Effects of Siderophores on the Growth of Pseudomonas Aeruginosa in Human Serum and Transferrin ROBERT ANKENBAUER, SOMPORN SRIYOSACHATI, and CHARLES D
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INFECTION AND IMMUNITY, JUlY 1985, p. 132-140 Vol. 49, No. 1 0019-9567/85/070132-09$02.00/0 Copyright C 1985, American Society for Microbiology Effects of Siderophores on the Growth of Pseudomonas aeruginosa in Human Serum and Transferrin ROBERT ANKENBAUER, SOMPORN SRIYOSACHATI, AND CHARLES D. COX* Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Received 15 October 1984/Accepted 27 March 1985 A combination of the siderophores produced by Pseudomonas aeruginosa, pyochelin and pyoverdin, dramatically stimulates the growth of this bacterium in medium containing human transferrin. The amount of growth stimulation observed when each siderophore was added alone was only slighly less than the amount observed with the combination. Siderophore-defective mutants of strain PAO1 were isolated to test the effects of siderophore production on growth in transferrin and human serum. The pyoverdin-proficient (Pvd+), pyochelin-deficient (Pch-) strain (IA5) grows just as well as the parent (PAO1), which produces both siderophores. On the other hand, the Pvd- Pch+ strain (211-5) has severely retarded growth, similar to that demonstrated by a mutant lacking production of both siderophores (IA1), but has an accelerated log phase compared with strain IAl at the later stages of the growth curve. However, the Pvd- Pch+ strain (211-5) had no observable advantage over the Pvd- Pch- strain, IAl, during incubation in human serum. The inability of P. aeruginosa strains to produce pyochelin in glucose-minimal medium may explain the poor growth of 211-5 in this medium and in human serum. The 211-5 strain grows much better than the IAl strain in the medium that allows pyochelin synthesis, but it still does not grow as well as the Pvd+ Pch- strain (IA5). Therefore, pyoverdin appears to be the most important siderophore for growth in human serum. Pseudomonas aeruginosa must be able to grow in mam- not known to exist for these siderophores or for enterochelin malian tissue to infect humans. Growth, in turn, is depend- and aerobactin. In all of these examples it is believed that ent upon the bacterial acquisition of iron from mammalian multiple siderophores are produced, because one of the iron-binding proteins (3). One of the most important iron- siderophores is critical for growth under severe iron depriva- binding proteins in host defense is transferrin (1). The most tion. In the case of E. coli, the siderophore that allows common mechanism by which bacteria compete with invasiveness is not the one possessing the highest binding transferrin for iron is through the activity of siderophores, coefficient. Therefore, siderophore activities in the presence bacterial products which bind iron and function in high-af- of ferritransferrin must be determined experimentally. finity iron transport (19). In this report we describe the The value of two siderophores to P. aeruginosa is not relative abilities of two siderophores, pyochelin (9, 10, 16) obvious. Pyochelin contains a salicyl ring bonded to a and pyoverdin (8, 17, 22, 31), to stimulate growth and iron thiazoline ring, which is, in turn, bonded to a terminal accumulation by P. aeruginosa during incubation with hu- N-methylthiazolidine ring (10). The structure of pyoverdinpa man transferrin or human serum. Two bacteria that produce from P. aeruginosa, reported by Wendenbaum et al. (31), is more than one siderophore, Escherichia coli and Azotobac- very similar to pseudobactin (29) and pyoverdinpf (23). It ter vinelandii, have been studied to some length. Certain contains a dihydroxyquinoline moiety (the chromophore), invasive strains of E. coli have been found to produce both and two N-hydroxyornithine residues, components which enterochelin (20) (also called enterobactin [24]) and are common to both pseudobactin and pyoverdinpf (8). Six aerobactin (2, 13, 28, 30). The expression of aerobactin has additional amino acids complete the structure. To under- been correlated with increased growth of E. coli in serum stand the effects of these two siderophores, mutants were (13, 27) and with the presence of a ColV plasmid (2, 28, 30). isolated with various synthetic capabilities for siderophores. The other bacterium, A. vinelandii, produces dihydroxyben- The growth capabilities of these mutants revealed that zoic acid, azotochelin, and azotobactin. Azotochelin is N,N'- pyoverdin synthesis is most important for growth of P. bis-(2,3-dihydroxybenzoyl)-L-lysine (6), and azotobactin is a aeruginosa in human transferrin or serum. Although dihydroxyquinoline bound to an octapeptide (12). pyochelin has impressive effects when added to these media, Siderophore synthesis is controlled in a sequential manner, it was synthesized sparingly in glucose-minimal medium progressing from dihydroxybenzoic acid to azotochelin and (GMM) and in human serum. Therefore, bacteria demon- then to azotobactin (21) and is governed by the increasing strated little growth advantage from pyochelin in these severity of iron limitation. Therefore, the organism appears media. to conserve energy in siderophore synthesis, expending it for the most complex, but most effective, siderophore, azotobactin, when it is essential. There is another example MATERIALS AND METHODS from a recent investigation in which Pseudomonas fluorescens produces both pyoverdinpf and ferribactin (17, Culture media. The medium for siderophore production, 22, 23). Pyoverdinpf is thought to be the most effective of the CAA, contained 0.5% Casamino Acids and 0.4 mM MgSO4. two, but the ferribactin is produced late in culture. There- Other minimal media contained 5 mM potassium phosphate fore, sequential synthesis dependent upon iron demand is buffer (pH 7.4), 5 mM K2SO4, 40 mM NH4Cl, and 0.5 mM MgSO4. Oxidizable substrates, arginine (AMM for arginine minimal medium) or glucose, were added to 20 mM. When * Corresponding author. arginine was used, the NH4Cl was omitted. These media 132 VOL. 49, 1985 EFFECTS OF SIDEROPHORES ON GROWTH OF P. AERUGINOSA 133 were also made into solid media by the addition of 1.5% agar. Chemical Co.) and was mixed with excess 55Fe in 40 mM To institute more stringent iron-limiting conditions, 0.9% Tris-hydrochloride and 20 mM sodium bicarbonate (Tris-bi- Gelrite (Kelco, San Diego, Calif.) was added to broth with 5 carbonate buffer, pH 7.4) to make 100% saturated mM MgSO4 to make solid medium. [55Fe]transferrin by the methods of Simonson et al. (26). This Isolation of mutants. P. aeruginosa PAO1 (ATCC 15692) mixture was dialyzed exhaustively against the same buffer was obtained from the American Type Culture Collection, until no 55Fe appeared in the dialysate. This iron substrate and mutants were derived from this parent strain. Mutagen- was added to culture medium at 6.8 p.g/ml and contained 0.21 esis was carried out with ethyl methanesulfonate by the ,uCi of 55Fe per ml. Bacterial accumulation of iron from procedure of Lin et al. (15). Strain PAO1 was grown in 1% culture medium was measured by passing 1-ml samples of tryptic soy broth (TSB) to an absorbance of 0.7 (600 nm). culture medium through 0.45-p.m pore size filters. The filters The cells were centrifuged from suspension, washed, and were washed with water and dried, and the amounts of iron suspended in 5 ml of the same medium. After 30 min of were determined by scintillation counting. Purified incubation at 37°C, 0.2 ml of ethyl methanesulfonate was siderophores were added to this medium at 10 p.g/ml, ap- added for 60 min. This procedure resulted in a 105-fold proximately the concentrations that could be found in CAA reduction in the number of viable cells. The surviving cells medium culture filtrates (7). Bacteria, prepared for inoculum were washed in sterile, distilled water and suspended in 1% by three passes through CAA culture medium, were washed TSB for overnight incubation in the dark. In some cases three times in water and added to medium at approximately selection was carried out in 0.5% CAA medium with sequen- 5 x 103 CFU/ml. Growth was assayed by measuring absorb- tial cycles of selection with D-cycloserine followed by ance at 600 nm or by diluting culture medium and determin- growth in 1% TSB (4). Siderophore-deficient mutants were ing viable bacteria by plate counts. Viable bacteria in sus- selected against on plating media containing 100 ,uM pensions were counted on agar surfaces (1% tryptic soy agar ethylenediamine-di-(o-hydroxyphenylacetic acid) and on or 1% peptone agar) after 24 h of incubation at 37°C. media containing 200 pLM ethylene glycol-bis(P-aminoethyl Siderophore purification and analysis. Pyochelin was ex- ether)-N,N-tetraacetic acid. Colonies that failed to grow in tracted from media into dichloromethane containing 10% the presence of selective agents, but grew on nonselective acetic acid (mixed 1:2 with medium). Pyochelin was purified media, were plated on 0.5% CAA agar medium and were on consecutive, preparative thin-layer plates as described observed under fluorescent lamps to screen for nonfluores- previously (9). The first silica thin-layer plate was developed cent colonies. One such colony, 211-5, has been determined in chloroform-acetic acid-ethanol (90:5:2.5), and the second to be pyoverdin defective (Pvd-), but proficient in pyochelin plate was developed in the same solvent in a 90:5:5 ratio. synthesis (Pch+). Strain IAl, Pvd- Pch-, was isolated after Pyochelin was eluted from the silica in dichloromethane, and ethyl methanesulfonate mutagenesis of strain 211-5 as a this solution was filtered and taken to dryness for determina- minute colony appearing on AMM agar containing 200 ,uM tion of the dry weight of the product. Screening mutants ethylene glycol-bis(3-aminoethyl ether)-N,N-tetraacetic involved the extraction of 5 ml of spent culture medium and acid after incubation at 37°C for 72 h. Each colony suspected chromatography of concentrated extracts on silica thin-layer of being a mutant was inoculated into 5 ml of CAA medium plates (Eastman Kodak Co.) in chloroform-acetic acid to be tested for siderophore synthesis.