and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), ...
Vol. 58, No. 1
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 42-47
0099-2240/92/010042-06$02.00/0
Characterization of Lactoferrin Binding by Aeromonas hydrophila F. ASCENCIO,"12 A.
LJUNGH,l AND T. WADSTROM1*
Department of Medical Microbiology, University of Lund, S-223 62 Lund, Sweden,1 and Department of Experimental Biology, Centro de Investigaciones Biologicas de Baja California Sur, La Paz, Baja California Sur 23,000, Mexico2 Received 20 June 1991/Accepted 17 October 1991
Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these ironcontaining compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium.
and colostrum lactoferrins)
The global distribution of Aeromonas species in many aquatic ecosystems indicates the successful adaptation of the bacterium to such environments (14, 36). Also, Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae are opportunistic pathogens of poikilothermic (2, 17) and homeothermic animals, including human beings (15). A. hydrophila is an important etiologic agent of gastrointestinal infections in human beings (11, 35) and also can invade the bloodstream through defective intestinal mucosa (2). A. hydrophila produces virulence factors which enable the bacterium to colonize the host and to obtain nutrients and growth factors in vivo (1, 26, 28). To obtain iron required for growth, tissue-invasive Aeromonas species have developed specific iron-sequestering mechanisms involving the production of extracellular hemolytic proteins (7, 19, 20, 25, 32, 33) and tryptophan- and phenylalanine-containing phenolate siderophores (6). In addition, Aeromonas salmonicida has been reported (8) to utilize iron in iron-containing proteins such as lactoferrin and transferrin by mechanisms that require cell contact. Despite progress in defining the iron uptake mechanisms of Aeromonas species, there is a paucity of published information about the expression of Aeromonas cell surface receptors which can bind iron-containing proteins of the host and which may be involved in the acquisition of iron by the bacterium. In this article, we report on the ability of A. hydrophila to bind lactoferrin, transferrin, ferritin, cytochrome c, and hemin.
riological Media Ltd., London. The bacteria usually were grown at 32°C overnight on blood agar base supplemented with 4% citrated horse blood, washed once with 0.02 M potassium phosphate buffer (pH 7.2) containing 0.15 M NaCl (PBS), and suspended in PBS to a density of 1010 cells per ml. Some experiments were performed with bacteria grown in various culture media (indicated), at various temperatures (15, 25, 32, 37, and 42°C), in minimal medium broth adjusted to 0.15 M NaCl (MMB) (27) containing various concentrations of Ca2+ and in iron-depleted MMB containing various concentrations of the iron chelator 2',2-dipyridyl. Binding and inhibition of `251-labeled lactoferrins and copper-containing proteins. All chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Lactoferrin from bovine and human milk (BLf and HLf, respectively) and from bovine colostrum, hemocyanin (HC), and hemolymph (HL) from Limulus polyphemus were labeled with 0.2 mCi of 1251, by using Iodo-beads as previously described (21). The binding assay was performed as previously described for fibronectin binding by Escherichia coli (12). Briefly, 50 pAl of '25N-labeled protein (ca. 25,000 cpm) solution in PBS containing 0.1% bovine serum albumin was incubated with 100 ,ul of an A. hydrophila suspension (109 cells) in a polystyrene centrifuge tube for 1 h at 20°C. Incubation mixtures not containing bacteria were used as background count controls. After adding 2 ml of ice-cold PBS containing 0.1% Tween 20, the mixtures were centrifuged (4,500 x g, 10 min, 4°C) and the radioactivity of the pellets was measured in a gamma counter. Some binding assays were performed in the presence of divalent cations (Ca2+, Mg2+, Mn2+, Fe2+) or EDTA (final concentration, 1 mM) and in the presence of iron chelators [Desferal, ethylenediamine di-(o-hydroxyphenyl) acetic acid, and 2',2-dipyridyl] at a final concentration of 1 mg/ml. The amount of 1251I-labeled protein bound to the bacteria was expressed as a percentage of the amount of radiolabeled protein incubated with 109 bacteria. Binding inhibition assays were performed by incubating bacterial suspensions (100 pAl containing 109 cells) at 20°C for
MATERIALS AND METHODS Bacterial strains and growth conditions. A total of 41 A. hydrophila strains were isolated from diseased fish and infected humans (5). All media and chemicals were purchased from Difco (Detroit, Mich.), except blood agar base no. 1 which was obtained from London Analytical & Bacte*
Corresponding author. 42
LACTOFERRIN BINDING BY A. HYDROPHILA
VOL. 58, 1992
1 h with 100 ,ug of unlabeled HLf and BLf with other iron-binding or iron-containing proteins (ferritin [horse spleen], human transferrin, sheep hemoglobin, myoglobin [horse skeletal muscle], cytochrome c [horse heart], and bovine hemin) and with copper-containing proteins (HC and HL) before incubating the bacteria with 125I-lactoferrin. The treated cells were washed with 2 ml of PBS, suspended in 100 ,u of buffer, and used in the binding assay described above. Inhibition values were calculated as the relative percentage of 125I-labeled protein bound by bacteria suspended in PBS without inhibitor. Inhibition higher than 10% was considered significant. Binding inhibition experiments were also performed with bacteria incubated with various monosaccharides (final concentration, 100 mM) and glycoproteins (fetuin [fetal calf serum], human orosomucoid, bovine serum glycoprotein [fraction VI], and bovine submaxillary asialomucin; final concentration, 0.1%) before incubation with labeled lactoferrin. Enzymatic treatment of bacteria. All of the enzymes were purchased from Sigma. A. hydrophila suspensions (100 [lI containing 109 cells) were incubated with 100 pug of pepsin, pronase E (from Streptomyces griseus), proteinase K (from Tritirachium album), trypsin (bovine pancreas), chymotrypsin (bovine pancreas), and neuraminidase (from Vibrio cholerae). The treatment conditions for each enzyme were those described in the Worthington Enzyme Manual (37). The cells were washed three times with ice-cold PBS, suspended in the same buffer to the original cell concentration, and used in the lactoferrin binding assay described above. Inhibition values were calculated as the relative percentage of 125I-labeled protein bound to untreated bacterial cells suspended in PBS. Assays for binding of ferritin, transferrin, cytochrome c, hemin, and Congo red. Bacteria grown on blood agar at 32°C were washed and suspended in PBS to a final concentration of 1010 cells per ml. Samples (100 [LI) of suspension were incubated, for various times at 37°C, with equal volumes of solutions of ferritin (1 mg/ml), transferrin (10 mg/ml), cytochrome c (1 mg/ml), hemin (100 p.g/ml), and Congo red (100 ,ug/ml), and the bacteria were sedimented by centrifugation (15,000 x g, 30 s). The supernatant fluids were assayed spectrophotometrically for unbound proteins and hemin (A492) and for Congo red (A540). Binding was manifested as a decrease in absorbance relative to the absorbances of the ferritin, transferrin, hemin, and Congo red solutions which were not incubated with bacteria. Assay for siderophores in culture supernatant fluids. The chrome azurol S substrate was prepared and the culture supernatant fluids were assayed for siderophores by the methods of Schwyn and Neilands (30). Briefly, samples (0.5 ml) of culture supernatant fluids were incubated at 22°C for 1 h in the dark with the chrome azurol S substrate (0.5 ml), and siderophore activity was estimated by measuring the A630 of the mixtures. The positive control was the iron chelator Desferal, and uninoculated broths and PBS were negative controls. Analysis of data. Samples were tested in duplicate, and each experiment was done at least three times. Background residual radioactivity from incubation mixtures without bacteria was less than 5%. Binding values below 10% of the total 125I-labeled glycoprotein added were considered to be negative. Standard deviations were less than 6%.
43
100'
C,
80
z
E
z
;
60
-I
40 I-
IN
w
20
00 0
20
60
100
140
180
TIME (min) FIG. 1. Displacement of cell-bound '25l-labeled lactoferrin from A. hydrophila A186 by unlabeled lactoferrin.
RESULTS
Characterization of lactoferrin binding. Approximately 40% of the 41 A. hydrophila strains examined bound 125I1 labeled lactoferrin. A. hydrophila A186 (isolated from a human gastrointestinal infection), which binds connective tissue proteins (5) and which exhibited a moderate capacity to bind lactoferrin, was selected for further characterization of lactoferrin binding. The percentages of binding of iron-depleted HLf, ironsaturated HLf, BLf, and bovine colostrum lactoferrin were 35, 26, 31, and 38%, respectively (data not shown). Sixtyfive percent of the 125I-BLf bound by A. hydrophila could be displaced by incubating the bacteria with 100 ,ug of unlabeled BLf for 1 h (Fig. 1). Incubation of the bacteria with unlabeled HLf, BLf, HC, and HL before exposure to labeled HLf and BLf inhibited binding (Table 1). However, other iron-containing glycoproteins (transferrin, ferritin, hemoglobin, myoglobin, and cytochrome c) did not inhibit binding of HLf and BLf. Binding of 125I-labeled BLf and HLf was significantly reduced by pepsin and neuraminidase treatment of the bacteria and, to a lesser extent by trypsin, pronase E, and proteinase K treatment (Table 2). To examine the importance of the iron complexed with the lactoferrin for lactoferrin binding by the bacterium, binding assays were performed in the presence of iron chelators. The iron chelator 2,2-dipyridyl inhibited more than 50% of the binding of lactoferrin by A. hydrophila (Table 2). In addition, the presence of Fe2+ or Ca2+, but not Mg2+, Mn2+, or EDTA, in the binding assay solutions (final concentration, 1 mM) enhanced the binding of 125I-lactoferrin by 110 and 71%, respectively (Table 3). The carbohydrate moieties of lactoferrin also seem to be involved with binding, since an excess of the monosaccharide precursors of the oligosaccharide moiety of lactoferrin (i.e., fucose, galactose, and mannose), as well as asialofetuin
44
APPL. ENVIRON. MICROBIOL.
ASCENCIO ET AL.
TABLE 3. Effect of divalent cations on the binding of 125I-lactoferrin by A. hydrophila A186
TABLE 1. Inhibition of binding of lactoferrin and coppercontaining proteins by A. hydrophila A186 % Inhibition of binding of 25[labeled: BLf HLf HC HL
Inhibitor
Iron-binding glycoproteins BLf HLf Ferritin Transferrin Metalloproteins Hemoglobin Myoglobin HL HC Cytochrome c Hemin Ovalbumin Carbohydrates Fucose Galactose N-acetyl-D-galactosamine Mannose Glycoproteins Asialofetuin Fetuin Submaxillary asialomucin Bovine serum glycoprotein Orosomucoid
62.2 55.6 0.0 3.2
65.2 51.5 0.0 5.3
66.2 52.7 0.0 6.3
70.0 57.9 1.6 13.0
5.6 0.0 3.0 28.0 9.5 56.0 16.0
0.0 11.0 11.0 18.0 n.d. 64.0 2.3
52.2 0.0 60.5 67.4 0.0 34.3 n.d.
46.0 8.7 56.5 64.4 10.6 34.2 n.d.
42.8 36.7 34.3 31.3
33.1 25.3 37.5 27.0
29.0 21.0 51.0 28.0
20.0 11.4 29.0 19.8
43.0 26.0 15.6 10.6 2.0
45.1 23.1 10.6 0.0 8.3
67.4 52.7 16.4 0.0 0.0
57.4 46.6 9.2 0.0 0.0
% Binding of 125I-labeled:
Addition to assay
None (control)
Ca2+ Fe2+ Mg2+ Mn2+ EDTA
HLf
BLf
35 60 74 37 34 33
31 53 67 33 31 30
activity exhibited depressed siderophore production (Table 4). Binding of ferritin, transferrin, cytochrome c, hemin, and Congo red by A. hydrophila. We have previously shown that A. hydrophila binds to transferrin immobilized on latex beads (3). However, the bacterium, which bound 1251-lactoferrin, did not commonly bind 125I-labeled transferrin, ferritin, cytochrome c, hemoglobin, or myoglobin (data not
100
HLf
BLf
80 c
|r 60 40 Cf 40
0
or fetuin (which have glycan branches similar to lactoferrin oligosaccharides) (33) inhibit lactoferrin binding (Table 1). In addition, A. hydrophila binds the copper-containing glycoproteins HC and HL which contain oligosaccharide moieties analogous to those of the lactoferrin (Table 1). Effect of culture conditions on lactoferrin binding. Lactoferrin binding by A. hydrophila A186 was greatly influenced by growth conditions such as type of culture medium (Fig. 2). Optimal binding of HLf, BLf, HC, and HL was observed at 15°C and decreased (31, 40, 41, and 44%, respectively) in a linear fashion from 15 to 42°C (data not shown). In addition, binding of HLf, BLf, HC, and HL was optimum at 4.5 mM CaCl (data not shown). Bacteria grown in various broth media expressed an inverse relationship between 125I-lactoferrin binding and siderophore activity. Bacteria grown in media which enhanced expression of binding
0
.0 20 a
T
CM
HC . 0 co
0
HL
80 60
0
2o
40
201 TABLE 2. Effect of enzymes and iron chelators on binding of lactoferrin and copper-containing proteins by A. hydrophila A186 Treatment
Enzymes Chymotrypsin Pepsin Proteinase Proteinase K Trypsin Neuraminidase Iron chelator Desferal EDDHAa 2',2-Dipyridyl a
EDDHA, ethylenediamine
% Inhibition of binding of
251I-labeled:
BLf
HLf
HL
6.6 36.4 16.0 12.7 14.3 36.4
14.9 34.6 15.8 19.0 16.6 33.0
28.8 27.3 40.5 9.5 21.6 24.2
26.6 14.4 49.0
13.0 6.4 57.2
14.0 3.5 38.5
di-(o-hydroxyphenyl)acetic acid.
0J Growth media FIG. 2. Lactoferrin binding and binding of copper-containing proteins by A. hydrophila A186 grown in various culture media. Abbreviations: BA, blood agar base supplemented with 4% citrated horse blood; McC, MacConkey agar; TSA, tryptic soy agar; T-80, tryptic soy agar supplemented with 2% Tween 80; DHBA, tryptic soy agar supplemented with 5% defibrinated horse blood; CFA, colonization factor antigen agar (10); BHI, brain heart infusion broth; TSB, tryptic soy broth; PW, peptone water; CFA-B, colonization factor antigen broth (10); CDB, chemically defined broth for Aeromonas spp. (27); CIB-4, broth for marine bacteria (4); MH, nutrient-rich broth for marine bacteria (4); M9/CA, minimal salts plus Casamino Acids broth (1); 2216, 2216 marine broth medium adjusted to 0.15 M NaCl (38).
VOL. 58, 1992
LACTOFERRIN BINDING BY A. HYDROPHILA 1.60
HLf 60
0.6
A
1.55
Qv
0
0.5
z z
45
IIv
50
a
0.~
.1.50
0.4
°b
04
0
a
0~~~~~
A
40
0.3 1.45 w 0----
30 -
-
----o
HC
0
0.2
o
-
I--,' _ . _
.
0
1.40
0.1
20
1.35
0.0
50
100
200
150
300
0
10
20
30
40 50 60
TIME (min)
2,2- DIPYRIDYL
(ug/ml)
FIG. 3. Binding of 125I-labeled lactoferrin and copper-containing proteins by bacteria grown in MMB depleted of iron by the addition of various concentrations of 2,2-dipyridyl.
shown). To rule out the possibility that the 125I-labeling procedure deleteriously affected certain domains on these proteins and eliminated their ability to be bound by the bacterium, we incubated A. hydrophila with unlabeled ferritin, transferrin, and cytochrome c, as well as with hemin and Congo red, and measured the adsorption of these compounds. A. hydrophila A186 bound unlabeled transferrin, ferritin, cytochrome, hemin, and Congo red (Fig. 4).
FIG. 4. Binding of ferritin, transferrin, cytochrome c, hemin, and Congo red by A. hydrophila A186. Binding conditions are described in Materials and Methods. Symbols: 0, ferritin; A, transferrin; O, cytochrome c; A, hemin; 0, Congo red.
HC
HL
9.3 9.7 26.2 26.8 27.0
11.0 13.2 37.2 35.3 39.5
(22). Similar mechanisms for iron acquisition from transferrin and lactoferrin have been reported for A. salmonicida (8), where direct contact between bacterial cell surface receptors and the iron-complexing protein is required. At the present time, it is not known whether one or both of the iron acquisition systems functions in vivo during Aeromonas infections. The results presented here indicate that binding of lactoferrin by A. hydrophila is specific and reversible. The carbohydrate moiety of the lactoferrin molecule is involved in the binding process as revealed by inhibition assays with homologous glycosides (Table 1). A. hydrophila A186 did not show significant differences in its ability to bind various 1251I-lactoferrin preparations isolated from different sources. Despite the fact that the complex oligosaccharide structures of the lactoferrin molecule vary from one source to another (18, 23, 31), it seems that they are recognized by A. hydrophila with the same binding affinity. Other iron-containing proteins, i.e., ferritin, transferrin, and cytochrome c, are also bound by A. hydrophila (Fig. 4). However, the observation that binding of BLf and HLf was not inhibited by these iron-containing proteins (Table 1) suggests that binding of the proteins involves different bacterial surface components than those required for lactoferrin binding. In addition, reduction of lactoferrin binding by pepsin and neuraminidase treatment of the bacterium suggests that A. hydrophila cell surface lactoferrin-binding components are proteins, and since neuraminidase treatment affects lactoferrin binding, sialic acid and/or its negative charge may be present in the lactoferrin-binding components and are involved with bind-
a TSB, tryptic soy broth; BHI, brain heart infusion broth; CDB, chemically defined broth for Aeromonas spp.; M9/CA, minimal salts plus Casanino Acids broth. b Siderophore activity is manifested as a decrease in A630. The absorbance values of the uninoculated media (negative controls) were 0.60 to 0.65, and the absorbance value for the positive control Desferal (100 p.g/ml) was 0.20. None of these uninoculated media interfered with the CAS assay. The experimental protocols are described in Materials and Methods.
ing. Concerning the possible participation of iron, complexed in the lactoferrin molecule, in the binding of lactoferrin by A. hydrophila, our results on (i) binding of iron-depleted and iron-saturated lactoferrin by the bacterium, (ii) binding of lactoferrin in the presence of iron chelators, and (iii) binding of lactoferrin by the bacterium when there is an excess of
DISCUSSION A. hydrophila and related species (A. caviae and A. sobria) have at least two mechanisms for acquiring host iron. The first is a siderophore (amonabactin)-dependent system for extracting iron from Fe-transferrin (6), and the second is a siderophore-independent system for the utilization of host heme compounds (i.e., hemoglobin-haptoglobin complexes) TABLE 4. Siderophore activity and binding of lactoferrin and iron containing proteins by A. hydrophila A186 grown in different media
sulpernatanta
Siderophore actiVityb (A630)
TSB BHI CDB M9/CA MMB
0.292 0.323 0.598 0.632 0.635
Culture fluid
% Binding of HLf BLf
11.3 11.5 31.0 40.8 47.2
9.1 10.1 25.2 32.5 35.8
"25I-labeled:
46
APPL. ENVIRON. MICROBIOL.
ASCENCIO ET AL.
Fe2" ions in the incubation mixture are contradictory. Since lactoferrin undergoes conformational changes, apparently in a redistribution of charges on the surface of the protein molecule when it has been saturated by iron (16), it seems that iron does not have any direct participation in the lactoferrin binding by A. hydrophila and that the findings observed are due to charge effects. However, iron is important for the expression of the lactoferrin-binding component(s) (Fig. 3). Deneer and Potter (9) have shown that Actinobacillus (Haemophilus) pleuropneumoniae expresses two outer membrane proteins which are involved in iron acquisition and which also bind hemin and Congo red (an aromatic dye structurally similar to the heme of the hemin molecule). On the basis of these observations, we also evaluated the ability of A. hydrophila cells to bind hemin and Congo red. Our results indicated that A. hydrophila binds these compounds (Fig. 4) and that hemin inhibits the binding of 125I-lactoferrin by A. hydrophila A186 (Table 1). A possible explanation for this observation is that hemin, which is a highly hydrophobic molecule (34), binds to A. hydrophila through hydrophobic interactions (as is the case with Porphyromonas gingivalis) (13) and nonspecifically inhibits lactoferrin binding. An alternative explanation is that hemin competes with lactoferrin for the same binding site. Further studies to elucidate the biochemical nature of the interaction between A. hydraphila surface components and lactoferrin or hemin are now in progress. Although the capacity of A. hydrophila to bind lactoferrin has not been correlated with virulence, as is the case for Neisseria meningitidis (29), it seems plausible that the bacterium might use such a source of host iron during in vivo growth, as is suggested by its ability to grow in the low-Fe environment found in vivo (7) and in vitro (Fig. 3). Further studies are needed to determine whether lactoferrin-binding components are important in Aeromonas infections. We also studied the effect of culture conditions on the capability of the bacterium to bind 1251I-lactoferrin, to evaluate the possible influence of environmental physicochemical factors on the ability of A. hydrophila to scavenge host Fe2+ from lactoferrin, and, in addition, the isolation, purification, and characterization of the lactoferrin-binding component(s) from the bacterium. Our results indicate that A. hydrophila grown in nutrient-poor media (MMB, minimal salts plus Casamino Acids broth) at low temperatures (15°C) and in medium depleted of iron expresses the highest percentage of binding of I251-lactoferrin (Fig. 2 and 3). On the other hand, bacteria grown in broth which enhances siderophore production do not bind I251-lactoferrin to the same extent as do bacteria grown in broth which suppresses siderophore production (Table 4).
4.
5.
6.
7. 8. 9. 10.
11.
12. 13.
14. 15. 16. 17.
18. 19. 20.
21. 22.
ACKNOWLEDGMENTS This work was supported by grant 16X04723 from the Swedish Medical Research Council and by grants from the Swedish Institute and from the Swedish Board for Technical Development. We thank A. Kreger for his valuable comments and discussions. REFERENCES 1. Allan, B. J., and R. M. W. Stevenson. 1981. Extracellular virulence factors of Aeromonas hydrophila in fish infections. Can. J. Microbiol. 27:1114-1122. 2. Altwegg, M., and H. K. Geiss. 1989. Aeromonas as a human pathogen. Crit. Rev. Microbiol. 16:253-286. 3. Ascencio, F., P. Aleljung, and T. Wadstrom. 1990. Particle agglutination assay to identify fibronectin and collagen cell
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29. Schryvers, A. B., and G. C. Gonzalez. 1989. Comparison of the ability of different protein sources of iron to enhance Neisseria meningitidis infection in mice. Infect. Immun. 57:2425-2429. 30. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for detection of siderophores. Anal. Biochem. 160:47-56. 31. Spik, G., G. Strecker, B. Fournet, S. Bouquelet, and J. Montreuil. 1982. Primary structure of the glycan from lactoferrin. Eur. J. Biochem. 121:413-419. 32. Titball, R. W., and C. B. Munn. 1983. Partial purification and properties of a haemolytic activity (T-lysin) from Aeromonas
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