Effect of EDTA on Salmonella enterica serovar Typhimurium involves a component not assignable to lipopolysaccharide release

H.-L. Alakomi1, M. Saarela1 and I. M. Helander1,2

1 VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Espoo, Finland
2 Department of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, PO Box 56, FIN-00014 University of Helsinki, Finland

Correspondence
H.-L. Alakomi
Hanna-Leena.Alakomi{at}vtt.fi


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The effect of EDTA on Salmonella enterica serovar Typhimurium was studied in different growth phases with cells grown with or without Ca2+ and Mg2+ supplementation. EDTA affected the outer membrane much more strongly in the early exponential phase than in the mid- or late exponential phase, as indicated by uptake of 1-N-phenylnaphthylamine (a nonpolar hydrophobic probe, Mr 219), and detergent (SDS) susceptibility. This effect was, however, not paralleled by LPS release (determined by measuring LPS-specific fatty acids or 14C-labelled LPS in cell-free supernatants, per a standardized cell density), which remained unchanged as a function of the growth curve. The conclusion from these results is that in the early exponential phase the effect of EDTA in S. enterica involves a component that is independent of LPS release.


Abbreviations: KDO, 3-deoxy-D-manno-octulosonic acid; NPN, 1-N-phenylnaphthylamine; OM, outer membrane


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Gram-negative outer membrane (OM) functions as a barrier for many external agents, protecting the cells from the detergent action of bile salts and degradation by digestive enzymes (Nikaido, 1989, 1996; Vaara, 1999). The effect is mainly due to the presence and features of lipopolysaccharide (LPS) molecules in the outer leaflet of the membrane, resulting in many Gram-negative bacteria having an inherent resistance to hydrophobic antibiotics (e.g. macrolides, novobiocins, rifamycins, actinomycin D), detergents (e.g. bile salts, SDS, Triton X-100) and hydrophobic dyes (e.g. eosin, methylene blue, brilliant green, acridine dyes) (Vaara, 1999). In Escherichia coli and Salmonella enterica, the LPS molecules consist of (1) a hydrophobic membrane anchor lipid part, termed lipid A, (2) a core oligosaccharide with multiple phosphoryl substituents, and (3) a structurally diverse polymer composed of oligosaccharide repeats, termed the O antigen, protruding outwards and providing the cell with a hydrophilic surface (for reviews see Helander et al., 1996; Raetz & Whitfield, 2002; Yethon & Whitfield, 2001a). In particular, negatively charged residues in the inner (lipid A-proximal) region of the LPS core oligosaccharide are critical to membrane integrity. These negative charges, provided by residues of 3-deoxy-D-manno-octulosonic acid (Kdo) and phosphate, allow neighbouring LPS molecules to be cross-linked by divalent cations (Mg2+, Ca2+), structurally reinforcing the OM (Nikaido, 1996). Since the initial research by Leive (1965) it has been known that chelating agents such as EDTA destabilize the OM of Gram-negative bacteria by sequestering the stabilizing divalent cations. Such destabilization leads to the release of substantial proportions, up to 40 %, of LPS (Leive, 1965, 1974; Hukari et al., 1986; Alakomi et al., 2000), whereby EDTA-treated bacteria become susceptible to agents that normally do not penetrate the OM and, as a consequence, do not affect the bacteria, as summarized by Vaara (1992, 1999). This phenomenon is often referred to as permeabilization.

The permeabilizing action of EDTA is usually considered to result from LPS release and from consequent perturbations of OM structure and function (Vaara, 1992), but there are still unexplained features of the mechanism. It is not known, for instance, why only a certain proportion of LPS is released. It was shown by Hukari et al. (1986) that the macromolecular quality (LPS chain length distribution) of EDTA-released LPS vs cell-bound LPS was identical. It can, however, be postulated that the releasable fraction differs from the non-releasable one in some structural aspect that is related to the stabilizing effect of divalent cations, perhaps at the level of anionic groups in the core oligosaccaharide and lipid A.

In our studies concerning permeabilization of Gram-negative bacteria we have utilized several methods to unravel the mechanisms underlying these phenomena. In addition to measuring LPS release, the sensitization of bacteria to lysozyme and detergents is used to measure alterations in OM function (Helander et al., 1997a, 1998; Alakomi et al., 2000). Yet another method is the application of a nonpolar hydrophobic probe, 1-N-phenylnaphthylamine (NPN). NPN fluoresces strongly in glycerophospholipid environments, but only weakly in aqueous environments (Träuble & Overath, 1973). This is utilized in fluorometric studies of the permeability of OMs, as increased fluorescence in suspensions of Gram-negative bacteria can be used as a measure of functional changes of the OM (Loh et al., 1984; Helander & Mattila-Sandholm, 2000).

By applying several independent methods to the study of EDTA-induced permeabilization in S. enterica we have noticed that mere LPS release does not account for OM alterations. The results leading to this conclusion are presented in this paper. These results have been in part presented in a poster by H.-L. Alakomi & I. M. Helander at the 6th Conference of the International Endotoxin Society, Paris, 24–27 August 2000.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chemicals.
Triton X-100 was from BDH; chicken egg white lysozyme (EC 3.2.1.17), HEPES, n-heptadecanoic acid methyl ester, 1-N-phenylnaphthylamine (NPN) and SDS were from Sigma-Aldrich; D-[1-14C]galactose (specific activity 49·4 µCi mmol-1, 1829 kBq mmol-1) was from Amersham Pharmacia Biotech; and EDTA was from Riedel-de-Haen. A stock solution of NPN (0·5 M) was prepared in acetone and diluted to 40 µM into 5 mM HEPES (pH 7·2) for the fluorometric assays.

Test strain and growth conditions.
Salmonella enterica serovar Typhimurium VTT E-95582T (ATCC 13311T) cells were grown in Luria–Bertani broth (LB) at 37 °C as described previously (Helander et al., 1997a). Experiments in LB were performed with either 2 mM CaCl2 or 2 mM MgCl2 addition or in LB without added salts. The amounts of calcium and magnesium in the LB without added salts were 0·26 mM and 0·23 mM, respectively (determined by atomic absorption spectrometry: AAS). The effect of the growth phase on EDTA sensitivity was studied by using cells harvested in different growth phases: early exponential (OD630 0·20±0·02), mid-exponential (OD630 0·50±0·02) and late exponential (OD630 0·70±0·02). After harvesting, the cells were washed in the appropriate buffer and resuspended to a standardized optical density. Efficacy of washing treatment to remove the excess salts was verified by AAS. No significant differences in the amounts of calcium and magnesium in the washed cell suspensions were observed (<0·04 mM and 0·04 mM respectively). Further details of cell treatments are given below under various experimental settings.

Permeability assays.
Two methods were utilized to determine permeability properties of the OM: (i) NPN uptake and (ii) sensitization to bacteriolysis induced by detergents or lysozyme.

(i) NPN uptake by bacterial suspensions was measured using black fluorotitre plates (cat. no. 9502 867, LabSystems) and the automated fluorometer Fluoroskan Ascent FL (LabSystems) as described earlier (Alakomi et al., 2000; Helander & Mattila-Sandholm, 2000). Briefly, cells grown to different growth phases were deposited by centrifugation at room temperature for 10 min at 1000 g, washed with 5 mM HEPES buffer (pH 7·2), and the suspension's optical density was adjusted to OD630 0·5±0·02 with the same buffer. After centrifugation as above the cells were suspended into 0·5 vol. 5 mM HEPES buffer. Aliquots (100 µl) of this cell suspension were pipetted into fluoroplate wells containing NPN (10 µM), and as test substances either EDTA (1·0 and 0·1 mM) or HEPES buffer (control) to make up a total volume of 200 µl. If desired, MgCl2 was added to the cell suspension before addition of NPN. Fluorescence was monitored within 3 min from four parallel wells per sample (excitation, 355 nm, half bandwidth 38±3 nm; emission, 402 nm, half bandwidth 50±5 nm). Each assay was performed at least three times.

(ii) Sensitization of target cells to the lytic action of lysozyme and the detergents SDS and Triton X-100 by EDTA was investigated according to the method described in detail by Helander et al. (1997a). Briefly, bacteria at standardized OD630 0·5±0·02 were subjected to treatments with EDTA (0·1 and 1·0 mM) for 10 min at room temperature and added to microtitre plate wells which already contained either lysozyme (10 µg ml-1), SDS (0·05 and 0·1 %), Triton X-100 (0·1 and 1·0 %) or buffer only. Turbidity of the cell suspensions was then monitored with the Multiskan MCC/340 spectrophotometer (LabSystems).

Results from the permeability assays were analysed statistically using two-tailed unpaired Student's t-test to determine differences.

LPS release.
EDTA-induced LPS/lipid release was studied in different growth phases and measured by two methods.

(i) Release of LPS and glycerophospholipids. The release of lipid components, including LPS, was assayed by fatty acid analysis (gas chromatography of fatty acid methyl esters) of cell-free supernatants after treatment of the bacterial suspensions with EDTA. Cells in different growth phases were collected by centrifugation, washed with 10 mM Tris/HCl (pH 7·2) and resuspended to OD630 0·5±0·02 in the same buffer containing either 0·1 or 1·0 mM EDTA (total volume, 10 ml). The control sample was suspended in buffer only. After a 10 min incubation at 37 °C with shaking (150 r.p.m.) the samples were distributed into Eppendorf tubes and centrifuged (13 000 g) in an Eppendorf microfuge for 1 min at room temperature. A total of 9·1 ml of cell-free supernatants per sample was collected, freeze-dried and processed for fatty acid analysis by saponification and methylation as described by Helander et al. (1997a).

(ii) Radiolabelling of lipopolysaccharide, release of [14C]Gal-LPS. Smooth S. enterica Typhimurium E- 95582T cells were grown in LB at 37 °C with shaking (200 r.p.m.) to the desired growth phase and supplemented with [14C]galactose (0·1 µCi ml-1) in order to label their LPS (Hukari et al., 1986). Labelling with [14C]galactose was performed for 5 min at 37 °C with shaking (200 r.p.m.). A 1 ml aliquot was then removed for checking the level of incorporation of radiolabel (Wallac 1410 Liquid Scintillation Counter, Pharmacia). The remaining cells were collected by centrifugation (1000 g, 10 min, 25 °C), and washed with 10 mM Tris/HCl buffer (pH 7·2) at room temperature. After centrifugation the cells were resuspended in the same buffer to OD630 0·5±0·02, divided into three portions and supplemented with either 1·0 or 0·1 mM EDTA; buffer was used as control. After adding the test substance, the assay suspensions were incubated at 37 °C for 10 min with shaking (100 r.p.m.). Samples (2x20 µl) were taken for radioactivity measurements and remaining cells were centrifuged twice (1500 g) at room temperature. After centrifugation, samples from the cell-free supernatants were taken for radioactivity measurements. The amount of radioactivity in the cell-free supernatant was taken as the measure of liberated LPS and the percentage value for LPS release was calculated by comparison to the radioactivity of a similar volume of untreated and uncentrifuged bacterial suspension. Whereas it is known that the buffer used, Tris, a bulky primary amine, alone at high concentrations (50 mM or higher, pH 7·2) can cause release of some LPS and to make bacterial cells somewhat more sensitive to various agents (Vaara, 1992), its concentration in these experiments was only 10 mM, and we observed no additional sensitivity due to the buffer.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
NPN uptake by cells in different growth phases
S. enterica serovar Typhimurium E-95582T grown in LB showed a typical growth curve with lag and exponential phases. The effect of the growth phase on EDTA sensitivity was studied by using cells harvested in different growth phases: early exponential (OD630 0·20±0·02), mid-exponential (OD630 0·50±0·02) and late exponential (OD630 0·70±0·02). With 2 mM CaCl2 or 2 mM MgCl2 supplementation no significant difference in the shape of the growth curve was observed (data not shown).

Fig. 1 shows results obtained in the NPN assay with cells grown in LB with salts and without salt additions. Fig. 1(a) shows that the NPN uptake by cells grown in LB did not differ significantly at different growth phases (fluorescence levels of control cells from 100 to 200 units). Upon treatment with EDTA, however, the cells showed increased NPN uptake, but this phenomenon was significant at the early exponential phase of growth only. Addition of 2 mM CaCl2 to the growth medium stabilized the OM, as indicated by the lower NPN uptake of the control cells (Fig. 1b, fluorescence levels of less than 100 units). In general, supplementation of cells with Ca2+ during growth considerably increased their NPN uptake induced by EDTA, especially in the mid- and late exponential phases. Accordingly, significant increases in NPN uptake by 1·0 mM EDTA were observed at all growth phases, cells in the earlier phases again exhibiting higher uptakes. An EDTA concentration of 0·1 mM was obviously not sufficient to destabilize and increase the permeability of the OM in early exponential cells (OD630 0·2) grown in LB supplemented with 2 mM CaCl2, indicating the presence of massive ionic interactions within the OM (Fig. 1b). In the presence of 2 mM added Mg2+ the results with 1 mM EDTA (Fig. 1c) were similar but not as high as with Ca2+ addition. However, the presence of 2 mM Mg2+ during cultivation was sufficient to destabilize and increase the permeability of the target cells by 0·1 mM EDTA already in the early growth phase. Addition of 1 mM MgCl2 to the buffer used in the NPN assay abolished the permeabilizing activity of EDTA (data not shown) and the NPN uptake of target cells was at the same level as in the corresponding control cells. The conclusion from these experiments was that the growth phase has a profound effect on the bacterium's sensitivity to EDTA as assayed by NPN uptake, with early exponential phase cells exhibiting particularly high sensitivity.



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Fig. 1. NPN uptake in suspensions of S. enterica serovar Typhimurium E-95582T cells harvested at different growth phases: early exponential (hatched columns), mid-exponential (white columns) and late exponential (shaded columns). Cells were cultivated in LB (a), or in LB supplemented with 2 mM CaCl2 (b) or with 2 mM MgCl2 (c). Upon treatment with 1 mM EDTA the cells grown in LB with or without salt additions showed increased NPN uptake. This phenomenon was significant in EDTA-treated early exponential phase versus late exponential phase cells (P<0·001, panel a; P<0·01, panel b).

 
Sensitization to lytic agents
Increased permeability of the OM is also manifested as an increased susceptibility to the bacteriolytic action of detergents and to the cell-wall-degrading action of lysozyme (Vaara, 1992). To further investigate the sensitivity of S. enterica Typhimurium cells in different growth phases we therefore tested the effect of EDTA on the susceptibility of cells to lysozyme- and detergent-induced cell lysis. The results are compiled in Tables 1 and 2. Table 1 shows that significant lysis by Triton X-100 did not occur in control cells, whereas SDS (anionic detergent probe) itself somewhat lysed the control cells. Treatment with EDTA, however, sensitized LB-grown cells to SDS (0·05 and 0·1 %). This sensitization was quantitatively similar in each growth phase. Table 2 shows that early exponential phase cells grown in LB supplemented with Ca2+ and pretreated with 0·1 mM EDTA were more resistant to lysis by 0·05 % SDS than cells grown without added Ca2+ (Table 1), a result in accordance with the similar finding in the NPN uptake experiment. These cells were equally sensitized to the action of 0·1 % SDS/1 mM EDTA in early and late exponential phases. Notably, the presence of 2 mM Ca2+ during cultivation rendered the early exponential phase cells sensitive to lysis by 1 % Triton X-100/1 mM EDTA. In conclusion, the results of the cell lysis experiments do not indicate major differences in functional properties of S. enterica Typhimurium OMs as a function of the growth phase.


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Table 1. Sensitization of S. enterica serovar Typhimurium E-95582T, grown in LB, to detergent-induced bacteriolysis after pretreatments with EDTA

 

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Table 2. Sensitization of S. enterica serovar Typhimurium E-95582T, grown in LB supplemented with 2 mM CaCl2, to detergent-induced bacteriolysis after pretreatments with EDTA

 
Release of LPS
Since EDTA seemed to weaken the OM markedly in the early exponential phase and since it is known to destabilize OMs by liberating LPS, we studied the amount of LPS and lipid material released by EDTA at different growth phases by analysing cell-free supernatants derived from cell suspensions for lipid components. Table 3 shows that EDTA liberated lipid material, including LPS, as indicated by the LPS-specific fatty acids C12 : 0, C14 : 0, C14 : 0(3OH), and glycerophospholipid, as indicated by fatty acids C16 : 0, C16 : 1, C18 : 1. Ca2+ supplementation during growth increased the total amount of liberated fatty acids. However, no significant difference was found in the amounts of LPS liberated by EDTA from cells grown to the early or late exponential phase. A similar result was obtained from experiments involving specific labelling of LPS ([14C]Gal-LPS) and analysis of EDTA-releasable [14C]Gal-LPS at three different growth phases (Table 4). The conclusion thus is that LPS release by EDTA is not dependent on growth phase in S. enterica Typhimurium E-95582T.


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Table 3. Liberation of lipid material from S. enterica serovar Typhimurium E-95582T

 

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Table 4. EDTA-induced [14C]LPS release from S. enterica serovar Typhimurium E-95582T in different growth phases

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results presented above demonstrate that the effect of EDTA on S. enterica cells involves a component that is independent of LPS release, the classical explanation for the mechanism of EDTA-induced permeabilization of Gram-negative bacterial OMs. This component was indicated by the significantly higher NPN uptake observed in early exponential phase cells as compared to late exponential phase cells. Release of LPS, as determined by measurement in cell-free supernatants of either LPS-specific fatty acids or radiolabelled LPS, per a standardized cell density, remained virtually unchanged along the growth curve. Another method to test permeabilization, i.e. sensitization to lytic agents, yielded mostly results paralleling those of the LPS release measurements. It can thus be concluded that in addition to its LPS-releasing mechanism, EDTA in the early exponential phase of growth acts upon cells by another mechanism that does not involve LPS release.

The mechanism underlying the sensitization to EDTA in the early exponential growth phase remains unknown at present, but its existence opens up interesting possibilities concerning stability-affecting properties of OM components. Since NPN fluorescence is associated with the presence of this hydrophobic probe in a glycerophospholipid environment (Träuble & Overath, 1973), it is evident that early exponential phase cells are very susceptible to the effect of EDTA in allowing access of NPN to glycerophospholipids either directly on the OM surface or via the periplasm. If differences in the stability properties of OM components are responsible for the effects described, there should be demonstrable differences in the structure of OM components as a function of the growth phase. Such differences could be expected to be found in the fine structure of LPS, especially in the degree of substitution of phosphate groups of lipid A and the core oligosaccharide. One could postulate, in addition to the EDTA-releasable fraction, the presence of a population of LPS whose interactions with neighbouring components are disturbed by EDTA to an extent that does not, however, result in LPS release. In order to prove this, the LPS obtained from distinct points of the growth phase should be scrutinized for phosphate substituents known to be critical for OM integrity (Helander et al., 1997b; Raetz & Whitfield, 2002; Yethon et al., 1998; Yethon & Whitfield, 2001b). In addition, LPS fractions which are releasable and non-releasable by EDTA should be studied for similar fine structures. In this context it is interesting to note the recent report of Kanipest et al. (2001), who demonstrated in E. coli that the critical parameter determining the presence or absence of phosphoethanolamine was the CaCl2 concentration in the medium. They observed a novel CaCl2-induced enzyme that modifies the outer Kdo moiety of E. coli LPS with a phosphoethanolamine group in the presence of 5–50 mM CaCl2. Such a modification would increase the average resistance of LPS molecules to the releasing action of EDTA, as molecules capped with phosphoethanolamine at Kdo are less prone to be stabilized by divalent cations. Our finding that the addition of Ca2+ ions to the growth medium in some cases stabilized the OM is in agreement with this. It is equally true, however, that similar addition of Ca2+ can stabilize the OM, as already suggested by Leive (1974); in our experiments this was observed in late exponential phase cells by NPN uptake.

Finch & Brown (1975) observed in Pseudomonas aeruginosa with low growth rates increased sensitivity to EDTA (cell lysis) when cells were grown either under carbon limitation or in Ca2+-enriched medium. Furthermore, increased resistance to EDTA in P. aeruginosa was reported when cells were grown in Mg2+-limited medium. According to Finch & Brown (1975) the removal of cations from the cell membrane is due to the greater affinity of the cations for EDTA than for cell membrane components. In addition, the higher stability constant for EDTA interaction with Ca2+ than with Mg2+ (10·7 and 8·7, respectively) also influences the activity of EDTA in the growth medium.

Studies of Kotra et al. (2000) employing atomic force microscopy demonstrated that the OM surface in E. coli is not uniform; i.e. LPS molecules form distinct patches with depressions in between. Furthermore, a non-uniform distribution of metal ions in the OM was implied, giving rise to local variations in the interactions between OM components. Our findings are in agreement with this view.

Another mechanism could be that efflux pumps that remove substances such as NPN from the periplasm are functionally impaired by EDTA, and that they are present in larger numbers in the early exponential phase of growth as compared to the late exponential phase. In this case the phenomenon is not one of permeabilization but rather a more indirect one.

Finally, our results demonstrate that NPN uptake assays should be carried out with cells that have been cultivated in standardized conditions, especially with respect to their growth phase.


   ACKNOWLEDGEMENTS
 
We thank Päivi Lepistö and Anna-Liisa Ruskeepää for excellent technical assistance. The authors are grateful to Helena Liukkonen-Lilja for the AAS measurements and Kyösti Latva-Kala for the GC measurements. This work was financially supported by the Academy of Finland (Project 44163).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Alakomi, H.-L., Skyttä, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K. & Helander, I. M. (2000). Lactic acid permeabilizes gram-negative bacteria by distrupting the outer membrane. Appl Environ Microbiol 66, 2001–2005.[Abstract/Free Full Text]

Finch, J. E. & Brown, R. W. (1975). The influence of nutrient limitation in a chemostat on the sensitivity of Pseudomonas aeruginosa to polymyxin and to EDTA. J Antimicrob Chemother 1, 379–386.[Medline]

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Helander, I. M., Alakomi, H.-L., Latva-Kala, K. & Koski, P. (1997a). Polyethyleneimine is an effective permeabilizer of gram-negative bacteria. Microbiology 143, 3193–3199.[Abstract]

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Hukari, R., Helander, I. M. & Vaara, M. (1986). Chain length heterogeneity of lipopolysaccharide released from Salmonella typhimurium by ethylenediaminetetraacetic acid or polycations. Eur J Biochem 154, 673–676.[Abstract]

Kanipest, M. I., Lin, S., Cotters, R. J. & Raetz, C. R. H. (2001). Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. J Biol Chem 276, 1156–1163.[Abstract/Free Full Text]

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Leive, L. (1974). The barrier function of gram-negative envelope. Ann N Y Acad Sci 235, 109–127.[Medline]

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Received 20 February 2003; revised 16 April 2003; accepted 28 April 2003.



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