Staphylococcus lugdunensis IsdG liberates iron from host heme

Abstract

Staphylococcus lugdunensis is often found as part of the normal flora of human skin but has the potential to cause serious infections even in healthy individuals. It remains unclear what factors enable S. lugdunensis to transition from a skin commensal to an invasive pathogen. Analysis of the complete genome reveals a putative iron-regulated surface determinant (Isd) system encoded within S. lugdunensis. In other bacteria, the Isd system permits the utilization of host heme as a source of nutrient iron to facilitate bacterial growth during infection. In this study, we establish that S. lugdunensis expresses an iron-regulated IsdG-family heme oxygenase that binds and degrades heme. Heme degradation by IsdG results in the release of free iron and the production of the chromophore staphylobilin. IsdG-mediated heme catabolism enables the use of heme as a sole source of iron, establishing IsdG as a pathophysiologically relevant heme oxygenase in S. lugdunensis. Together these findings offer insight into how S. lugdunensis fulfills its nutritional requirements while invading host tissues and establish the S. lugdunensis Isd system as being involved in heme-iron utilization.

Fig. 1.

S. lugdunensis can utilize heme for growth and encodes a putative Isd system. (A) Growth comparison of S. lugdunensis grown in a medium alone, an iron-depleted medium, or an iron-depleted medium supplemented with 5 μM heme. (B) Genomic organization of the isd locus within S. lugdunensis, with four predicted transcriptional start sites designated by bent arrows. Putative Fur binding sites are marked by stippled boxes. All assignments are based on the annotated S. lugdunensis N920143 genome (18a) (accession number FR870271). (C) Amino acid alignment of S. aureus IsdG and S. lugdunensis IsdG, with nonconserved amino acids shown in boldface. Amino acids within the conserved catalytic triad are indicated by asterisks. (D) Nucleotide sequence alignment of the S. aureus Fur box consensus sequence, the S. aureus isdG Fur box sequence, and the S. lugdunensis isdG Fur box sequence. Nucleotides that differ from the consensus sequence are shown in boldface.

Fig. 2.

S. lugdunensis IsdG binds and degrades heme. (A) Increasing amounts of heme (2 to 40 μM) were added to a sample of purified IsdG (10 μM) and to a reference sample. (Inset) The difference between the absorbances (Δabs.) of the protein-heme complex and free heme at 413 nm is plotted against the total heme concentration. A 10 μM protein sample was used. (B) Spectra of 40 μM IsdG-heme complex were taken at the time of addition of 1 mM ascorbate (0 m; curve shown in blue) and 15, 30, 60, 90, and 120 min (curve shown in red) thereafter. Reactions were performed in the presence of catalase at a 0.5:1 (catalase to hemoprotein) molar ratio.

Fig. 3.

The catalytic triad is functionally conserved in S. lugdunensis IsdG. Point mutations were made within the NWH catalytic triad of S. lugdunensis IsdG by converting each of the three amino acids to alanine. Proteins were expressed and purified similarly to those of the wild type and were assessed for enzymatic activity. Spectra of 40 μM IsdG-heme complex were taken at the time of addition of 1 mM ascorbate (0 m; curve shown in blue) and 15, 30, 60, and 90 min (curve shown in red) thereafter.

Fig. 4.

HPLC analysis of the S. lugdunensis IsdG heme degradation product. HPLC comparison of established heme degradation products indicates that S. lugdunensis IsdG degrades heme to staphylobilin. (A) S. aureus staphylobilin purification monitored at 465 nm. (B) S. lugdunensis IsdG-mediated heme degradation product monitored at 465 nm. (C) Biliverdin purification monitored at 405 nm.

Fig. 5.

Tandem LC-HR-ESI-MS analysis of the S. lugdunensis IsdG heme degradation product and S. aureus staphylobilin. (A) ESI-MS-MS of fragment ion selection for m/z 599.3 for the S. lugdunensis IsdG-mediated heme degradation product. (B) ESI-MS-MS of fragment ion selection for m/z 599.3 for S. aureus staphylobilin.

Fig. 6.

S. lugdunensis isdG is iron regulated. S. lugdunensis was grown overnight with the indicated supplements, and protoplasts were lysed and normalized by total-protein concentration. (A) Immunoblot analysis of the effect of iron chelation by 2,2′-dipyridyl (Dip) on the IsdG expression level. (B) Difference (expressed as the fold change) between cytoplasmic IsdG levels in increasing concentrations of Dip and those in medium alone. (C) Immunoblot analysis of the effect of exogenous heme on the expression of IsdG when S. lugdunensis is grown in an iron-depleted medium. (D) Difference (expressed as the fold change) between the IsdG levels of cells grown in an iron-depleted medium with increasing concentrations of exogenous heme and those in cells grown in an iron-depleted medium alone.

Fig. 7.

S. lugdunensis IsdG complements the heme utilization defect of an S. aureus heme oxygenase mutant. Shown is a comparison of the growth of S. aureus, S. aureus ΔisdGI, and S. aureus ΔisdGI expressing S. lugdunensis isdG in trans from the pOS1plgt plasmid. All strains contain a pOS1plgt plasmid and were grown in minimal medium with supplemental heme as the only iron source.

Fig. 8.

Two distinct staphylococcal clades within a phylogenetic tree of annotated IsdG-family heme oxygenases. Shown is a midpoint-rooted phylogenetic tree of IsdG/I amino acid sequences from bacterial taxa with characterized heme oxygenases. The phylogeny was reconstructed using ML, and the tree with the highest log likelihood (lnL = −3,131.92126) is shown. A nonparametric bootstrap (500 replicates) was used to determine node support. Bootstrap values are shown at each node. Branch lengths are drawn to scale and are measured in amino acid substitutions per site.