Siderophore Production by Staphylococcus aureus and Identification ...

INFECTION AND IMMUNITY, May 1997, p. 1944–1948 0019-9567/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 65, No. 5

Siderophore Production by Staphylococcus aureus and Identification of Iron-Regulated Proteins RENE J. COURCOL,1* DOMINIQUE TRIVIER,1 MARIE-CHRISTINE BISSINGER,1 GUY R. MARTIN,1 AND MICHAEL R. W. BROWN2 Bacteriology Laboratory, A. Calmette Hospital, 59037 Lille, France,1 and Microbiology Research Group, Department of Pharmaceutical and Biological Sciences, Aston University, Birmingham B4 7ET, United Kingdom2 Received 4 October 1996/Returned for modification 15 November 1996/Accepted 5 February 1997

Siderophore activity of Staphylococcus aureus was detected in an iron-restricted chemically defined medium. The molecular mass of this siderophore, called aureochelin, was 577 Da. Surface-associated proteins of 120, 88, 57, 35, and 33 kDa were mainly expressed under iron restriction conditions. Results showed a relationship between siderophore production and the existence of the 120- and 88-kDa proteins. Western blotting of surface-associated proteins revealed that these proteins were recognized both by patients sera and polyclonal rabbit serum.

Iron availability is of major importance in bacterial pathogenesis, as nearly all bacteria require this essential nutrient. In response to the low level of available iron in vivo, bacteria induce high-affinity iron uptake systems which consist of lowmolecular-weight iron chelators, called siderophores, and a number of iron-regulated membrane proteins. Siderophore production could be an essential virulence factor in the pathogenic process (6, 25, 26). Despite the importance of staphylococci in human flora and the involvement of the genus Staphylococcus in pathology, little is known about the production of siderophore by Staphylococcus aureus. We previously reported (3, 4) that S. aureus produced siderophore by using the universal chrome azurol S assay of Schwyn and Neilands (19). Recently, Lindsay et al. (12) reported that S. aureus can acquire iron from transferrin for growth. Furthermore, iron depletion is known to have a profound effect on the composition and surface structure of microorganisms (2, 20). Under that condition, few investigators have examined protein profiles of S. aureus (3, 11, 21, 24). Using an iron-restricted medium, we report for the first time (i) the existence of a new staphylococcal siderophore that we call aureochelin and (ii) the identification of several iron-regulated proteins expressed in a chemically defined medium (CDM) recognized by both patient and rabbit sera. Siderophore synthesis and characterization. Glassware and medium were prepared as previously described (4, 17). Medium without iron (CDM2Fe) contained less than 0.04 mM Fe31. For medium with iron (CDM1Fe), FeSO4 was added to give a concentration of 60 mM. S. aureus strains were clinical isolates obtained from either blood cultures (strains LAT, MAT, and VOS) or cystic fibrosis patients (strain DES) or else a laboratory strain (S. aureus Cowan 1). An overnight culture in CDM2Fe was used as an inoculum and calibrated to give 5 3 106 CFU/ml of culture. Strains were all grown for 48 h at 378C in 200-ml shake flask cultures in an orbital shaker (172 rpm). Growth was monitored both spectrophotometrically (A470) and by colony counts, using a pour plate procedure. Siderophore in culture supernatants was detected by the

method previously described by Arnow (1) or a Cza`ky method modified by Gibson and Magrath (5) to detect either hydroxamate or phenolate compounds in the CDM2Fe culture supernatants, respectively. Negative results were obtained with both methods. Siderophore was detected with the Schwyn and Neilands reagent (19) and was expressed in terms of either arbitrary units (100 arbitrary units equals total removal of iron from the dye reagent) or equivalent of Desferal (micromolar). S. aureus DES was grown in CDM2Fe over 5 days of incubation. Figure 1 shows the kinetics of growth and the siderophore production. Siderophore activity was detected with the Schwyn and Neilands reagent in culture supernatants from 24 h onward, which was the time related to the end of the log phase as shown in Fig. 1. Siderophore was detected only when the number of bacteria was over 108 ml21 (day 1). Over 5 days of growth, siderophore production increased after the stationary phase was reached. In another experiment (data not shown), addition of iron ranging from 0.1 to 0.9 mM in CDM led to a reduction of the siderophore production from 4.02 to 0.97 mM Desferal equivalents, respectively, over 24 h of incubation and an increase in cell yield. The quantity of siderophore was less than 0.25 mM Desferal equivalents when the iron concentration was equal to 1.0 mM in CDM2Fe. As we have previously observed (3), Lindsay and Riley (11) reported that S. aureus has an intrinsically low requirement for iron. In our study, siderophore production was reduced by addition of low concentrations of iron. Siderophore production was population dependent: it was likely that siderophore was synthesized within the lag phase and the first hours of the exponential phase to initiate growth. Although the chrome azurol S assay was independent of siderophore structure, considering that it was universal, it was not sensitive enough to detect early production of iron chelator which should have preceded or accompanied the exponential growth. Moreover, siderophore production was also time dependent: a prolonged incubation resulted in an increased quantity of siderophores. The present data and others published in previous papers (24, 25) did not agree with those published by Lindsay and Riley (11). These authors reported maximal levels of the chelating activity after 16 h onward, which was the stationary phase. However, our results show that when the stationary phase was reached, siderophore production still increased. Two hypoth-

* Corresponding author. Mailing address: Laboratoire de Bacte´riologie-Hygie `ne, Hopital A. Calmette, 59037 Lille ce ´dex, France. Phone: (33).3.20.44.49.08. Fax: (33).3.20.44.48.95. 1944

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FIG. 1. Kinetics of growth (bacterial counts expressed in CFU/milliliter; ■) and siderophore production (siderophore expressed in micromolar Desferal equivalents; h) of S. aureus DES in CDM2Fe.

eses might be suggested to explain this phenomenon at the steady state: either bacteria were still able to synthesize siderophore or the increase was due both to siderophore synthesis by new bacteria and to release of intracellular siderophore during cell lysis (24). On the other hand, Lindsay and Riley observed with S. aureus NCTC 8531 that the late log phase started approximately at 10 h and the stationary phase started after 16 h. These growth times obtained in minimal and irondepleted media were shorter than ours, suggesting that the iron restriction of their medium was not sufficiently great. Difficulties in removing iron from media have been reported (8). Furthermore, Lindsay and Riley obtained at 40 h a maximum level of 110 mM desferrioxamine equivalents, which was half of the amount that we detected under our conditions at the same time. However, this significant discrepancy might be due to the capacity of their strain to produce siderophore. Siderophore was extracted from the culture supernatants according by the method of Rogers (18). The crude siderophore extract was loaded on plastic-backed cellulose plates (Merck-Cle´venot, Nogent-sur-Marne, France). A solvent system containing chloroform-acetic acid-methanol was used in the ratio 80/10/10. The thin-layer chromatography plates were sprayed with either the Schwyn and Neilands or the Swain reagent’s showing a faint and labile pink spot or a blue spot, respectively. The Swain reagent indicated the presence of a phenolic moiety (23). Strains produced one spot giving an Rf of 0.75 for all strains tested. As a control, thin-layer chromatography plates were sprayed with ninhydrin. No amino acids were detected from the crude siderophore extract. From preparative plates, the purified siderophore was obtained by scraping the areas reacting with the Swain reagent. The extracts produced from S. aureus LAT and DES were ready for mass spectrophotometry analysis. It was carried out, using impact electronic mode after direct inlet, on a model 59827A mass spectrometer from Hewlett-Packard (Orsay, France). The energy of electrons was 70 eV. The molecular mass of aureochelin was 577

Da (Fig. 2), which was different of those of siderophores produced by S. hyicus, 481 and 448 Da for staphyloferrins A and B, respectively (7, 9, 15). The chemical structure of aureochelin is currently being investigated. However, reactivity of siderophore extract with the Swain reagent, which detects phenolic moieties, supports the hypothesis that aureochelin can be classified as a phenolate-catecholate compound. Preliminary results obtained by nuclear magnetic resonance analysis are in agreement with this potential chemical structure. Iron chelator production by staphylococci on the surface of nutrient agar has been previously reported (13, 14, 16). In the present study, biological activity of siderophore extracts was determined by using a bioassay agar plate with S. aureus DES and LAT. Siderophore extracts were solubilized with methanol for bioassay experiment. Sterile filter paper strips were impregnated either with 100 ml of purified extracts from CDM1Fe and CDM2Fe supernatants, enterochelin (gift to Michael R. W. Brown), or methanol. S. aureus test strains were seeded (inoculum of 104 ml21) on the surface of CDM2Fe agar plates. Plates were incubated at 378C for 48 h. Results were considered positive when a halo of growth surrounding the paper strips was visualized. Results obtained are shown in Fig. 3. No growth halos were detected when paper strips were loaded either with methanol or with ethyl acetate extracts of the CDM1Fe culture supernatant. In contrast, growth halos were observed with ethyl acetate extracts of CDM2Fe culture supernatants obtained with the two strains tested. These extracts stimulated the growth of small inocula of staphylococci. Growth was inhibited around the paper strip impregnated with enterochelin. Our bioassay results were in agreement with those reported by Marcelis et al. (13). These authors did not observe functional interchangeability of iron chelators between staphylococci and enterobacteriaceae. This assumption suggests that iron chelator structures produced by gram-positive bacteria differ from those of gram-negative bacteria. Reexamining the previous study, Maskell (14) agreed with the prelim-

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FIG. 2. Mass spectrometry analysis of the compound produced by S. aureus in CDM2Fe. A major peak is observed with an apparent molecular mass of 577 Da.

inary findings of Miles and Khimji (16) and found opposite results on the ability of staphylococci to utilize enterochelin. Enterobacterial iron chelators appeared to be effective with staphylococci. This effectiveness was not observed in our bioassay plate. Staphylococcal iron-regulated proteins. The effect of iron on S. aureus proteins was investigated after growth in CDM2Fe and CDM1Fe and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Cell wall proteins were prepared as described by Sprott et al. (22). Proteins were separated either on SDS–11 or 6% polyacrylamide gel slabs (10). Gels were stained with Coomassie brilliant blue R. The cell wall contained a large variety of different cell surface-associated proteins (Fig. 4). Proteins of

120, 88, and 57 kDa were iron-repressible proteins and were expressed mainly under iron restriction conditions. These proteins were never observed with the reference strain S. aureus Cowan 1 (data not shown). Proteins of 120, 88, 57, 35, and 33 kDa were iron-repressible proteins and were expressed mainly under iron restriction conditions. Two strains of S. aureus which were found as high and low siderophore producers at 24 h were investigated to determine a putative relationship between siderophore production and expression of the 120and 88-kDa proteins (data not shown). With the high siderophore producer strain (strain VOS, 278 mM Desferal equivalents), the two proteins were strongly expressed on SDSPAGE whereas they were weakly expressed with the low siderophore producer strain (strain MAT, 36.5 mM of Desferal

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FIG. 3. Bioassay plate with S. aureus DES. Paper strips were impregnated with the following extracts: CDM1Fe culture supernatant of strain LAT (plate A), CDM2Fe culture supernatants of strains LAT and DES (plates B and C, respectively), methanol (plate D), and enterochelin (plate E).

equivalents). In this experiment, bacterial counts were similar (1.98 3 109 and 2.0 3 109 CFU/ml, respectively). As protein profiles of S. aureus grown in iron-depleted medium have not been studied extensively (2, 11), these insufficient data do not allow us to define clearly the role of the iron-regulated proteins in iron metabolism. Two iron-repressible proteins (120 and 88 kDa), not described by Lindsay and Riley (11), seem especially interesting due to their expression and relationship with siderophore production. Their presence was variable from one experiment to another and according to the strains tested. We found that a high siderophore activity from a high-producing strain correlated with a strong expression of the proteins of 120 and 88 kDa and vice versa. One

FIG. 4. SDS-PAGE (8% gel) profiles of cell surface-associated proteins from S. aureus DES. Lane a, CDM1Fe; lane b, CDM2Fe; lane c, molecular mass markers in kilodaltons; lane d, prestained molecular weight markers. Arrowheads indicate proteins with molecular masses of 120, 88, 57, 35, and 33 kDa.

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FIG. 5. Cell surface-associated proteins from S. aureus DES. Immunoblotting experiments were performed with polyclonal rabbit (A) and human (B) sera. Lane a, CDM1Fe; lane b, CDM2Fe; lane C, prestained molecular weight markers. Arrowheads indicate the 120- and 88-kDa proteins.

possible explanation of these findings is that chromosomal genes could control low-level synthesis of a siderophore, while virulent strains could harbor plasmids encoding the synthesis of a second siderophore produced in larger amounts. This explanation is in agreement with the findings of Meiwes et al. (15), who reported that S. hyicus produced two compounds with siderophore activity. Consequently, more than one siderophore could be produced by S. aureus and therefore isolated. An explanation of the different proteins observed by other authors is that the pattern of bacterial iron-regulated proteins obtained with EDDA or Desferal is not always identical to that obtained using a host with naturally occurring iron-binding protein (6). Immune response. Immunoblotting experiments were performed to determine whether any of the surface-associated proteins were recognized by polyclonal rabbit and human sera. Hyperimmune serum was produced in male New Zealand rabbit (Janvier, le Genest, France). Preimmune bleeds confirmed the absence of antistaphylococcal antibodies. Strain LAT was injected intradermally with 109 live bacteria in 1 ml of 0.85% saline solution mixed with 1 ml of incomplete Freund adjuvant (Difco Laboratories, Detroit, Mich.) to obtain immune serum. Injections by the same route were repeated after 3 weeks. Human sera were collected from patients with staphylococcal septicemia. Normal human serum was collected from a healthy donor. Proteins separated by SDS-PAGE were transferred by electroblotting onto nitrocellulose membrane (Amersham, Les Ulis, France). After transfer, membranes were blocked then probed with either polyclonal rabbit serum or patient sera diluted 1:500 and 1:250, respectively, for 1 h. Proteins were detected by using either goat anti-rabbit or anti-human immunoglobulin IgG (heavy plus light chain)–horseradish peroxidase conjugate (Bio-Rad Laboratories) diluted 1:3,000. Reactive bands were visualized with 3-39 diaminobenzidine (Sigma Chemical Co.). More proteins appeared when bacteria were grown in CDM2Fe (Fig. 5). Particularly, the surface-associated proteins of 120 and 88 kDa were strongly recognized by either the homologous serum from the patient or nonhomologous sera from other patients having staphylococcal septicemia, as well as with polyclonal rabbit serum. Serum collected from healthy donor blood did not react with these proteins (data not shown). In conclusion, the exact mechanism by which S. aureus can obtain Fe31 from the medium is yet unknown, but it might be that aureochelin, a new siderophore isolated from S. aureus,

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can be used directly by bacteria. Exploitation of siderophore as a vector of antimicrobial agents could offer some promises against methicillin-resistant S. aureus. Much information about the host-microbe interaction are provided by blotting surface proteins with patient serum. These reactions between staphylococcal antigens expressed in iron-restricted medium and patient antibodies underline the fact that these antigens are likely expressed in vivo and that S. aureus grows in patients under iron-restricted conditions. Further studies are needed to characterize these new surface antigens contributing to a better understanding of the mechanisms involved in the pathogenicity of S. aureus. This work was supported by the Conseil Re´gional Nord-Pas de Calais, Centre Hospitalier et Universitaire de Lille, and Direction de la Recherche et des Etudes Doctorales. R.J.C. was supported by a grant from INSERM (France) and MRC (United Kingdom), and D.T. was supported by a grant from the Conseil Re´gional Nord-Pas de Calais and Centre Hospitalier et Universitaire de Lille. We are most grateful to Claude Vandeperre for photography. REFERENCES 1. Arnow, L. E. 1937. Colorimetric determination of the components of 3,4dihydroxyphenylalanine-tyrosine mixtures. J. Biol. Chem. 118:531–537. 2. Brown, M. R. W., H. Anwar, and P. A. Lambert. 1984. Evidence that mucoid Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron-restricted conditions. FEMS Microbiol. Lett. 21:113–117. 3. Courcol, R. J., P. A. Lambert, M. R. W. Brown, P. G. Domingue, and G. R. Martin. 1988. Influence of iron-depletion on cellular proteins, antigenic profiles and siderophores of Staphylococcus aureus, abstr. D-184, p. 101. In Abstracts of the 88th Annual Meeting of the American Society for Microbiology 1988. American Society for Microbiology, Washington, D.C. 4. Courcol, R. J., P. A. Lambert, P. Fournier, G. R. Martin, and M. R. W. Brown. 1991. Effect of iron depletion and subinhibitory concentrations of antibiotics on siderophore production by Staphylococcus aureus. J. Antimicrob. Chemother. 28:663–668. 5. Gibson, F., and D. I. Magrath. 1969. The isolation and characterisation of a hydroxamic acid aerobactin formed by Aerobacter aerogenes 62-1. Biochim. Biophys. Acta 454:285–297. 6. Griffiths, E., H. Chart, and P. Stevenson. 1988. High-affinity iron uptake systems and bacterial virulence, p. 121–137. In J. A. Roth (ed.), Virulence mechanisms of bacterial pathogens. American Society for Microbiology, Washington, D.C. 7. Haag, H., H. P. Fiedler, J. Meiwes, H. Drechsel, G. Jung, and H. Za ¨hner. 1994. Isolation and biological characterization of staphyloferrin B, a compound with siderophore activity from staphylococci. FEMS Microbiol. Lett. 115:125–130. 8. Kadurugamuwa, J. L., H. Anwar, M. R. W. Brown, G. H. Sand, and K. H. Ward. 1987. Media for study of growth kinetics and envelope properties of

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