Departamento de Biología Molecular (Unidad Asociada al Centro de Investigaciones Biológicas, CSIC), Facultad de Medicina, Universidad de Cantabria, Cardenal Herrera Oria s/n, 39011-Santander, Spain1
Author for correspondence: Manuel I. González Carreró. Tel: +34 42 201943. Fax: +34 42 201945. e-mail: carrerom{at}unican.es
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ABSTRACT |
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Keywords: iron acquisition, catechol, macrophage, 2,3-DHBA
Abbreviations: 2,3-DHBA, 2,3-dihydroxybenzoate; CAS, chrome-azurol-S; DIP, 2,2-dipyridyl; EDDA, diethylenediamine di(o-hydroxyphenylacetic acid); NTA Fe(III), ferric nitriletriacetate
The GenBank accession number for the sequence reported in this paper is AF361942.
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INTRODUCTION |
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Brucella abortus is an intracellular parasite able to proliferate within macrophages, thereby successfully avoiding the bactericidal effects of phagocytes. Our knowledge about the mechanisms used by B. abortus for its intracellular survival has improved considerably in the last few years. Recent findings indicate that B. abortus is able to survive and replicate in phagosomes of the phagocytic cells by preventing the fusion between phagosomes and lysosomes (Frenchick et al., 1985 ; Pizarro-Cerda et al., 1998
).
When grown under iron-limited conditions, B. abortus secretes 2,3-dihydroxybenzoate (2,3-DHBA), a simple catecholic compound. 2,3-DHBA was considered the only B. abortus siderophore responsible for Fe55 uptake and able to relieve the growth inhibition caused by the strong iron chelator EDDA (Lopez-Goñi et al., 1992 ). Production of other catecholic or hydroxamate siderophores in Brucella has not yet been reported. The role of 2,3-DHBA in protecting Brucella from the antibacterial activity of macrophages has been studied by the addition of exogenous 2,3-DHBA to B. abortus-infected murine macrophages in culture. In these experiments the number of intracellular brucellae recovered from 2,3-DHBA supplemented cells was higher than that of bacteria recovered from untreated macrophages (Leonard et al., 1997
). This observation suggests that production of this catechol could provide intracellular brucellae with an efficient mechanism for acquiring iron in an iron-restricted environment that exists within the phagosome, thereby contributing to bacterial proliferation in the host. However, a recent report indicated that production of 2,3-DHBA was not required for replication of Brucella in murine macrophages or for the establishment of a chronic infection in the BALB/c mouse model (Bellaire et al., 1999
).
In this work we report the isolation and characterization of a mutant B. abortus strain which, unlike the parental strain, was siderophore-negative in chrome-azurol-S (CAS) plates and unable to grow in iron-restricted medium, although it secreted 2,3-DHBA. The growth restriction observed under these conditions was overcome by the addition of iron in a soluble form or of a catecholic extract from culture supernatants of the parental strain grown in the presence of subinhibitory concentrations of EDDA. The mutant strain altered in siderophore production was deficient in iron assimilation but did multiply in macrophages.
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METHODS |
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Isolation of mutants defective in siderophore production.
pSUP2021, an ampicillin-resistant plasmid containing Tn5, and unable to replicate in Brucella, was introduced into B. abortus 2308 by conjugation with E. coli S17-1(pSUP2021) as described previously (Sangari & Agüero, 1991 ). Transconjugants containing Tn5 were initially selected on BA containing nalidixic acid and kanamycin and purified on MMB plates with the same antibiotics. Isolated colonies from MMB plates were transferred to the blue CAS medium to screen for siderophore-deficient mutants. Colonies that were negative in this assay were tested for susceptibility to ampicillin to confirm the loss of the plasmid and were further tested for their growth on MMB plates containing different concentrations of the iron chelator EDDA.
Growth of B. abortus in iron-deprived medium.
B. abortus strains were cultured at 37 °C in liquid MMB medium containing different concentrations of EDDA. Samples were taken at different time intervals and growth was determined by measuring A590. In all these experiments the glass material was previously washed overnight with a 1 M HCl solution followed by three washes with ultrapure water (18 M) to remove traces of iron.
Bioassays for siderophores.
The presence of siderophore was detected with a cross-feed assay, consisting of checking the growth of siderophore-negative mutants in iron-deprived solid medium around filter disks containing different substances. When the strain to be cross-fed was E. coli AN193 entA, half strength TSB plates containing 150 µM DIP were used, and in the case of BAM41 the medium used was MMB agarose with 150 µM EDDA. In both cases cells from 1 ml early-stationary-phase low-iron cultures were collected by centrifugation, resuspended in 10 µl sterile saline and seeded onto the indicator plates. Five microlitres of the supernatants were spotted onto a Millipore filter (0·22 µm) and allowed to dry before the disk was applied to the plate. The plates were then checked for the presence of a halo of growth after 4872 h at 37 °C. To assay ethyl acetate extracts or pure dissolved substances, they were placed on sterile filter paper disks and dried for 10 min in the dark at room temperature to allow the solvent to evaporate. The size of the halo of growth represents the amount/strength of the siderophore present in the sample.
Preparation of ethyl acetate extracts and analysis by TLC.
Early-stationary-phase B. abortus cultures in MMB were centrifuged (4000 r.p.m. for 10 min at 4 °C) and the supernatants were filter-sterilized through Millipore 0·22 µm filters. Quantification of catechols in these supernatants was carried out by the colorimetric test of Arnow (1937) , using 2,3-DHBA as control. Catechol-containing supernatants were acidified to pH 2·0 by addition of 6 M HCl and catechols were extracted by addition of ethyl acetate (20 ml per 100 ml supernatant). The extracts were pooled and concentrated at low temperature in a vacuum system.
TLC of ethyl acetate extracts was carried out on silicagel G plates (Merck). Chromatograms were developed with benzene/acetic acid/water (125:72:3, by vol.). 2,3-DHBA and its derivatives were first detected by fluorescence under UV light. The presence of iron-reacting substances was revealed by spraying the plate with 0·12 M FeCl3 in 0·1 M HCl. When required, the compounds were recovered from the chromatograms by elution in aqueous acetic acid (15%, v/v), the resulting solutions were made up to 0·5 M HCl and the compounds were extracted with ethyl acetate.
Recombinant DNA techniques.
Chromosomal DNA from B. abortus strains was extracted by the guanidium thiocyanate method (Pitcher et al., 1989 ). Construction of plasmids, restriction enzyme analysis, agarose gel electrophoresis and Southern hybridizations were carried out by standard protocols (Sambrook et al., 1989
). A 3·5 kb HindIII fragment from Tn5 was used as a hybridization probe and labelled with digoxigenin with the DIG-High Prime Kit (Roche Diagnostics). The DNA sequence flanking transposon mutants was cloned using arbitrary PCR (Caetano-Annoles, 1993
), as described by OToole & Kolter (1998)
, and sequenced. Database searches and alignments were performed at the National Center for Biotechnology Information (NCBI), using the BLAST network service.
Infection and intracellular viability assay of B. abortus in J774 cells.
J774 murine macrophages were cultured in RPMI medium with 2 mM L-glutamine, penicillin (100 U ml-1), streptomycin (0·1 mg ml-1) and 10% fetal calf serum (FCS) at 37 °C under 5% CO2 and 100% humidity. Confluent monolayers were trypsinized and 2x105 cells per well were incubated for 24 h before infection in 24-well tissue culture plates. B. abortus cells used for infection were opsonized with decomplemented antiserum for 30 min at 37 °C. Infection of macrophages was carried out for 45 min at 37 °C in RPMI without serum and antibiotics at a multiplicity of infection of 100. After infection the wells were washed five times with sterile phosphate-buffered saline (PBS) and further incubated in RPMI with 2 mM L-glutamine, 10% FCS and 40 µg gentamicin ml-1 to kill extracellular bacteria. The number of intracellular viable B. abortus was determined at different times post-infection. To this end, the monolayers were washed twice with sterile PBS and treated for 5 min with 1 ml 0·1% Triton X-100 (Sigma) in deionized water. Lysates were serially diluted and plated onto BA plates for determination of c.f.u.
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RESULTS |
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Catechols were extracted from supernatants with ethyl acetate, concentrated and analysed by TLC. This analysis indicated the presence of a catechol molecule (Arnow-positive) with an Rf identical to that of 2,3-DHBA both in the mutant BAM41 and in the parental strain, 2308. Strain 2308 presented, in addition, a second Arnow-positive spot that exhibited a lower Rf and was less abundant than 2,3-DHBA (Fig. 3).
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Phenotypic complementation of mutant BAM41
To further characterize the Brucella siderophore-deficient mutant, we tried to restore its growth in EDDA-chelated MMB medium with different substances spotted on filters placed on the agar surface. 2,3-DHBA (3 µl, 10 mM) was unable to restore the growth of mutant BAM41. Ethyl acetate extracts from B. abortus 2308 culture supernatants, but not from BAM41, did complement the growth defect in the mutant. The two catechols in the 2308 supernatant were separately purified from a TLC plate and used in the complementation assay. Only the slow-migrating catechol was able to restore the growth of the mutant (Table 2).
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DISCUSSION |
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The appearance of large amounts of 2,3-DHBA or salicylic acid in culture supernatants is a phenomenon commonly observed for bacteria producing phenolate siderophores in response to iron deprivation. However, those compounds are considered to be low-affinity siderophores (Actis et al., 1986 ), unable to compete with stronger iron-chelating compounds. Moreover, recent theoretical studies do not consider 2,3-DHBA to be capable of acting as a bacterial siderophore (Chipperfield & Ratledge, 2000
). These facts question the role of 2,3-DHBA as a siderophore in B. abortus and suggest that Brucella either produces a stronger siderophore, different from 2,3-DHBA, or it uses a different iron assimilation system.
We have observed that B. abortus 2308 was able to decolorize CAS plates, indicating that this strain secretes a siderophore. To discover the role of this siderophore in intracellular survival we have isolated a Tn5 mutant of B. abortus 2308, named BAM41, which was negative in the CAS assay and thus considered to be siderophore-deficient. The minimal inhibitory concentration of EDDA for this mutant was lower than that of the parental strain, 2308. The correlation between absence of siderophore activity in the CAS assay and inability to grow in an iron-deprived medium indicated that the transposon insertion in this mutant was probably affecting genes involved in siderophore biosynthesis.
The growth of BAM41 in a low-iron medium was stimulated by filtered supernatants from a 2308 culture grown in an iron-deprived medium, and also by ethyl acetate extracts from the same culture, indicating that an iron-related growth factor was present in both samples. Analysis of ethyl acetate extracts of B. abortus 2308 low-iron culture supernatants by TLC showed the presence of two different catechol species, one with an Rf identical to that of 2,3-DHBA and the other with a lower Rf. The concentration of the low Rf catechol in culture supernatants was always considerably lower than that of 2,3-DHBA. When isolated from TLC plates, only the compound with the low Rf, but not 2,3-DHBA, was able to complement the growth of the mutant in an iron-deprived medium and to decolorize CAS plates. These results indicated that B. abortus 2308 produced a second catecholic substance with a higher affinity for iron than the formerly reported 2,3-DHBA. Since this new compound, capable of decolorizing CAS medium, could be an efficient siderophore for B. abortus, we propose to call it brucebactin. The presence of cathecol compounds in addition to 2,3-DHBA had been described by Lopez-Goñi et al. (1992) . Brucebactin could be identical to the cathecol with an Rf of 0·02 described in that report. However, this catechol was not further assayed and the possibility that it could be a siderophore was discarded by the authors.
An alternative explanation for the inability of BAM41 to grow in iron-deprived media is that the mutation in this strain affected siderophore transport instead of siderophore biosynthesis. However, siderophore transport mutants usually present a large halo in CAS plates (Schwyn & Neilands, 1987 ) and this was not the case for BAM41. Moreover, the fact that this strain could be cross-fed by B. abortus 2308 ethyl acetate supernatant extracts indicated that siderophore transport was not affected in this mutant.
The results obtained in a cross-feed assay with an E. coli entA mutant confirmed that the fast-moving catechol was 2,3-DHBA. Cross-feed experiments and TLC also demonstrated that BAM41 produced more 2,3-DHBA than the parental strain 2308. This would be the expected behaviour of a mutant blocked in the late steps of the biosynthesis of a complex catecholic siderophore structurally based on 2,3-DHBA. Genes encoding the enzymes for this process are usually organized in two clusters. The first encodes the enzymes for the production of 2,3-DHBA from chorismic acid. The second cluster encodes a multienzyme complex responsible for the condensation of 2,3-DHBA with amino acids or other molecules to produce the final catecholic siderophore (Ratledge & Dover, 2000 ). On these grounds, BAM41, which did not produce the final catechol, but produced 2,3-DHBA, might well be affected in the condensation step.
Genetic analysis revealed that BAM41 contained a single Tn5 copy inserted in a gene homologous to E. coli entF (Rusnak et al., 1991 ). The product of this gene (EntF) together with EntB and EntE constitute a multienzyme complex responsible for the biosynthesis of the catecholic siderophore enterobactin in E. coli (Gehring et al., 1998
). The Brucella gene presumably should form part of an operon responsible for brucebactin biosynthesis.
Attempts to determine the conditions for optimal brucebactin production indicated that the better yields were always obtained in iron-limited media at the beginning of the stationary phase of growth. Attempts to isolate high amounts of brucebactin for structural and chemical analyses were unsuccessful because of the instability of the purified compound.
We conclude that 2,3-DHBA can be used by Brucella as a low-affinity siderophore when iron in the medium is readily soluble, but this substance cannot work as an efficient siderophore when iron is complexed by EDDA or high-affinity physiological chelators such as transferrin or lactoferrin, as theoretically predicted by Chipperfield & Ratledge (2000) . In this case Brucella must compete for iron by secreting another compound with similar or stronger affinity for iron (responsible for CAS decoloration). Moreover, concentrations of 2,3-DHBA higher that those secreted by BAM41 in iron-deprived medium were unable to decolorize CAS plates, indicating the lack of siderophore activity of this catechol.
Results presented here and previous evidence (Bellaire et al., 1999 ) have shown that siderophores appear not to play a relevant role in the intracellular survival of B. abortus. This finding has also been reported for other intracellular pathogens such as Salmonella (Benjamin et al., 1985
), probably because iron availability in the phagosome is high enough for this pathogen or because other effective iron assimilation mechanisms are used by Salmonella. On the other hand, it has been shown that 2,3-DHBA improves the survival of B. abortus in the macrophage (Leonard et al., 1997
). Considering the poor ability of 2,3-DHBA as a siderophore, this effect could be due to another circumstance different from iron assimilation. Brucella needs an acidic phagosome in the first hours of infection to survive (Porte et al., 1999
). In this low pH compartment, iron is readily soluble and thus highly bioavailable. Apart from this, catecholic siderophores present less affinity for iron at acid than at neutral pH (Emery, 1978
). Therefore, given the pH of Brucella-containing phagosomes, and taking into account the available results, we may conclude that the role of catecholic compounds in the phagosomal survival of B. abortus, if any, should be different from iron sequestration.
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ACKNOWLEDGEMENTS |
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Received 5 September 2001;
revised 26 October 2001;
accepted 29 October 2001.