follows: Fe, FeCl₃; NE, dl-norepinephrine; E, epinephrine; D, dopamine; and LD, l-DOPA. The numbers indicate final concentrations (μM). All data points represent average values for at least three independent biological replicates, and error bars indicate the corresponding standard deviations from the means.

Fig. 3.

Western immunoblot for detection of expression of SstD in S. aureus. The indicated strains were grown in TMS medium containing 20% human serum. Further experimental details are outlined in Materials and Methods. The SstD lipoprotein of approximately 38 kDa is identified.

Fig. 4.

Catecholamine-dependent growth stimulation of staphyloferrin-deficient S. aureus in medium containing either human serum (A and C) or transferrin (B) requires the SstABCD transporter. For panels B and C, although all tested catecholamines promoted growth of an Sst-proficient S. aureus strain equivalent to that with norepinephrine, for clarity, the results are graphed only for norepinephrine. In panel C, the sstABCD operon, expressed from plasmid pSB5 with the endogenous iron-regulated sst promoter, complemented the Δsst growth deficiency of staphyloferrin-deficient S. aureus in serum in the presence of catecholamines. pLI50 is the vehicle control, and additions to the media were as follows: Fe, FeCl₃; NE, dl-norepinephrine; E, epinephrine; D, dopamine; and LD, l-DOPA. The numbers indicate the final concentration (μM). All data points represent average values for at least three independent biological replicates, and error bars indicate the corresponding standard deviations from the means.

Fig. 5.

SstD binds ferrated catecholamines and catechol siderophores. (A) SstD was purified in preparation for ligand binding studies; see Materials and Methods for details. Fluorescence quenching was used to determine binding affinities of SstD for ferrated catecholamines (B) and ferrated catechol siderophores (C). E, epinephrine; NE, norepinephrine; D, dopamine; LD, l-DOPA; EB, enterobactin; S4, salmochelin S4; DHBA, 2,3-dihydroxybenzoic acid; BB, bacillibactin; PB, petrobactin.

Fig. 6.

Contributions of siderophore biosynthesis and transport to S. aureus infection in immunocompetent BALB/c mice. Experimental details are found in Materials and Methods. Strains evaluated are as indicated, and bacterial burdens in organs were evaluated 4 days following challenge. Each symbol represents an individual mouse, and groups of 10 mice were challenged. Each horizontal line indicates the average log₁₀ CFU/organ for the group. Statistically significant data, determined by Student's t test (P < 0.05), are shown for comparisons of groups of mice infected with mutant bacteria versus those infected with wild-type bacteria, unless otherwise indicated.

Fig. 7.

Staphyloferrin production continues in the absence of the ability to transport staphyloferrins. (A) Growth curves for S. aureus Newman and derivatives in TMS medium that was not treated with Chelex 100. (B) Chrome azurol S reagent was used to assay culture supernatants for siderophore output throughout growth (shown in panel A) at the indicated time points. All data points represent average values for at least three independent biological replicates, and error bars indicate the corresponding standard deviations from the means.