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Isolation and characterization of staphyloferrin A, a compound with siderophore activity from Staphylococcus hyicus DSM 20459
FEMS Microbiology Lcuers 67 (19~0) 201-206 Fuhlished by Elsevier
201
FEMSLE 03883
Isolation and characterization of staphyloferrin A, a compound with siderophore activity from Staphylococcushyicus DSM 20459 J. Meiwes i, H.-P. Fiedler i H. H a a g i. H. Z~ihner 1, S. K o n e t s c h n y - R a p p 2 and G . J u n g 2 J Mikrobiologi¢ I. and : Organische Chemic. Unu.¢rMlilt T'~bmgen. "l'tibmgen, F.R.G.
Received 19 September 1989 Revisionrec¢iveA9 October 1989 Accepled l l October 108q Key words: Staphylococcus h.;,ic~t~ DSM 20459, Siderophore. Staphyloferrin
1. S U M M A R Y A highly hydrophilic compound was isolated from low iron culture broth of Staphylococcus hyicus DSM 20459 which exhibits siderophose activity to the producer and 37 other staphylococci. The previously unknown metabolit¢ was designated staphyloferrin A and consists of two molecules of citric acid, each linked to D-ornitine by an amide bond. Using an ion-pair HPLC-system we detected staphyloferrin A and a second iron regulated compound (staphyloferrin B) in the culture fluid of several Staphylococcus strains. We found no evidence that staphylococci synthesize catecholor hydroxamate-type sideropbores.
2. I N T R O D U C T I O N Virtually all aerobe microorganisms have an absolute requirement for iron. Because of the insolubitity of iron under aerobic conditions this
Correspondent~ to: H. Z~ganer,UniversiOt Ttihingen, Biologic
II, Mik~ohiolosie I, Auf tier Morgenstelle 28, D-7400 Ttibingen. F.R.G.
element often is growth limiting. In host tissues iron is tightly hound to macromolecules such as transferrin, thus unavailable for pathogenic microorganisms. High affinity iron uptake systems have been evolved for assuring the survival of microorganisms exposed to these conditions. The best known high affinity uptake system depends on the secretion of iron specific ligands, termed siderophores, and the specific recognition of the corresponding iron complexes by specialized membrane receptors and transport systems. Both components of the system are coordinately induced and expressed according to the iron concentration [1]. For many microorganisms a correlation between siderophore production and virulence has been established [2], however not much is known about the iron supply of staphylococci in contrast to their importance as virulent microorganisms. The growth of Staphylococcus aureus in human serum depends on the iron concentration as shown by Schade [3]. Marcelis et aL [4] and Maskell [5] reported on the secretion of siderophores by staphylococci but no attempts have been made to isolate and identify these siderophores. Recently the expression of cytoplasma membrane proteins by Staphylococcus aureus [6] and Staphylococcus epidermidis [7] under iron limitation has been
0378-1097/90/$03,50 © 1990 Federation of European Microbiological Societies
202 reported. T h e s e p r o t e i n s are p o s s i b l y p a r t of a s i d e r o p h o r e m e d i a t e d iron u p t a k e system. W e h a v e investigated the s i d e r o p h o r e p r o d u c tion o f s t a p h y l o c o c c i u s i n g the c h r o m e a z u r o l S test ( C A S - t e s t ) [8] a n d a n i r o n n u t r i t i o n b i o a s s a y . A n e w s i d e r o p h o r e f o u n d t h e r e b y w a s i s ol a t e d a n d p a r t i a l l y characterized.
3. M A T E R I A L S
AND METHODS
3.1. B a c t e r i a l s t r a i n s Bacterial s t r a i n s u s e d in this s t u d y are listed i n T a b l e 1. 3, 2. C u l t u r e conditions A l l g l a s s w a r e w e r e i n c u b a t e d for 12 h w i t h 10 mM EDTA and rinsed 5 times with double-distilled w a t e r p r i o r to use. T h e s t r a i n s were s t o r e d at -20°C i n B H I m e d i u m w i t h 50% glycerol. T h e p r e c u l t u r e s w e r e g r o w n for 12 h at 3 7 ° C o n G A a g a r m e d i u m [9]. A : n o d i f i e a t i o n of the m e d i u m of C o v e et al. [10] w a s u s e d for the s i d e r o p h o r e p r o d u c t i o n . T h e m e d i u m c o n t a i n e d : {A}: ( m g / I , f i n a l c o n c e n t r a t i o n ) L-alanine, 200; L-arginine, 400; L-aspartic acid, 20; g-cystine, 50: L-Cysteine, 50; L-ghitamic acid 1000; L-glycine, 800; L-histid i n e h y d r o c h l o r i d e , 400; g - h y d r o x y p r o l i n e , 200; L-isoleucine, 100; L-leucine, 200; L-lysine h y d r o c h l o r i d e , 200; L-methionine, 600: L-phertylalanine, 600; L-proline, 200; L-Serine, 4001 L-threonine, 400; L - t r y p t o p h a n , 100; L-tyrosine, 200, L-valine, 200; a d e n i n e , 10; g u a n i n e , 10 a n d uracil 10. {B}: ( m R / I , f in al c o n c e n t r a t i o n ) KH2PO4, 1000; ( N H 4 ) 2 S O 4 , 20~0i HaC1, 4000; M g S O 4 - T H 2 0 , 100.0; C a C I 2 • 5 H 2 0 , 5.0 a n d tricine, 1 7 9 0 0 ; p H 8.5 a d j u s t e d w i t h 3 N N a O H . {C}: g l n f o s e 10 g / L T h e s o l u t i o n s w e r e c o m b i n e d after s t e r i l i z a t i o n a n d 5 m l o f a sterile filtered v i t a m i n s t oc k s o l u t i o n |10] w a s a d d e d . I n o r d e r to p r o v i d e very l o w i r o n c o n t e n t o f the m e d i u m , a five fold c o n c e n t r a t e d s o l u t i o n o f all c o m p o n e n t s e x c l u d i n g d i v a l e n t cations and vitamins was prepared and treated w i t h C h e l e x I 0 0 . S u b s e q u e n t l y the l a c k i n g c o m p o n e n t s were a d d e d , the final v o l u m e w a s a d j u s t e d w i t h d o u b l e - d e s t i l l e d w a t e r a n d the m e d i u m w a s filter sterilized ( M i l l i p a k T M 20, 0,22 p m , M i l l i p o r e , E s c h b o r n , F,R,G.)~
Table l Production of staphyloferrin A and Ig by staphylococct in Chelex 100 trealed medium with and without supplementation of 1 ram D-omithine Strain
with D-omithine staphyloferrln: A B [mg/I]
S. aureus ATCC 12600 S. aureus SA 113 S. aureus KL 207 S. a~etts DSM 799 S~ aureus DSM 203"/2 S. capitis DSM 20326 S. carnosu.v TM 300 S. cohnii DSM 20260 S. epidermidis DSM 1"/98 S. epidermidix DSM 20042 S. epidermidis ATCC 14990 S. epidermidJs DSM 3GgS/T'.'. 3298 S. gall/nahum DSM 4616/Tti 3928 S. haemolyticm DSM 20263 S. haemolytic~ DSM 20265 S. hominis DSM 20322 S. hylrvs DSM 20454 S. intermedius DSM 20373 S. saprophytictts DSM 20229 S. semri DSM 20352 S. simulans DSM 20322 S, sirnvlans MK 148 S, warneri DSM 20036 S. xylos~ DSM 20266 S. aureus 315886 * S. aureus R 253 * S. aureus G 4 * S. aareus G 3 * S. St 653" S . J 22" S. G 4 ? * S. K 105" S, R 2 ? I * S. R 273" S, K S I * S. K 8 6 " S. K 9 6 "
without 13-ornithine A B [mg/I I
147 < 44 38 S8 86 18 14 ]4 60 6 42 31
+ + + +
27
+
4
+
94
< + <
20 7 < < 10 < < 5 < < 2 < < < < < < 34 < 4
< + <
43 14 41 43 33 80 36 6 41 33 48
42 72 72 19 9 tl0 30 28
"t < < <
+ + + ÷
+
<
+
< < < < + + < <
11 < < < < < < <
< < + < < + < <
< < < + 4< + + < + + < < < < + <
< + < + + < + + < < < < < < < ~: <
* Clinical isolates, obtained from F. Schumacher.Pendreau (Universit~t K~ln, Instltul fiir Hysiene, K/~ltt, F.R.G,). + = production of staphyloferrin B; - ffi strain has grown very poor in the production medium; < ffi staphyloferfin not title,table,
F o r c u [ t u r i n g w e u s e d E r l e n m e y e r f l a s k s (100 ml) with one extrusion containing 20 ml medium a n d a 4 - 1 s t i r r e d - t a n k r e a c t o r ( B i o s t a t S, B r a u n ,
203 Melsungen, F.R.G.). The Erlenmeyer flasks were incubated on a rotary shaker (RC I, Infors AG, Basel, Switzerland) operating at 160 rpm and 36°C. For cultivation in the stirred tank reactor the aeration rate was 1 v~am and the stirring rate was 500 rpm. In this case the medium was made without tricine, and during fermentation the pH was maintained at 7.5 _+0.1 with 3 N N a O H and 3 ]q H2SO4. For inoculation 5% of a 12 h preculture in NB medium was used. The broth was harvested and analyzed after 18 h of cultivation.
3.3. Isolation of staphyloferrin A After removal of the cells by centrifugation the supernatant (4 1) was passed through a column (5 × 25 cm) containing Ambedite 401 S (CI-). Non- or weakly adsorbed components were washed out with 1% formic acid (2 1). Staphyloferrin A was eluted with 3 N formic acid (1 1). The volume was reduced to 40 ml in vacuo using a rotary evaporator. The pH of the resultant concentrate was adjusted to 7.0 with 10 N N a O H at 4 ° C . To this solution ethanol was added to a final concentration of 90% ( v / v ) a r d the suspension was stored at 4 ° C for I h. The precipitate was harvested by centrifugation (8000 × g), dissolved in 50 ml destilled water and methanol added to a final concentration of 70% (v/v). The resultant suspension was centrifuged at 8000 × g and the precipitate dissolved in 50 nd destilled water. The methanol precipitation was repeated twice. The siderophore containing supernatants were combined and concentrated in wry=no to 40 ml. This solution (20 ml) was chrumatographed on a column (6 X 70 cm) containing Biogel P2 (200-400 mesh), equifibrated with 10 mM ammonium acetate buffer (pH 4): elution rate was set at 50 m l / h . The staphyloferrin A containing fractions were pooled, concentrated and rechromatographed under the same conditions. Subsequently the sidercphore concentrate was passed through the same column as described above but pH was changed to 5.0. The active fractions were concentrated and chromatographed on a column (2.8 x 40 cm) filled with Fractogel TSK H W 40 (F) (Merck, Darmstadt, F.R.G.) equilibrated with water-methanol 9 : 1. Finally the staphyloferrin A containing fractions were pooled, concentrated in
vacuo and lyophilized to dryness. The yield was about 150 m 8 / I of culture supernatant.
3.4. Bioassay The bioassay was carried out by a modification of the method described by Miles and Khimji [11]. A modified production medium described above was used. The tricine was replaced by TES (50 raM) and the pH adjusted to 7.5 30 mg of deferrated E D D A (ethylenediamine di-(o-hydroxyphenyl-acetic acid)) [12] and 15 g of agar (Bacto) were added to solution B prior to sterilization. 10 ml of the melted agar (45°C) were inoculated with a 12 h preculture (100/~l) of the desired strain in NB medium and poured into a petri-dish. A sterile paper disc, loaded with test solution (20 .~1) was placed on the solidified agar and the plate incubated at 3 7 ° C for 18 h. A halo of bacterial growth surrounds those discs bearing siderophore activity or iron.
3.5. Ion-pair HPLC The chromatographic system consisted of a HP-1090M liquid chromatograph equipped with a diode array detection system and a work station (Hewlett-Packard, Waldbronn, F.R.G.). FeCl 3 (1 mM) was added to the siderophore containing solutions and the samples were cleared by centrifugation at 1 2 0 0 0 × g . 10 /tl-samples of these solutions were injected onto a HPLC column (125 × 4.6 mm i.d.) fitted with a precolumn (20 x 4.6 m m i.d.), each packed with 5 ?~m Shandon Hypersil ODS (Groin, Hersenberg, F.R.G.); detection was at 340 rim. The samples were separated by gradient ehition; solvent A was 10 mM sodiumphosphate buffer (pH 7.3) with 5 m M tetrabutylammonium-hydrogensulfate (TBA), solvent B was aceton;trile. The gradient was from 15 to 28% B in 3 rain, from 28 to 37% B in the following 4 rnin and raised to 60% B in 1 min. The flow-rate was 2 ml/min.
3.6. Characterization of staphylo/errin A A sample of desferri-staphyloferrin A was hydrolyzed with 6 N HCI (12 h, I10°C). Amino acid analysis was carried out according to Fiedler and Plaga [13]. For configurational analysis of the amino acids gas chromatography on the chiral
204 phase Chirasil-Val [14] was used. Citric acid was detected by using an enzyme test kit (Bcehringer, F.R.G.) depending on citrate-oxalacetate-lyase (EC 4.1.3.6).
4. RESULTS A N D DISCUSSION Using the CAS-test [8] we were able to detect iron chelating activity in iron deficient (about 0.5 # M iron) but not in iron sufficient (10 p~M) centrifuged culture broths of some staphylococci. For isolation of the siderophore we have chosen Staphylococcus hyieus DSM 20459 [15]. This strain produced staphyloferrin A very well in the stirred tank reactor in contrast to the other strains which require a very low iron concentration. It is not possible to maintain such low iron concentration in the stirred tank reactor made of steel component parts. On the other hand pretreatment of the medium with Chelex 100 decreased growth and production of the iron chelating activity of Staphylococcus hyicus DSM 20459. Addition of 1 mM D-omithine increased the production of the siderophore (staphyloferrin A) by a factor of 19, as shown in detail elsewhere [16]. The Csaky method [17] and the methods of Arnow [18] and Rioux [19] were used to detect hydroxamates and catechols, respectively, but both were found to be absent in the culture broth of all strains listed in table 1. The iron chelating metabolite of Staphylococcus hyicus DSM 20459 was purified in the iron free form and was obtained as coloudcss powder (ammonium salt). The ferrated staphyloferrin A has a slightly yellow eolour, The U V / V I S spectra recorded during the HPLC-run (Fig. 1) are lacking charge transfer bands between 420 and 500 nm, indicative of iron(Ill) eomplexation by eatechol or hydroxamate type ligands [1]. This rules out the presence of these functional groups in staphyloferrin A. In the hydrolysate of staphyloferrin A citric acid and ornithine were found. Ornithine was shown to possess D-conflguration, The detailed structure elucidation revealed that staphyloferrin A is Na,NS-di-(l-oxo-3-hydroxy-3,4-dlcarbo xylbutyl)-D-ornithine (Fig. 2) |16], a so far unknown metabolite. Thus staphyloferdn A is a new
200
3OO
40O
wavelength
SC0
[nm] Fig. L U V / V I S spectra of staphyloferrin A and B recorded
during a HPLC-run. Inset: HPLC-run of cullure broth from Staphylococcushyic~ DSM 20459 monitored a! 340 nm. Staph A : staphyloferrin A; Slaph B : staphyloferrin B. siderophore of the carboxylate type like rhizobactin [20] and a-hydroxyisovaleric acid [21]. We were able to measure the concentration of staphyloferrin A in the culture broth and during the isolation by ion-pair HPLC. In the culture broth of Staphylococcus hyicus DSM 20459 a second component (staphyloferrin B) was detected by this HPLC-system (Fig. 1), which too was pro. duced only under iron limited conditions. The isolation and characterization of staphyloferrin B is still in progress, but we can exclude the occurfence of cateehol or hydroxamate moieties. The summary in Table ! shows 34 strains producing staphyloferrin A, when supplemented with D-oruithine. Without supplementation only 1| strains were found to produce detectable amounts of the siderophor¢ whereas staphyloferrin B is produced by 18 and 16 strains, respectively. (staphyloferrin B could be measured only qualitatively because so far no pure staphyloferrin B was available for calibrating the HPLC-analysis). COOH
/~HH
~
NH ,0 OH COOH ~C00H
--~iOH D COOH OH
Fig.2.St~c~reo[~aphylo~in A.
205 T h e utilization o f staphyloferrin A by the prod u c e r a n d all other strains, listed in T a b l e 1. was tested by the E D D A bioassay. In all cases growth halos a p p e a r e d a r o u n d p a p e r discs loaded with 50 n m o l of ferrated staphyloferrin A o r 100 nmol of FeC13 as a control. T h i s indicates that the iron transport system d e p e n d i n g on staphyloferrin A as a siderophore is widely spread a m o n g the staphylococci, though not all strains seem to prod u c e staphyloferrin A. T h e role of staphyloferrin A as a virulence factor remains to he investigated as well as the function and structure of staphyloferrin B.
ACKNOWLEDGEMENTS W e t h a n k F. Gi~tz for c o m m e n t s on the manuscript, the D e u t s c h e Forschungsgemeinschaft ( S F B 323) and the Bundesministerium ffir F o r s c h u n g und Technologie ( B C T 05014) for financial support. J.M. was supported by a D E C H E M A grant. S.K.-R. thaks the Studieastiftung des Deutschen Volkes for a doctorate scholarship.
REFERENCES [1] Winkelmann, G. (1986) in Biotechnology Vol. 4 (Rehm, H.-J. and Reed, G., eds). pp. 215-243. 12] Braun, V., Fischer, E., Hantke, K. and Rotering, H, (19871 in 38. Coloquium Mosbach, Molecular Basis of Viral and
Microbial Pathogenesls (Ro~t. R. and Goebel, W., eds.), pp. 151-159, Springer. Berlin, Heidelberg, New York. [3] Schade, A.L (1963) Biochem. Z. 338,140-148. [4] Marcelis, J.H., Den Daas-Slaagl. HJ. and HoogkampKorstanje, J.A.A. (1978) Anionic van Leeuwenhoeck 44, 257-267. [5] MaskelL J.P. {1980) Antoni© van Leeuwenhoeck 46, 343-351. [6] Domingu¢. P.A~G.. Lambert. P.A. and Brown. M.R.W. (1989) FEMS Microbiol. Let¢. 59, 265-268. [7] Williams, P.. Denyer. S.P. and Finch. R.G. (1988) FEMS Microbiol. Letl. 50. 29-33. [8] Schwyn. B. and Neilands. J.B. (1987) Anal. Biochem. 160. 47-56. [9] HOmer, T., Z~hner, H., Kellner, R. and ,lung, G. (1989) Appl. Microbiol. Biotechnol. 30. 219-225. [10] Cove, ,l.H, Holland, K.T. and Cunli[fe, W,J. (1980) J. Appl. Bacteriol. 49, 29-37. [11] Mile& A.A. and Khimji, P.L. 0975) `l. Med. Microbiol. 8. 477-41~1.
[121 Rogers. HJ. (1973) Infect. Immunol. 7. 0A5-456. [131 Fiedler. H.P. and Plaga. A. (1987) J. Chromalogr. 386. 229-241. 114] Frank. H.. Nicholson. G.J. and Bayer. E. 0978) `l. Chromatogr. 167.18/-196. [I.51 Devriese. L.A.. Hajek. V.. Oeding. P.. Meyer. S.A. and Schleifer. K.H. 11978) Int. SysL Bacteriol. 28. 482-490. [161 Konetschny-Rapp. $.. Meiwes. J.. Jung. G. and Ziihner. H. (in preparation). [1"~] Csaky, T.Z. (1948) Acta Chem. Scand. 2. 450-454. [18] Arnow, L.IL (1937) J. Biol. Chem. 118, 531-537. [19] Rioux, C., Jordan, D.C. and Rauray, ,l.B.M. (1983) Anal. Biochem. 133,163-169. ~20] Smilh, M.J., Shoolery, J.N., Schwyn, B., Holden, L and Neilands, J.B. {1985) J. Am. Chem. Soc. 107, 1739-1743. [21] Evanylo, LP.. Kadis, S. and Maudsley, ,l.R. (1984) Can..1. Microbiol. 30, 1046-1051.
201
FEMSLE 03883
Isolation and characterization of staphyloferrin A, a compound with siderophore activity from Staphylococcushyicus DSM 20459 J. Meiwes i, H.-P. Fiedler i H. H a a g i. H. Z~ihner 1, S. K o n e t s c h n y - R a p p 2 and G . J u n g 2 J Mikrobiologi¢ I. and : Organische Chemic. Unu.¢rMlilt T'~bmgen. "l'tibmgen, F.R.G.
Received 19 September 1989 Revisionrec¢iveA9 October 1989 Accepled l l October 108q Key words: Staphylococcus h.;,ic~t~ DSM 20459, Siderophore. Staphyloferrin
1. S U M M A R Y A highly hydrophilic compound was isolated from low iron culture broth of Staphylococcus hyicus DSM 20459 which exhibits siderophose activity to the producer and 37 other staphylococci. The previously unknown metabolit¢ was designated staphyloferrin A and consists of two molecules of citric acid, each linked to D-ornitine by an amide bond. Using an ion-pair HPLC-system we detected staphyloferrin A and a second iron regulated compound (staphyloferrin B) in the culture fluid of several Staphylococcus strains. We found no evidence that staphylococci synthesize catecholor hydroxamate-type sideropbores.
2. I N T R O D U C T I O N Virtually all aerobe microorganisms have an absolute requirement for iron. Because of the insolubitity of iron under aerobic conditions this
Correspondent~ to: H. Z~ganer,UniversiOt Ttihingen, Biologic
II, Mik~ohiolosie I, Auf tier Morgenstelle 28, D-7400 Ttibingen. F.R.G.
element often is growth limiting. In host tissues iron is tightly hound to macromolecules such as transferrin, thus unavailable for pathogenic microorganisms. High affinity iron uptake systems have been evolved for assuring the survival of microorganisms exposed to these conditions. The best known high affinity uptake system depends on the secretion of iron specific ligands, termed siderophores, and the specific recognition of the corresponding iron complexes by specialized membrane receptors and transport systems. Both components of the system are coordinately induced and expressed according to the iron concentration [1]. For many microorganisms a correlation between siderophore production and virulence has been established [2], however not much is known about the iron supply of staphylococci in contrast to their importance as virulent microorganisms. The growth of Staphylococcus aureus in human serum depends on the iron concentration as shown by Schade [3]. Marcelis et aL [4] and Maskell [5] reported on the secretion of siderophores by staphylococci but no attempts have been made to isolate and identify these siderophores. Recently the expression of cytoplasma membrane proteins by Staphylococcus aureus [6] and Staphylococcus epidermidis [7] under iron limitation has been
0378-1097/90/$03,50 © 1990 Federation of European Microbiological Societies
202 reported. T h e s e p r o t e i n s are p o s s i b l y p a r t of a s i d e r o p h o r e m e d i a t e d iron u p t a k e system. W e h a v e investigated the s i d e r o p h o r e p r o d u c tion o f s t a p h y l o c o c c i u s i n g the c h r o m e a z u r o l S test ( C A S - t e s t ) [8] a n d a n i r o n n u t r i t i o n b i o a s s a y . A n e w s i d e r o p h o r e f o u n d t h e r e b y w a s i s ol a t e d a n d p a r t i a l l y characterized.
3. M A T E R I A L S
AND METHODS
3.1. B a c t e r i a l s t r a i n s Bacterial s t r a i n s u s e d in this s t u d y are listed i n T a b l e 1. 3, 2. C u l t u r e conditions A l l g l a s s w a r e w e r e i n c u b a t e d for 12 h w i t h 10 mM EDTA and rinsed 5 times with double-distilled w a t e r p r i o r to use. T h e s t r a i n s were s t o r e d at -20°C i n B H I m e d i u m w i t h 50% glycerol. T h e p r e c u l t u r e s w e r e g r o w n for 12 h at 3 7 ° C o n G A a g a r m e d i u m [9]. A : n o d i f i e a t i o n of the m e d i u m of C o v e et al. [10] w a s u s e d for the s i d e r o p h o r e p r o d u c t i o n . T h e m e d i u m c o n t a i n e d : {A}: ( m g / I , f i n a l c o n c e n t r a t i o n ) L-alanine, 200; L-arginine, 400; L-aspartic acid, 20; g-cystine, 50: L-Cysteine, 50; L-ghitamic acid 1000; L-glycine, 800; L-histid i n e h y d r o c h l o r i d e , 400; g - h y d r o x y p r o l i n e , 200; L-isoleucine, 100; L-leucine, 200; L-lysine h y d r o c h l o r i d e , 200; L-methionine, 600: L-phertylalanine, 600; L-proline, 200; L-Serine, 4001 L-threonine, 400; L - t r y p t o p h a n , 100; L-tyrosine, 200, L-valine, 200; a d e n i n e , 10; g u a n i n e , 10 a n d uracil 10. {B}: ( m R / I , f in al c o n c e n t r a t i o n ) KH2PO4, 1000; ( N H 4 ) 2 S O 4 , 20~0i HaC1, 4000; M g S O 4 - T H 2 0 , 100.0; C a C I 2 • 5 H 2 0 , 5.0 a n d tricine, 1 7 9 0 0 ; p H 8.5 a d j u s t e d w i t h 3 N N a O H . {C}: g l n f o s e 10 g / L T h e s o l u t i o n s w e r e c o m b i n e d after s t e r i l i z a t i o n a n d 5 m l o f a sterile filtered v i t a m i n s t oc k s o l u t i o n |10] w a s a d d e d . I n o r d e r to p r o v i d e very l o w i r o n c o n t e n t o f the m e d i u m , a five fold c o n c e n t r a t e d s o l u t i o n o f all c o m p o n e n t s e x c l u d i n g d i v a l e n t cations and vitamins was prepared and treated w i t h C h e l e x I 0 0 . S u b s e q u e n t l y the l a c k i n g c o m p o n e n t s were a d d e d , the final v o l u m e w a s a d j u s t e d w i t h d o u b l e - d e s t i l l e d w a t e r a n d the m e d i u m w a s filter sterilized ( M i l l i p a k T M 20, 0,22 p m , M i l l i p o r e , E s c h b o r n , F,R,G.)~
Table l Production of staphyloferrin A and Ig by staphylococct in Chelex 100 trealed medium with and without supplementation of 1 ram D-omithine Strain
with D-omithine staphyloferrln: A B [mg/I]
S. aureus ATCC 12600 S. aureus SA 113 S. aureus KL 207 S. a~etts DSM 799 S~ aureus DSM 203"/2 S. capitis DSM 20326 S. carnosu.v TM 300 S. cohnii DSM 20260 S. epidermidis DSM 1"/98 S. epidermidix DSM 20042 S. epidermidis ATCC 14990 S. epidermidJs DSM 3GgS/T'.'. 3298 S. gall/nahum DSM 4616/Tti 3928 S. haemolyticm DSM 20263 S. haemolytic~ DSM 20265 S. hominis DSM 20322 S. hylrvs DSM 20454 S. intermedius DSM 20373 S. saprophytictts DSM 20229 S. semri DSM 20352 S. simulans DSM 20322 S, sirnvlans MK 148 S, warneri DSM 20036 S. xylos~ DSM 20266 S. aureus 315886 * S. aureus R 253 * S. aureus G 4 * S. aareus G 3 * S. St 653" S . J 22" S. G 4 ? * S. K 105" S, R 2 ? I * S. R 273" S, K S I * S. K 8 6 " S. K 9 6 "
without 13-ornithine A B [mg/I I
147 < 44 38 S8 86 18 14 ]4 60 6 42 31
+ + + +
27
+
4
+
94
< + <
20 7 < < 10 < < 5 < < 2 < < < < < < 34 < 4
< + <
43 14 41 43 33 80 36 6 41 33 48
42 72 72 19 9 tl0 30 28
"t < < <
+ + + ÷
+
<
+
< < < < + + < <
11 < < < < < < <
< < + < < + < <
< < < + 4< + + < + + < < < < + <
< + < + + < + + < < < < < < < ~: <
* Clinical isolates, obtained from F. Schumacher.Pendreau (Universit~t K~ln, Instltul fiir Hysiene, K/~ltt, F.R.G,). + = production of staphyloferrin B; - ffi strain has grown very poor in the production medium; < ffi staphyloferfin not title,table,
F o r c u [ t u r i n g w e u s e d E r l e n m e y e r f l a s k s (100 ml) with one extrusion containing 20 ml medium a n d a 4 - 1 s t i r r e d - t a n k r e a c t o r ( B i o s t a t S, B r a u n ,
203 Melsungen, F.R.G.). The Erlenmeyer flasks were incubated on a rotary shaker (RC I, Infors AG, Basel, Switzerland) operating at 160 rpm and 36°C. For cultivation in the stirred tank reactor the aeration rate was 1 v~am and the stirring rate was 500 rpm. In this case the medium was made without tricine, and during fermentation the pH was maintained at 7.5 _+0.1 with 3 N N a O H and 3 ]q H2SO4. For inoculation 5% of a 12 h preculture in NB medium was used. The broth was harvested and analyzed after 18 h of cultivation.
3.3. Isolation of staphyloferrin A After removal of the cells by centrifugation the supernatant (4 1) was passed through a column (5 × 25 cm) containing Ambedite 401 S (CI-). Non- or weakly adsorbed components were washed out with 1% formic acid (2 1). Staphyloferrin A was eluted with 3 N formic acid (1 1). The volume was reduced to 40 ml in vacuo using a rotary evaporator. The pH of the resultant concentrate was adjusted to 7.0 with 10 N N a O H at 4 ° C . To this solution ethanol was added to a final concentration of 90% ( v / v ) a r d the suspension was stored at 4 ° C for I h. The precipitate was harvested by centrifugation (8000 × g), dissolved in 50 ml destilled water and methanol added to a final concentration of 70% (v/v). The resultant suspension was centrifuged at 8000 × g and the precipitate dissolved in 50 nd destilled water. The methanol precipitation was repeated twice. The siderophore containing supernatants were combined and concentrated in wry=no to 40 ml. This solution (20 ml) was chrumatographed on a column (6 X 70 cm) containing Biogel P2 (200-400 mesh), equifibrated with 10 mM ammonium acetate buffer (pH 4): elution rate was set at 50 m l / h . The staphyloferrin A containing fractions were pooled, concentrated and rechromatographed under the same conditions. Subsequently the sidercphore concentrate was passed through the same column as described above but pH was changed to 5.0. The active fractions were concentrated and chromatographed on a column (2.8 x 40 cm) filled with Fractogel TSK H W 40 (F) (Merck, Darmstadt, F.R.G.) equilibrated with water-methanol 9 : 1. Finally the staphyloferrin A containing fractions were pooled, concentrated in
vacuo and lyophilized to dryness. The yield was about 150 m 8 / I of culture supernatant.
3.4. Bioassay The bioassay was carried out by a modification of the method described by Miles and Khimji [11]. A modified production medium described above was used. The tricine was replaced by TES (50 raM) and the pH adjusted to 7.5 30 mg of deferrated E D D A (ethylenediamine di-(o-hydroxyphenyl-acetic acid)) [12] and 15 g of agar (Bacto) were added to solution B prior to sterilization. 10 ml of the melted agar (45°C) were inoculated with a 12 h preculture (100/~l) of the desired strain in NB medium and poured into a petri-dish. A sterile paper disc, loaded with test solution (20 .~1) was placed on the solidified agar and the plate incubated at 3 7 ° C for 18 h. A halo of bacterial growth surrounds those discs bearing siderophore activity or iron.
3.5. Ion-pair HPLC The chromatographic system consisted of a HP-1090M liquid chromatograph equipped with a diode array detection system and a work station (Hewlett-Packard, Waldbronn, F.R.G.). FeCl 3 (1 mM) was added to the siderophore containing solutions and the samples were cleared by centrifugation at 1 2 0 0 0 × g . 10 /tl-samples of these solutions were injected onto a HPLC column (125 × 4.6 mm i.d.) fitted with a precolumn (20 x 4.6 m m i.d.), each packed with 5 ?~m Shandon Hypersil ODS (Groin, Hersenberg, F.R.G.); detection was at 340 rim. The samples were separated by gradient ehition; solvent A was 10 mM sodiumphosphate buffer (pH 7.3) with 5 m M tetrabutylammonium-hydrogensulfate (TBA), solvent B was aceton;trile. The gradient was from 15 to 28% B in 3 rain, from 28 to 37% B in the following 4 rnin and raised to 60% B in 1 min. The flow-rate was 2 ml/min.
3.6. Characterization of staphylo/errin A A sample of desferri-staphyloferrin A was hydrolyzed with 6 N HCI (12 h, I10°C). Amino acid analysis was carried out according to Fiedler and Plaga [13]. For configurational analysis of the amino acids gas chromatography on the chiral
204 phase Chirasil-Val [14] was used. Citric acid was detected by using an enzyme test kit (Bcehringer, F.R.G.) depending on citrate-oxalacetate-lyase (EC 4.1.3.6).
4. RESULTS A N D DISCUSSION Using the CAS-test [8] we were able to detect iron chelating activity in iron deficient (about 0.5 # M iron) but not in iron sufficient (10 p~M) centrifuged culture broths of some staphylococci. For isolation of the siderophore we have chosen Staphylococcus hyieus DSM 20459 [15]. This strain produced staphyloferrin A very well in the stirred tank reactor in contrast to the other strains which require a very low iron concentration. It is not possible to maintain such low iron concentration in the stirred tank reactor made of steel component parts. On the other hand pretreatment of the medium with Chelex 100 decreased growth and production of the iron chelating activity of Staphylococcus hyicus DSM 20459. Addition of 1 mM D-omithine increased the production of the siderophore (staphyloferrin A) by a factor of 19, as shown in detail elsewhere [16]. The Csaky method [17] and the methods of Arnow [18] and Rioux [19] were used to detect hydroxamates and catechols, respectively, but both were found to be absent in the culture broth of all strains listed in table 1. The iron chelating metabolite of Staphylococcus hyicus DSM 20459 was purified in the iron free form and was obtained as coloudcss powder (ammonium salt). The ferrated staphyloferrin A has a slightly yellow eolour, The U V / V I S spectra recorded during the HPLC-run (Fig. 1) are lacking charge transfer bands between 420 and 500 nm, indicative of iron(Ill) eomplexation by eatechol or hydroxamate type ligands [1]. This rules out the presence of these functional groups in staphyloferrin A. In the hydrolysate of staphyloferrin A citric acid and ornithine were found. Ornithine was shown to possess D-conflguration, The detailed structure elucidation revealed that staphyloferrin A is Na,NS-di-(l-oxo-3-hydroxy-3,4-dlcarbo xylbutyl)-D-ornithine (Fig. 2) |16], a so far unknown metabolite. Thus staphyloferdn A is a new
200
3OO
40O
wavelength
SC0
[nm] Fig. L U V / V I S spectra of staphyloferrin A and B recorded
during a HPLC-run. Inset: HPLC-run of cullure broth from Staphylococcushyic~ DSM 20459 monitored a! 340 nm. Staph A : staphyloferrin A; Slaph B : staphyloferrin B. siderophore of the carboxylate type like rhizobactin [20] and a-hydroxyisovaleric acid [21]. We were able to measure the concentration of staphyloferrin A in the culture broth and during the isolation by ion-pair HPLC. In the culture broth of Staphylococcus hyicus DSM 20459 a second component (staphyloferrin B) was detected by this HPLC-system (Fig. 1), which too was pro. duced only under iron limited conditions. The isolation and characterization of staphyloferrin B is still in progress, but we can exclude the occurfence of cateehol or hydroxamate moieties. The summary in Table ! shows 34 strains producing staphyloferrin A, when supplemented with D-oruithine. Without supplementation only 1| strains were found to produce detectable amounts of the siderophor¢ whereas staphyloferrin B is produced by 18 and 16 strains, respectively. (staphyloferrin B could be measured only qualitatively because so far no pure staphyloferrin B was available for calibrating the HPLC-analysis). COOH
/~HH
~
NH ,0 OH COOH ~C00H
--~iOH D COOH OH
Fig.2.St~c~reo[~aphylo~in A.
205 T h e utilization o f staphyloferrin A by the prod u c e r a n d all other strains, listed in T a b l e 1. was tested by the E D D A bioassay. In all cases growth halos a p p e a r e d a r o u n d p a p e r discs loaded with 50 n m o l of ferrated staphyloferrin A o r 100 nmol of FeC13 as a control. T h i s indicates that the iron transport system d e p e n d i n g on staphyloferrin A as a siderophore is widely spread a m o n g the staphylococci, though not all strains seem to prod u c e staphyloferrin A. T h e role of staphyloferrin A as a virulence factor remains to he investigated as well as the function and structure of staphyloferrin B.
ACKNOWLEDGEMENTS W e t h a n k F. Gi~tz for c o m m e n t s on the manuscript, the D e u t s c h e Forschungsgemeinschaft ( S F B 323) and the Bundesministerium ffir F o r s c h u n g und Technologie ( B C T 05014) for financial support. J.M. was supported by a D E C H E M A grant. S.K.-R. thaks the Studieastiftung des Deutschen Volkes for a doctorate scholarship.
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