Growth promotion of the opportunistic human pathogen, Staphylococcus lugdunensis, by heme, hemoglobin, and coculture with Staphylococcus aureus

Abstract

Staphylococcus lugdunensis is both a commensal of humans and an opportunistic pathogen. Little is currently known about the molecular mechanisms underpinning the virulence of this bacterium. Here, we demonstrate that in contrast to S. aureus, S. lugdunensis makes neither staphyloferrin A (SA) nor staphyloferrin B (SB) in response to iron deprivation, owing to the absence of the SB gene cluster, and a large deletion in the SA biosynthetic gene cluster. As a result, the species grows poorly in serum-containing media, and this defect was complemented by introduction of the S. aureus SA gene cluster into S. lugdunensis. S. lugdunensis expresses the HtsABC and SirABC transporters for SA and SB, respectively; the latter gene set is found within the isd (heme acquisition) gene cluster. An isd deletion strain was significantly debilitated for iron acquisition from both heme and hemoglobin, and was also incapable of utilizing ferric-SB as an iron source, while an hts mutant could not grow on ferric-SA as an iron source. In iron-restricted coculture experiments, S. aureus significantly enhanced the growth of S. lugdunensis, in a manner dependent on staphyloferrin production by S. aureus, and the expression of the cognate transporters by S. lugdunensis.

Keywords: Heme; hemoglobin; iron acquisition; staphylococci; staphyloferrins..

Figure 1

Physical maps of the sfa-hts and isd loci in Staphylococcus lugdunensis. (A) Staphyloferrin A (SA) biosynthetic and uptake locus. Shown is the homologous locus in S. aureus versus that which is present in all sequenced genomes of S. lugdunensis. Note that the SA biosynthetic locus in S. lugdunensis carries a deletion that eliminates two genes completely (sfaA and sfaD), along with the promoter for the remaining two genes (sfaB and sfaC). The deleted region is indicated between the dashed lines. Asp23, alkaline shock protein 23. (B) Shown is the ∼65-kb region of the S. lugdunensis strain HKU09-01 genome (spanning orfs SLGD_00056 to SLGD_00116) with the tandemly duplicated isd-sir locus. The duplicated region is shown between the dashed vertical lines. Abbreviations are as follows, with predicted or hypothetical functions: ABC, component of an ATP-binding cassette transporter; ATP-A,B,C, K+-ATPase components A, B, and C, respectively; mem, membrane protein; FMN, FMN binding protein; NAT,N-acetyltransferase; marR, MarR-type regulator; hyp, hypothetical; Red, reductase; Pase, phosphatase.

Figure 2

Staphylococcus lugdunensis grows poorly in iron-restricted growth media. (A) Growth kinetics comparing S. lugdunensis to that of Staphylococcus aureusWT and staphyloferrin A (sfa) and staphyloferrin B (sbn)-deficient mutants in C-TMS with 20% serum. (B) The growth deficiencies of S. lugdunensis and the S. aureus staphyloferrin-deficient mutant in iron-restricted media are complemented with addition of 100 μmol/L FeCl₃. All data points represent average values for at least three independent biological replicates, and error bars represent standard deviation from the mean.

Figure 3

Introduction of a complete sfa locus from Staphylococcus aureus into Staphylococcus lugdunensis leads to SA production and growth in iron-restricted media. (A) Chrome azurol S (CAS) assay demonstrates that culture supernatants of S. lugdunensis lack siderophore activity, whereas introduction of the plasmid carrying the S. aureus sfaA-D genes into S. lugdunensis allows for the production and secretion of siderophore into culture supernatants. (B) Growth of S. lugdunensisWT strain containing either vehicle (pLI50) or plasmid carrying the staphyloferrin A biosynthetic genes (sfaA-D) in C-TMS plus 20% serum. (C) Agar plate bioassays demonstrating that the culture supernatant of S. lugdunensis does not promote the growth of iron-restricted S. aureus strains, while the supernatant of S. lugdunensis carrying the sfa gene cluster, which generates SA, can promote the growth of iron-restricted S. aureus in an hts (encodes the SA transporter)-dependent manner. Ferric citrate was used as a positive control in the experiment. All data points represent average values for at least three independent biological replicates, and error bars represent standard deviation from the mean.

Figure 4

Expression of Staphylococcus lugdunensis HtsA and SirA homologues is iron-regulated. (A) Identification of Fur-boxes upstream of the htsA,sirA and isdC genes in S. lugdunensis. Numbers represent the number of identical bases between the 19-bp Fur boxes of S. aureus and S. lugdunensis. (B) Western blots demonstrating iron-regulated expression of HtsA and SirA, and confirmation of mutations and complementation, where pRMC2 is the vehicle control. Cultures were grown in C-TMS with (+Fe) or without (−Fe) addition of FeCl₃ (25 μmol/L). Mutant samples were all grown in C-TMS without addition of iron.

Figure 5

Plate bioassays demonstrate that Staphylococcus lugdunensis HtsABC and SirABC are required for uptake of staphyloferrin A and staphyloferrin B, respectively. Water and ferric citrate were used as negative and positive controls, respectively. Supernatant extracts supplied were those of Staphylococcus aureus mutants that secrete SA (sbn mutant), SB (sfa mutant) or neither SA or SB (sfa sbn mutant). All data points represent average values for at least three independent biological replicates, and error bars represent standard deviation from the mean.

Figure 6

Coculture experiments demonstrate that Staphylococcus aureus-produced siderophores can enhance the iron-restricted growth of Staphylococcus lugdunensis. (A) Picture of colonies of S. aureus RN6390 (large and white) and S. lugdunensis (smaller and yellow) growing on a TSB plate after 24 h of incubation at 37°C, followed by 48 h of incubation at room temperature. (B) Growth of individual strains in C-TMS + 20% serum was monitored for CFU/mL at 12 and 24 h timepoints. (C) Growth of strains in cocultures with the pairs of strains grouped as indicated. All data points represent average values for at least three independent biological replicates, and error bars represent standard deviation from the mean. The Student's unpaired t-test was used to define statistical significance for the CFU values between strains as indicated by the brackets. *P < 0.0001.

Figure 7

Growth of Staphylococcus lugdunensisWT and its isogenic Δisd mutant using iron, hemoglobin or heme as a sole iron source. Growth of the Δisd-sir mutant was compared to that of WT strain HKU09-01 at 12-, 24-and 36-h timepoints in RPMIC containing FeSO₄ (A) varying concentrations of human hemoglobin, (B) or varying concentrations of bovine hemin, (C) as the sole iron source. All data points represent average values for at least three independent biological replicates, and error bars represent standard deviation from the mean. Statistical significance was determined using the Student's unpaired t-test; *P < 0.05; **P < 0.01; ***P < 0.0001.