Vanadium interferes with siderophore- mediated iron ... - CiteSeerX

Microbiology (2000), 146, 2425–2434

Printed in Great Britain

Vanadium interferes with siderophoremediated iron uptake in Pseudomonas aeruginosa Christine Baysse,1 Daniel De Vos,1† Yann Naudet,1 Alain Vandermonde,1 Urs Ochsner,2 Jean-Marie Meyer,3 Herbert Budzikiewicz,4 Matthias Scha$ fer,4 Regine Fuchs4 and Pierre Cornelis1 Author for correspondence : Pierre Cornelis. Tel : j32 2 3590221. Fax : j32 2 3590390. e-mail : pcornel!vub.ac.be

1

Laboratory of Microbial Interactions, Department of Immunology, Parasitology and Ultrastructure, Flanders Interuniversity Institute of Biotechnology and Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium

2

University of Colorado Health Sciences Center, Microbiology, Box B-175, 4200 E Ninth Avenue, Denver, CO 80202, USA

3

Laboratoire de Microbiologie et de Ge! ne! tique, Universite! Louis Pasteur, UPRES-A 7010, F-67000 Strasbourg, France

4

Institut fu$ r Organische Chemie der Universita$ t zu Ko$ ln, Greinstrasse 4, D-50939 Ko$ ln, Germany

Vanadium is a metal that under physiological conditions can exist in two oxidation states, V(IV) (vanadyl ion) and V(V) (vanadate ion). Here, it was demonstrated that both ions can form complexes with siderophores. Pseudomonas aeruginosa produces two siderophores under iron-limiting conditions, pyoverdine (PVD) and pyochelin (PCH). Vanadyl sulfate, at a concentration of 1–2 mM, strongly inhibited growth of P. aeruginosa PAO1, especially under conditions of severe iron limitation imposed by the presence of non-utilizable Fe(III) chelators. PVD-deficient mutants were more sensitive to vanadium than the wild-type, but addition of PVD did not stimulate their growth. Conversely, PCH-negative mutants were more resistant to vanadium than the wild-type strain. Both siderophores could bind and form complexes with vanadium after incubation with vanadyl sulfate (1 :1, in the case of PVD ; 2 :1, in the case of PCH). Although only one complex with PVD, V(IV)–PVD, was found, both V(IV)– and V(V)–PCH were detected. V–PCH, but not V–PVD, caused strong growth reduction, resulting in a prolonged lag phase. Exposure of PAO1 cells to vanadium induced resistance to the superoxide-generating compound paraquat, and conversely, exposure to paraquat increased resistance to V(IV). Superoxide dismutase (SOD) activity of cells grown in the presence of V(IV) was augmented by a factor of two. Mutants deficient in the production of FeSOD (SodB) were particularly sensitive to vanadium, whilst sodA mutants deficient for Mn-SOD were only marginally affected. In conclusion, it is suggested that V–PCH catalyses a Fenton-type reaction whereby the toxic superoxide anion ON2 is generated, and that vanadium compromises PVD utilization.

Keywords : Pseudomonas, vanadium, siderophores, oxidative stress

INTRODUCTION

Vanadium is a transition metal which, at neutral pH, can exist in two oxidation states, V(IV) (vanadyl ion ; cationic species, VO+), and V(V) (vanadate ion ; anionic species, H VO−) #(Rehder, 1991, 1992). As a # % .................................................................................................................................................

† Present address : Brandwonden Centrum, Militair Hospitaal Koningin Astrid, B-1120 Brussels, Belgium. Abbreviations : CAS, chrome azurol ; EDDHA, ethylenediamine-di(ohydroxyphenylacetic acid) ; ESI, electrospray ion ; PCH, pyochelin ; PVD, pyoverdine ; SOD, superoxide dismutase. 0002-4135 # 2000 SGM

result, vanadium can affect diverse biological processes. Vanadate resembles phosphate (HPO#−), and conse% quently can take its place in phosphate-metabolizing enzymes (Rehder, 1991, 1992). Another important biological characteristic of both vanadate and vanadyl ions is that they can form complexes with carboxylate, catecholate and hydroxamate ligands, which are present in different siderophores (Keller et al., 1991 ; Rehder, 1991). Although this last characteristic is well known, surprisingly, there has been no study done so far on the influence of vanadate or vanadyl ions on the siderophore-mediated iron(III) uptake systems in Gramnegative bacteria. Such an investigation is justified if one 2425

C. BAYSSE and OTHERS

Table 1. Bacterial strains and plasmids used in this study Strain or plasmid Strains P. aeruginosa ATCC 27853 PA6 PAO1 PAO 6609 PAO 66024 PAO 6284

Characteristics

Source of type II PVD Source of type III PVD Wild-type ; source of type I PVD PVD− PCH+ PVD− PCH+ pchA (PVD+ PCH− Sal−)

PAO 6285 PAO 128-5

pchD : Ω (PVD+ PCH− Sal+) pvdB pchD (PVD− PCH− Sal+)

PAO 128-6 PAO sodA : : Gmr PAO sodA : : Gmr ∆sodB : : Tcr FRD1 FRD1 sodA FRD1 sodB

pvdB pchA (PVD− PCH− Sal−) Gmr sodA mutant of PAO1 Gmr Tcr sodAsodB double mutant of PAO1 Wild-type cystic fibrosis isolate sodA mutant of FRD1 sodB mutant of FRD1

FRD1 sodAsodB

sodAsodB mutant of FRD1

E. coli DH5α-MCR

Reference

Meyer et al. (1997) Meyer et al. (1997) ATCC 15692 ; Meyer et al. (1997) Hohnadel et al. (1986) Hohnadel et al. (1986) C. Reimmann (University of Lausanne, Switzerland) Serino et al. (1997) C. Reimmann (University of Lausanne, Switzerland) Serino et al. (1995) This study This study Goldberg & Ohman (1984) Hassett et al. (1997b) D. J. Hassett (University of Cincinnati, USA) Hassett et al. (1997b) Bethesda Research Laboratories

SM10

Host for subcloning of plasmids F− lacZ∆M15 recA1 hsdR17 supE44 ∆(lacZYA argF) Kmr, mobilizer strain

Plasmids pCRII-2.1 pEX100T pBR322 pUC19

Apr Kmr TA cloning vector for PCR fragments Apr oriT mob sacB Apr Tcr, E. coli cloning vector Apr ColE1, E. coli cloning vector

Invitrogen Schweizer & Hoang (1995) Bethesda Research Laboratories Yanisch-Perron et al. (1985)

considers the importance of siderophores for iron acquisition by pathogenic bacteria (Crosa, 1997). Furthermore, it is known that, at neutral pH, V(IV) can be readily oxidized to V(V), generating the toxic superoxide anion (O−) (Liochev & Fridovich, 1987). It has also been # complexation of vanadium by the sideroshown that phore desferrioxamine enhances its redox cycling capacity in vitro (Stern et al., 1992). Despite this, the antibacterial activity of vanadium–siderophore complexes has never been investigated. Under conditions of iron limitation, Pseudomonas aeruginosa produces two siderophores, pyochelin (PCH), a thiazolin derivative (Cox et al., 1981), and pyoverdine (PVD), a fluorescent siderophore, which is composed of a chromophore (a quinoline derivative) and a peptide arm (Budzikiewicz, 1993, 1997). PVD has a much higher affinity for Fe(III) than PCH, which is consistent with PVD-negative mutants being unable to grow in the presence of the strong iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and being avirulent in a mouse infection model (Meyer et al., 1996). The production of siderophores and of the proteins required for their uptake needs to be tightly regulated in order to avoid wastage of 2426

Simon et al. (1983)

energy and accumulation of iron, which can be toxic for the cell because free Fe+ can generate toxic oxygen # radicals via the Fenton reaction. Iron uptake in P. aeruginosa is controlled at different levels via a complex interaction of regulators (Vasil & Ochsner, 1999). The ferric uptake regulator (Fur) is a general regulator, which, together with Fe+ as co# gene, repressor, represses the transcription of the pvdS which encodes a sigma factor needed for the transcription of PVD biosynthetic genes. Fur also controls the transcription of pchR, a gene that encodes an activator for the transcription of the PCH biosynthetic genes. Using an elegant approach, Ochsner & Vasil (1996) found that Fur regulates a large array of genes in P. aeruginosa, including genes for putative siderophore receptors, haem uptake, alternative sigma factors, twocomponent regulatory systems, regulators and other unknown genes. Another gene repressed by Fur is sodA, which encodes the Mn-superoxide dismutase (SOD) of P. aeruginosa (Hassett et al., 1997a). Two SODs are produced by P. aeruginosa, a Mn-SOD induced under low-iron conditions, and an Fe-SOD, which is the predominant form (Hassett et al., 1993). These enzymes catalyse the dismutation of O− to H O (hydrogen # # #

P. aeruginosa and vanadium

peroxide) and O ; the peroxide, in turn, can be converted to H O# and O by catalases. Both reactive # oxygen species #are the product of the Fenton reaction catalysed by free Fe+. Knowing that vanadium can substitute for iron in# siderophores and in some ironproteins such as transferrin (Keller et al., 1991 ; Saponja & Vogel, 1996), we investigated whether vanadyl ions could interfere with siderophore-mediated iron uptake systems and\or induce an oxidative stress in P. aeruginosa. METHODS Strains and growth conditions. The P. aeruginosa strains used

in this study are listed in Table 1. Cultures were grown in Luria–Bertani medium (LB) or in the iron-poor Casaminoacids medium (CAA) (Cornelis et al., 1992). Cultures were grown in a Bioscreen apparatus (Life Technologies) using the following parameters : shaking for 10 s every 3 min ; reading every 20 min ; temperature, 37 mC ; volume of culture, 300 µl. As inoculum, an overnight culture of PAO1 in CAA was diluted in order to achieve a final optical density at 600 nm of 0n0001. Each culture was replicated three times and each experiment was performed in triplicate. Vanadyl sulfate (VOSO ;5H O) was prepared as a 100 mM # Filter-sterilized FeCl (0n1 M stock solution and kept %at 4 mC. $ in 0n1 M HCl) was added to the medium after autoclaving at the concentrations indicated in the text. EDDHA was prepared as a 100 mg ml−" solution (pH adjusted to 9) and filtersterilized. Detection and quantification of PVD in culture supernatants.

PVD was detected in culture supernatants by measuring the absorbance at 400 nm. The concentration of PVD was estimated spectrophotometrically at 400 nm using a molar absorption coefficient, ε , of 2 i 10% M−" cm−" (Ho$ fte et al., %!! 1993) and normalized to the OD value of the culture. '!! To determine the effect of vanadium on PVD production, PAO1 cells were grown for 12 h in CAA containing 0, 0n5, 1n0 or 1n5 mM VOSO and then diluted to start a new culture in % mC using the Bioscreen apparatus. Each CAA for 48 h at 37 experiment was performed in triplicate. The PVD content in the supernatant was determined by measuring A . %!! Chrome azurol (CAS) detection of complex formation between siderophores and vanadium. A vanadium\CAS sol-

ution was prepared by a method adapted from the original protocol described by Schwyn & Neilands (1987). Six millilitres of 10 mM hexadecyltrimethylammonium bromide (HDTMA) was first diluted to 20 ml with water, to which 5n5 ml of 0n5 mM VOSO ;5H O (in 10 mM HCl) was added. # CAS was added slowly under To this solution, 7n5 ml of% 2 mM constant agitation. Finally, 35 ml piperazine solution were added (4n3 g piperazine in 30 ml water and 5 ml pure HCl) and 5-sulfosalicylic acid to a final concentration of 4 mM. The final volume was adjusted to 100 ml. This purple vanadium\ CAS solution could be poured as a gel after addition of 1 % (w\v) agarose. Siderophore and vanadium–siderophore complex purification. PVD (succinamide isoform) from P. aeruginosa

PAO1 was isolated from CAA growth supernatants by the chloroform\phenol method followed by chromatography on CM-Sephadex and elution with 0n1 M pyrimidine\acetic acid buffer pH 5, as previously described (Hohnadel & Meyer, 1988). Eighty micromoles of the pure compound solubilized in

2 ml de-ionized water was supplemented with a slight excess of VOSO ;5H O (100 µmol) and then filtered through a # Sephadex %G25 column (2n5 i 45 cm) eluted with de-ionized water. The major peak showing a brownish colour was collected and lyophilized to obtain the dark brown compound further analysed as a 1 : 1 V–PVD complex. PCH was extracted with chloroform from pH-3-acidified growth supernatants and purified by Sephadex-LH20 chromatography as described by Meyer et al. (1989). Complexation with vanadium was achieved by mixing 90 µmol PCH in methanol solution with 100 µmol VOSO ;5H O. The red-brown complex that formed instantaneously% was #purified by chromatography on Sephadex-LH20 (1n5 i 30 cm column, elution with methanol). Mass spectrometric analysis of vanadium–siderophore complexes. Mass spectra were obtained with a Finnigan MAT

(Bremen) 900 ST instrument equipped with an electrospray ion (ESI) source, an electrostatic and a magnetic analyser, and an ion trap system. Plate assay for sensitivity to paraquat. CAA or LB plates containing 0, 0n5 or 1n0 mM VOSO ;5H O were inoculated % #and dried for 1 h. with 10( cells (1 ml) from pre-cultures Resistance to paraquat was analysed according to Hassett et al. (1995). SOD activity measurements. Total SOD activity present in P.

aeruginosa crude extracts was measured using the pyrogallol auto-oxidation inhibition assay described by Marklund & Marklund (1974). Briefly, 30 µl pyrogallol (20 mM), 30 µl diethylenetriaminepentaacetic acid (DTPA ; 0n1 M) and 100 µl freshly prepared crude cell extract (300 µg total protein) were added to 1n5 ml 0n1 M Tris\cacodylic acid pH 7n8 ; the final volume was adjusted to 3 ml with water. The change in A %#! was monitored at 25 mC for 20 min. In the control autooxidation sample, the crude extract was replaced by 50 mM sodium phosphate buffer (cell lysis buffer ; pH 7). P. aeruginosa cell-free extracts were prepared from overnight cultures in CAA or in CAA containing 0n5 or 1n0 mM VOSO as described by Clare et al. (1984) except that, instead of using% a French press, cells were lysed by sonication. Construction of sodA and sodAsodB mutants of P. aeruginosa PAO1. A 1150 bp fragment containing the sodA region

was PCR-amplified using the primers 5h-GATGTGGCGCTGGAAAACAC and 5h-GCCAGTCGATCACGTTGTAG. The PCR product was cloned into pCRII-2.1 (Invitrogen), and a 1n7 kb gentamicin resistance (Gmr) cassette was cloned into the unique HincII site within the sodA gene. The 3n2 kb sodA : : Gmr construct was excised from the pCRII-2.1 polylinker with PvuII and ligated into the SmaI site of the gene replacement vector pEX100T, which allows sacB counterselection (Schweizer & Hoang, 1995). Escherichia coli SM10 containing pEX100T-sodA : : Gmr was used as the donor strain in a bi-parental mating with P. aeruginosa PAO1. Transconjugants were selected on BHI agar containing gentamicin (75 µg ml−") and irgasan (50 µg ml−"), and subsequently plated on LB agar containing gentamicin (75 µg ml−") and 5 % (w\v) sucrose. Successful double-crossover events resulting in sodA : : Gmr mutants were monitored by the loss of a pEX100T-encoded carbenicillin resistance (Cbr) marker. The insertion of the Gmr cassette into sodA was confirmed by PCR across the sodA : : Gmr region using the primers above. This yielded a 2n8 kb product for sodA : : Gmr mutants compared to a 1n15 kb product for PAO1 wild-type (data not shown). A 1380 bp PCR product containing the sodB region was amplified with primers 5h-TGATGGTGGCGGCCATGATG 2427

C. BAYSSE and OTHERS

and 5h-ATCGCCATTTCCCGGTCGAG and ligated into pCRII-2.1. The PCR product was excised with HindIII and XbaI and cloned into pUC19. A 540 bp NcoI–HincII fragment containing most of the sodB coding region was excised, the ends of the sodB flanking regions were filled in with Klenow enzyme and ligated to a 1n4 kb tetracycline resistance (Tcr) cassette which had been obtained by cutting pBR322 with EcoRI and StyI followed by end-polishing. The 2n6 kb ∆sodB : : Tcr construct was excised from the pUC19 polylinker with PvuII and ligated into the SmaI site of pEX100T. E. coli SM10 harbouring the resulting plasmid, pEX100T∆sodB : : Tcr, was used as the donor strain in a biparental mating with P. aeruginosa sodA : : Gmr. The mating and the subsequent isolation of sodAsodB mutants were performed under anaerobic conditions using Campy Pak jars with palladium catalyst (Becton-Dickinson) in the presence of 1 % (w\v) potassium nitrate as an alternative electron acceptor during anaerobic growth. Candidate sodA : : Gmr sodB : : Tcr double mutants were initially selected on BHI agar containing 1 % (w\v) KNO , tetracycline (150 µg ml−") and irgasan (50 µg ml−"), followed $by sacB counter-selection on LB agar containing 5 % sucrose, 1 % KNO and tetracycline (150 µg ml−"). $ The successful replacement of the sodB gene by the Tcr cassette was verified by PCR across the sodB region using the above primers, resulting in a 2n2 kb PCR product for the sodA : : Gmr sodB : : Tcr double mutant compared to a 1n38 kb product for the sodA : : Gmr single mutant (data not shown). PCR was performed using Taq polymerase and custom-made primers (Bethesda Research Laboratories) in a Perkin-Elmer Cetus thermal cycler, with 30 cycles of denaturing (1 min, 94 mC), annealing (1 min, 54 mC) and extending (1 min per kb of DNA, 72 mC). The PCR products were purified in lowmelting-point agarose gels, routinely cloned into pCRII-2n1 (Invitrogen) and sequenced with Sequenase 2n0 (United States Biochemical) and M13 primers or custom-made 18-mer oligonucleotides.

RESULTS Effect of iron on the growth of P. aeruginosa PAO1 in the presence of vanadium

Table 2 shows the effect of the addition of increasing concentrations of VOSO (1n0, 1n5 and 2 mM) on the % growth of PAO1 under iron-limiting or iron-sufficient conditions. The following parameters were analysed :

the duration of the lag phase, the slope of the exponential phase (OD min−"), the maximal optical density (OD '!! '!! the time to reach the maximal OD , and max) reached, '!! the OD after 48 h of growth. Addition of 10 µM FeCl to the '!! CAA medium resulted, as expected, in strong$ stimulation of growth as judged by the maximal OD '!! reached and the increase in the slope. When iron was present, the addition of increasing concentrations of VOSO resulted in a concentration-dependent increase % phase (up to 35 h for the highest concentration in the lag of 2 mM), but once growth resumed, both the slope and the maximal OD reached were relatively unchanged. '!! iron, increased concentrations of In the absence of VOSO not only caused a prolongation of the lag phase, % decreased the slope and decreased the maximal but also OD . In the presence of 2 mM VOSO , no growth was '!! % observed in CAA medium without added iron, even after 72 h. Effect of Fe(III) chelators on the growth of P. aeruginosa in the presence of vanadium

The effects of adding the strong, non-metabolizable Fe(III) chelator EDDHA, the cognate purified PVD, and a heterologous PVD from P. aeruginosa ATCC 27853 for which PAO1 has no receptor (Hohnadel & Meyer, 1988), on the growth of PAO1 in CAA medium was tested after 24 h. As expected, addition of the cognate PVD (50 µM) in CAA medium stimulated growth (0n49 OD units compared with 0n36 OD units for the '!!control), whilst addition of EDDHA '!! (0n5 mg ml−") CAA or the heterologous PVD (50 µM) had little effect on growth (0n29 and 0n40 OD units, respectively). In the presence of a subinhibitory'!!concentration of vanadium (1 mM), the OD after 24 h (0n33 OD units) was '!!to the CAA control. Strikingly, '!! reduced compared the combination of 1 mM vanadium and EDDHA resulted in complete suppression of growth (0n09 OD units), '!! was an effect which persisted even when incubation extended to 48 h (results not shown). Interestingly, addition of the cognate PVD in the presence of 1 mM VOSO did not stimulate growth (0n22 OD units), % '!! case of whilst addition of the non-cognate PVD, as in the

Table 2. Effect of vanadium on growth of P. aeruginosa PAO1 in CAA medium .................................................................................................................................................................................................................................................................................................................

CAA medium was supplemented (j) or not supplemented (k) with 10 µM FeCl . Results shown are means of five experiments. $ Cells were grown at 37 mC in a Bioscreen apparatus as described in Methods. All cultures received the same initial inoculum. … VOSO4 (mM) … FeCl3 Lag phase (min) Slope (OD min−") '!! OD max '!! Time to OD max (min) '!! OD after 48 h '!! , No growth. 2428

0

1n0

1n5

2n0

V

T

V

T

V

T

V

T

230 3n1i10−% 0n43 3010 0n43

300 3n1i10−$ 1n56 1030 0n95

350 1n4 10−% 0n38 3240 0n38

700 3n8i10−$ 1n36 1580 1n16

1040 0n8i10−% 0n25 3240 0n23

1490 4n2i10−$ 1n35 2420 1n25

  0n09  0n09

2120 3n4i10−$ 1n29 3020 1n25

P. aeruginosa and vanadium

found to be completely unaffected by the presence of 1n5 mM VOSO , a concentration that severely com% promised the growth of the wild-type strain. The PCH− Sal+ and the PCH− Sal− mutants showed an intermediate level of resistance to vanadium. These results led us to conclude that the production of PVD contributes to resistance to vanadium whilst, conversely, the production of PCH enhances the toxic effect of vanadium.

OD600

1·0

0·1

Effect of vanadium on PVD production 0·01

1000

2000

3000

4000

Time (min) .................................................................................................................................................

Fig. 1. Growth of P. aeruginosa PAO1 wild-type ($), and PVD− mutants PAO 6609 (=) and PAO 66024 ( ) in CAA medium containing 1 mM VOSO4. Growth was measured in a Bioscreen apparatus at 37 mC as described in Methods. Results shown are mean values from triplicate cultures.

OD600

1·0

0·1

We wanted to investigate if pre-incubation of the cells with vanadium would affect PVD production since we had observed that the characteristic fluorescence due to PVD production disappeared when cells were grown in CAA medium containing 0n5 mM VOSO . Compared to the control condition (CAA medium, no %VOSO , 100 % PVD production), pre-culturing in the presence% of 0n5, 1n0 and 1n5 mM VOSO for 12 h caused a reduction in % medium of 20, 40 and 42 %, PVD production in CAA respectively, whilst cell growth, as determined by OD , '!! was identical under each condition used. Conversely, PCH production was not affected by pre-exposure of the cells to 0n5 mM VOSO . The PCH content of supernatants from cultures of% the PVD− mutant 6609 grown in CAA was determined by liquid CAS assay (Schwyn & Neilands, 1987) and was found to be identical whether the pre-culture was performed in the presence or in the absence of 0n5 mM VOSO . % Vanadium complexation by PVD and PCH

0·01

1000

2000

3000

4000

Time (min) .................................................................................................................................................

Fig. 2. Growth of P. aeruginosa PAO1 wild-type ($), PVD+ PCH− mutants PAO 6284 (>) and 6285 ( ), and PVD− PCH− mutants PAO 128-5 ( ) and PAO 128-6 (=) in CAA medium and in the presence of 1n5 mM VOSO4. Growth was measured in a Bioscreen apparatus at 37 mC as described in Methods. Results shown are mean values from triplicate cultures.

EDDHA, resulted in complete growth suppression (0n09 OD units). '!! Inhibition of growth of wild-type PAO1 and siderophore-negative mutants by vanadium

Fig. 1 shows the growth curves of wild-type PAO1 and two PVD-negative mutants. These mutants are nonfluorescent under UV, do not produce PVD and are unable to grow in the presence of the Fe(III) chelator EDDHA. These two mutants grew in the presence of 1 mM vanadium only after a prolonged lag phase. Conversely, all mutants defective in the production of PCH showed an increased resistance to vanadium, compared to the wild-type, although to different extents (Fig. 2). The two PVD+ PCH− mutants especially were

Spent medium of PVD-producing strains grown in the presence of vanadium showed a characteristic brown colour that was not observed in spent medium of PVDnegative mutants. Furthermore, the fluorescence under UV, characteristic of free PVD, could not be detected in cultures of P. aeruginosa grown in the presence of 0n5 mM VOSO . This prompted us to investigate % PCH could complex vanadium. For whether PVD and both purified siderophores, a very clear shift of the UVvisible peaks was observed after addition of 1 mM VOSO , an indication that a complex was formed. The % peaks were observed : purified free PVD at following pH 5n2, 365 and 385 nm ; PVD plus VOSO , 405 nm ; purified PCH in methanol, 218 and 248 nm ;% PCH plus VOSO , 232 and 273 nm. Using another approach to detect %vanadium binding by both siderophores, a vanadium-CAS assay was developed whereby iron was replaced by vanadium. Addition of both siderophores to a vanadium\CAS plate resulted in a rapid colour change due to the de-complexation of vanadium–CAS (Fig. 3). Mass spectrometric analysis of the vanadium–siderophore complexes

After binding with vanadium, the PCH– and PVD–V complexes were purified as described in Methods and 2429

C. BAYSSE and OTHERS

OD600

1·0

0·1

0·01

1000

2000

3000

4000

Time (min) .................................................................................................................................................

Fig. 4. Growth of P. aeruginosa PAO1 in CAA ($), CAA plus 125 µM V–PVD complex (#) and CAA plus 50 µM purified V–PCH complex ( ). Growth was measured in a Bioscreen apparatus at 37 mC as described in Methods. Results shown are mean values from triplicate cultures.

.................................................................................................................................................

Fig. 3. Vanadium/CAS plate showing complexation of vanadium by PVDs of P. aeruginosa PAO1, ATCC 27853 and PA6 (top row, left to right), EDDHA (bottom left) and PCH (bottom right). The concentration of chelators was 15 mM and the volume applied was 10 µl. The plate was scanned after 30 min at room temperature.

analysed by ESI-MS. In the case of PVD, a 1 : 1 complex with V(IV) was demonstrated, whilst in the case of PCH, a 2 PCH : 1 V complex was found. Interestingly, for PCH, both V(IV) and V(V) were detected in the complexes. The molecular ion region of the PVD (succinamide side chain)–V complex showed two peaks, viz. m\z 1397, corresponding to [PVDk2H+jVO+] j H+ and m\z 1429, corresponding to [PVDk2H+jVO#+jCH OH] j H+. This is in agreement with the replacement of$ two H+ by V(0). V(IV)O#+ has only four co-ordination sites free to accommodate two of the three bidentate ligands of PVD (catecholate and two N-formyl-N-hydroxyOrn). One site is occupied by the oxygen atom. To complete the octahedral structure, the remaining site binds one molecule of CH OH, the solvent used for the $ ESI measurements. In an aqueous medium it is probably replaced by H O. That the mass difference of 32 units is # CH OH (and not, for example, to O ) actually due to # was confirmed by a$ determination of the exact mass difference between the two ions. The PCH–V complex solution contains mainly (approx. 85 %) ions with the composition [PCHk2H+jVO#+] j K+ (m\z 428) (form 1). Again V(IV)O#+ replaces two H+. The neutral complex 1 is then ionized by attachment of K+. The isotope pattern confirms the composition. A second component (approx. 9 %) corresponds according to its exact mass and isotope pattern to [PCHk2H+jVO +]− j K+ (m\z 483) (form 2). In this case V(V) has been#incorporated. The negative complex 2 needs the attachment of two K+ to give a positive ion 2430

in the mass spectrometer. The last approximately 6 % consisted of the uncomplexed anion of PCH, [PCHkH+]− j 2K+ (m\z 401) (form 3), again confirmed by exact mass measurements and the isotope pattern. As frequently observed in ESI mass spectrometry, cluster ions were also seen, such as [1j3]−j 2K+ (m\z 790), [1j1] j K+ (m\z 817), [1j2]− j 2K+ (m\z 872), [1j1j1] j K+ (m\z 1206) and [1j1j2]− j K+ (m\z 1261). Effect of V–PVD and V–PCH complexes on growth of P. aeruginosa PAO1

Purified vanadyl-siderophores were tested for their inhibitory effect on growth of P. aeruginosa PAO1 in CAA medium. V–PVD up to 125 µg ml−" did not affect the growth of PAO1, whilst, conversely, V–PCH had a very striking inhibitory effect on growth (Fig. 4). Addition of V–PCH resulted in a prolongation of the lag phase that lasted for more than 50 h before growth resumed. Effect of vanadium on resistance of P. aeruginosa to paraquat

The strong inhibitory activity of V–PCH, and the fact that both V(IV) and V(V) were detected in association with PCH, were strongly suggestive of V–PCH undergoing a redox cycle which could result in the generation of toxic superoxide anions (O−), as already described for # 1990 ; Britigan et al., ferripyochelin (Coffman et al., 1997). On the other hand, it is well known that V(IV) can auto-oxidize in neutral aqueous solutions, yielding O− and vanadate (Liochev & Fridovich, 1987). Cells # grown in the presence of VOSO were found to be more % resistant to the superoxide-generating agent paraquat, as expressed by the decrease in the diameter of the growth inhibition zone caused by paraquat on CAA or LB plates (Table 3). This resistance increased as a function of the concentration of vanadium present in the

P. aeruginosa and vanadium Table 3. Effect of pre-incubation with VOSO4 on the sensitivity of P. aeruginosa PAO1 to paraquat .....................................................................................................................................................................................................................................

The diameter of the growth inhibition zone around a drop of paraquat on a Petri plate (Hasset et al., 1995) was measured after 18 h (LB) or 48 h (CAA) incubation at 37 mC. Values are means (p0n2) of three separate experiments. Medium

Pre-incubation with 0n5 mM VOSO4

No VOSO4

VOSO4 (0n5 mM)

VOSO4 (1 mM)

– j – j

2n4 2n0 1n9 1n4

2n0 2n0 1n6 1n2

1n3 1n3 1n3 1n3

CAA CAA LB LB

Table 4. Effect of pre-incubation with VOSO4 on the growth of P. aeruginosa PAO1 in the presence of VOSO4

1·0 (a)

.................................................................................................................................................

Cells were grown overnight in CAA containing 1 mM VOSO % and 100 µM FeCl at 37 mC and diluted to inoculate cultures in $ the Bioscreen apparatus as described in Methods. Results are shown only for cultures grown in CAA plus FeCl (100 µM). $ Values are means of five experiments.

0 0 1n0 1n0 1n5 1n5

Induction with 1 mM VOSO4

Lag phase (min)

OD600 min−1

– j – j – j

220 220 240 240 280 280

4n3i10−$ 4n3i10−$ 2n1i10−$ 3n4i10−$ 1n2i10−$ 1n7i10−$

0·01

1000

2000

3000

4000

OD600

VOSO4 (mM)

0·1

1·0 (b)

medium. Pre-incubation of the cells with 0n5 mM VOSO also caused a decrease in the growth inhibition caused% by paraquat. In addition, we noticed that on CAA plates containing 1 mM VOSO a ring of increased % paraquat growth growth was visible at the limit of the inhibition zone (not shown). Altogether, these results strongly suggest that vanadium can induce an oxidative response and a defence mechanism in P. aeruginosa. Since both paraquat and vanadyl ions can generate superoxides, it is most likely that vanadyl ions can induce a SOD activity in P. aeruginosa. To test this hypothesis, total SOD activity was measured by the inhibition of the auto-oxidation of pyrogallol in crude extracts of P. aeruginosa PAO1 cells before and after pre-incubation with 0n5 mM VOSO . The total SOD % before and 20 % activity in the conditions used was 10 % after exposure to VOSO (expressed as percentage of % inhibition of pyrogallol auto-oxidation). Such a doubling of SOD activity was consistently observed, confirming that vanadyl ions indeed induce an oxidative stress and an adaptive response in P. aeruginosa. Pre-exposure of bacterial cells to subinhibitory vanadium concentrations induced a higher level of resistance, resulting in an increased growth rate, but

0·1

0·01

500

1000

1500

2000

2500

3000

Time (min) .................................................................................................................................................

Fig. 5. (a). Growth of P. aeruginosa PAO1 wild-type in CAA ($), sodA mutant in CAA ( ), sodAsodB mutant in CAA (>), PAO1 wild-type in CAA plus 1 mM VOSO4 (#), sodA in CAA plus 1 mM VOSO4 ( ) and sodAsodB mutant in CAA plus 1 mM VOSO4 (=). (b) Growth of wild-type strain FRD1 ($), FRD1 sodA (#), FRD1 sodB (=) and FRD1 sodAsodB ( ) in CAA plus 1 mM VOSO4. Growth was measured in a Bioscreen apparatus at 37 mC as described in Methods. Results shown are mean values from triplicate cultures.

only when iron was present during the pre-culture and the culture (Table 4). Similar results were obtained in LB medium (results not shown). 2431

C. BAYSSE and OTHERS

We also observed that PAO1 cells plated on P-agar containing 1 mM VOSO produced more pyocyanin % than on P-agar alone, an indication that the level of FeSOD might be elevated in the cells exposed to vanadium (Hassett et al., 1995). Effect of mutations in sodA and sodB genes on the resistance of P. aeruginosa to vanadium

P. aeruginosa produces two SODs, one Mn-co-factored (SodA), and another Fe-co-factored (SodB) (Hassett et al., 1993). As already mentioned, resistance to vanadium was higher in the presence of iron (Table 2), indicating that SodB could be the major contributor to the resistance since the production of this haem-containing enzyme was shown to be increased under conditions of iron sufficiency (Hassett et al., 1993). Furthermore, it is known that SodB contributes more to resistance to superoxide induced by paraquat than SodA (Hasset et al., 1995). SOD mutants from PAO1 and a cystic fibrosis strain, FRD1 (Hassett et al., 1997b), were grown in CAA or in CAA containing 1 mM VOSO . The results are shown % in Fig. 5(a) for strain PAO1 (wild-type, and sodA and sodAsodB mutants), and in Fig. 5(b) for strain FRD1 (wild-type, and sodA, sodB and sodAsodB mutants). The growth of both sodA mutants (PAO1 and FRD1) was relatively unaffected by the presence of 1 mM VOSO compared to the respective wild-type strains. % the growth of the sodB (FRD1) mutant was However, strongly inhibited in the presence of vanadium. The double sodAsodB mutants grew either very poorly (in the case of FRD1) or not at all (PAO1). These results again indicated that the resistance to vanadiumgenerated superoxides is largely due to SodB. DISCUSSION

One of the main concerns for the treatment of bacterial infections is the increasing proportion of antibioticresistant strains. Metals, such as silver and cerium, are already used in topical applications because of their antibacterial activity (Klasen, 2000). Vanadium salts are known to exert, among others, anti-diabetic effects (Srivastava, 2000). Few reports mention the antibacterial effects of vanadium salts. In the case of Streptococcus pneumoniae, both vanadate and vanadyl compounds were found to have a strong antibacterial activity (Fukuda & Yamase, 1997). Although these authors found that vanadium salts had a negligible activity towards other bacteria, including Staphylococcus aureus, E. coli and P. aeruginosa, it is important to mention the fact that they grew these bacteria only in the iron-rich Mueller–Hinton medium. In this study, we demonstrate that vanadium salts inhibit the growth of P. aeruginosa, especially in an iron-limited medium. In an iron-replete medium, we observed that vanadium caused a prolongation of the lag phase without affecting the exponential phase. Conversely, under conditions of iron limitation, the lag phase was less affected by the presence of vanadium in the medium whilst the growth rate was 2432

severely reduced. Although we do not at present have an explanation for this phenomenon, it suggests that vanadium can affect different functions depending on the iron content of the cell. The inhibitory effect of vanadium was strongly enhanced by the presence of non-utilizable iron(III) chelators such as EDDHA or a heterologous, noncognate PVD. This could mean that vanadium interferes with siderophore-mediated iron uptake in P. aeruginosa. One way by which vanadium could affect iron uptake is via the formation of stable vanadyl– or vanadate– siderophore complexes that, eventually, could be taken up by the cell, increasing the intracellular concentration of vanadium. Alternatively, the vanadium–siderophore complexes could block the uptake of iron–siderophores in a competitive way. The competition of vanadium with iron for binding by siderophores could explain why the uptake of iron via the siderophores is compromised. However, we do not at this stage know whether the vanadyl–siderophores are actively taken up by the cells. We demonstrate here that vanadium can indeed form complexes with the two siderophores of P. aeruginosa, PCH and PVD. This is not surprising since it is well established that vanadium can be liganded by hydroxamates, carboxylates and catecholates (Keller et al., 1991 ; Rehder, 1991). Others have also demonstrated the fact that PVD, as well as PCH, can complex copper, zinc or manganese (Poppe et al., 1987 ; Chen et al., 1994 ; Visca et al., 1992 ; Bouby et al., 1999). Visca et al. (1992) suggested that PCH could play a role in the acquisition of metals other than Fe(III), such as Co(II) and Mo(VI). Likewise, azotochelin, a catecholate siderophore, has been suggested to participate in the uptake of molybdenum by Azotobacter vinelandii (Duhme et al., 1998). Here we show that the V–PCH complex, but not the V–PVD complex, has a strong antibacterial effect. Such an effect was not observed by Visca et al. (1992) for PCH complexed with other metals, including Mo(VI) and Cu(II). Interestingly, two forms of V–PCH complexes were found, one with V(IV) and one with V(V). This result suggests that V–PCH can undergo an oxidative cycle that can result in the generation of superoxide radicals (O−). It is well known that ferripyochelin can # a redox cycle, resulting in the production undergo such of cell-damaging active oxygen species (Coffman et al., 1990 ; Britigan et al., 1997). Stern et al. (1992) also demonstrated that V(IV)–desferrioxamine has a redoxcycling activity and generates active oxygen species. In another study on A. vinelandii siderophores, Cornish & Page (1998) demonstrated that, among the three catecholate siderophores produced by this bacterium, only aminochelin, which has the lowest affinity for iron(III), was unable to sequester iron and prevent a Fenton reaction. Interestingly, the same authors also observed that increased aeration resulted in a parallel increase in the production all three catecholate siderophores of A. vinelandii, aminochelin, azotochelin and

P. aeruginosa and vanadium

protochelin. Protochelin is a tri-catecholate with the highest affinity for iron and is produced only under conditions of extreme oxygen stress. Like protochelin, PVD from P. aeruginosa can form a 1 : 1 complex with iron and protect the cells from oxidative damage due to the Fenton reaction (Coffman et al., 1990). Indeed, these authors showed that O− could reduce and release iron # Fe–PVD. bound to Fe–PCH but not The fact that PVD-negative mutants are more affected by vanadium can be explained by their higher production of PCH (Ho$ fte et al., 1993), which results in an increase in the toxicity of vanadium. Another intriguing observation we report is the repression of PVD production when wild-type cells were grown in the presence of vanadium. We can assume that vanadium did not interfere with the normal Furmediated regulation since iron-repressed outer-membrane proteins are normally produced in CAA medium containing vanadium and their production is repressed when iron is also present (results not shown). We propose that V–PCH molecules undergo an oxidative cycle, resulting in the production of reactive oxygen species that, in turn, induce a response in the form of increased SOD activity. We observed that a preincubation with vanadium salts increases the resistance of PAO1 cells both to vanadium and to the redoxcycling agent paraquat, and causes an increase in SOD activity. Analysis of sod mutants indicated that MnSOD, encoded by sodA, only marginally participates in the resistance towards vanadium in iron-limiting conditions, whereas SodB plays a major role. SodA is known to be regulated by the Fur repressor (Hassett et al., 1996, 1997a), whilst not much is known about the regulation of SodB production in P. aeruginosa except that its production is optimal under iron-sufficient conditions (Hassett et al., 1992). The pronounced growth inhibitory effect of vanadium on iron-starved cells could be the result of insufficient production of the iron-containing superoxide-detoxifying enzyme SodB under these conditions. Further studies should include experiments designed to investigate whether vanadium uptake by the cells is increased by siderophores. Also, the search for vanadium-resistant or vanadium-susceptible mutants in P. aeruginosa should provide useful information about the mechanisms involved in the homeostasis of this metal, and the interaction between siderophores and oxidative stress in this bacterium.

ACKNOWLEDGEMENTS We thank Dr Cornelia Reimman for sending us the PCH mutants, and Dr Dan Hassett for sending us the FRD1 strain and the different sod mutants as well as for interesting discussions. We also thank Dr Theresa Pattery for carefully reading this manuscript. The Bioscreen apparatus was acquired thanks to the FWO (krediet aan navorser). This work was also supported by the Jean and Alphonse Forton Fund.

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Received 23 March 2000 ; revised 20 June 2000 ; accepted 29 June 2000.