APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1990, p. 1926-1931
Vol. 56, No. 6
0099-2240/90/061926-06$02.00/0 Copyright © 1990, American Society for Microbiology
Particle Agglutination Assays To Identify Fibronectin and Collagen Cell Surface Receptors and Lectins in Aeromonas and Vibrio Species F. ASCENCIO,"2 P.
ALELJUNG,l AND T. WADSTROM1*
Department of Medical Microbiology, University of Lund, Solvegatan 23, S-223 62 Lund, Sweden,' and Department of Experimental Biology, Centro de Investigaciones Biologicas de Baja California Sur, La Paz, Baja California Sur, Mexico2 Received 30 November 1989/Accepted 25 March 1990
A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.
Aeromonas salmonicida is an important fish pathogen, while Aeromonas hydrophila and a number of Vibrio species are pathogens for both fish and other warm-blooded animals, including humans (8, 9, 22, 28). Most of these microorganisms produce a variety of hemolysins or cytolytic toxins and some (such as Vibrio cholerae) also produce cytotoxic enterotoxins such as choleragen (18, 24, 26). Studies by Atkinson and Trust (1) have shown that A. hydrophila produces a variety of surface hemagglutinins. Faris et al. (4) and others (11, 16, 27) showed that V. cholerae and other vibrios can produce several hemagglutinins. Moreover, Wiersma et al. (29) showed that V. cholerae binds specifically to one serum and connective tissue protein, i.e., fibronectin. We have recently developed a particle agglutination assay (PAA) for rapid screening of bacterial cell surface receptors with biospecific affinity to fibronectin (Fn), fibrinogen (Fg), and collagen (Cn) (21). We now report on the evaluation of such Fn, Fg, and types I, II, and IV Cn (Cn-I, Cn-II, Cn-IV) PAAs, as well as similar tests with various glycoprotein PAA reagents for rapid screening of cell surface receptors in strains of various Aeromonas and Vibrio species isolated from diseased fish, human stools, and the environment.
ferrin from rabbit plasma (T-6136), ovalbumin grade V (A-5503), bovine fraction VI glycoproteins (G-3259), crude mucin from porcine stomach (type II [M-2378]), asialomucin from bovine submaxillary gland (A-0789), fetuin, fraction IV (F-3004), and asialofetuin from fetal calf serum (type I [A-4781]) were purchased from Sigma Chemical Co., St. Louis, Mo. All other chemicals used were purchased from different commercial sources and were of analytical grade. Latex suspensions (control 764522) and the bacteriological media used were purchased from Difco Laboratories, Detroit, Mich. Bacterial strains and culture conditions. Strains used in this study were isolated from diseased fish, human infections, and the environment (a complete list of the bacteria strains used can be obtained from the corresponding author). Strains were grown on blood agar for 24 h for all binding assays. Bacterial colonies were suspended and washed once in 0.02 M potassium phosphate buffer (pH 6.8). Bacterial cells were resuspended in the same buffer to approximately 1010 cells per ml and immediately used in various binding assays. The same cell suspensions were used for the salt aggregation test (SAT). Preparation of standard latex reagents. Standard latex reagents were prepared and used as previously described (21). Briefly, 1 ml of latex particle suspensions (bead diameter of 0.8 ,um) was mixed with 3.0 ml of 0.17 M glycineNaOH buffer (pH 8.2) and centrifuged at 4,500 x g for 5 min, and the cell pellets were resuspended in 3.0 ml of the same buffer. Highly purified protein (100 ,ug) was added, and the mixtures were kept at 30°C for 12 h on a horizontal shaker at 50 rpm. The mixtures were centrifuged (9,200 x g for 5 min at 20°C), and the supernatants were discarded. Pellets were suspended in 2.0 ml of the glycine buffer containing 0.01% ovalbumin and 0.01% Merthiolate and kept at 4°C for 12 h. A gelatin reagent (gelatin PAA) was prepared as described above with a slight modification. Gelatin (200 ,ug) was added to 0.2 ml of glycine buffer, dissolved by warming at 45°C for
MATERIALS AND METHODS Chemicals. Porcine plasma Fn was a kind gift from Biolnvent International AB, Lund, Sweden. Highly purified human Fg (batch 61857) was kindly supplied by Kabi, Stockholm, Sweden. Orosomucoid (a-1-glycoprotein concentrate from human plasma, batch FVII 12) was a kind gift from the Scottish National Blood Transfusion Association Protein Fractionation Center, Edinburgh. Dextran (Dx)-palmitate, DEAE-Dx, and polyethylene glycol (PEG)-palmitate were kind gifts from Gote Johansson, Department of Biochemistry, University of Lund. Cn-IV from human placenta (C-7521; lot 52F-3848), gelatin (G-2500; lot 64C 0077), trans*
Corresponding author. 1926
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10 min in a water bath, and then added to latex suspensions. The reagents were thoroughly suspended before use. PAA. PAA was performed as previously described (21). Briefly, latex reagents (20 ,ul) were placed on a glass slide, and equal volumes of bacterial cell suspensions were added. The two drops were gently mixed, and the agglutination reaction was read after 2 min. The reactions were scored (PAA value) from strongly positive (3) to weakly positive (1) or negative (0) as previously described (21). Strains were tested for autoaggregation by mixing 1 drop of bacterial cell suspension with 1 drop of potassium phosphate buffer. Bacterial cell suspensions of Staphylococcus aureus Cowan 1 and Newman were used as positive controls and strain Wood 46 was used as a negative control for the Fn, Cn, Fg, and gelatin PAAs (21). Particle agglutination inhibition assay. PAA reactivity was blocked with soluble gelatin and bovine serum albumin (BSA) (at final concentrations of 0.12, 0.25, 0.5, and 1.0 mg/ml) or with glycoproteins and carbohydrates (0.1% fetuin, bovine serum glycoprotein, bovine and porcine mucin, orosomucoid, or 0.1 M N-acetyl-D-galactosamine). Briefly, 100 p,l of inhibitor was preincubated with an equal volume of a bacterial cell suspension as in the standard PAA for 30 min at 20°C and then mixed with the PAA reagents. Test reactions were scored as in the standard PAA test (21). SAT. SAT was performed as previously described (18). Bacterial cells were suspended in 0.001 M sodium phosphate buffer (pH 7.0), washed once in this buffer, and diluted to 1010 bacteria per ml. From a series of ammonium sulfate solutions of different molarities (0.02 to 4.0 M, pH 6.8), four concentrations, 0.02, 0.2, 2.0, and 4.0 M, were chosen as representing breakpoints for groups with different hydrophobicity. Bacterial suspensions (20 ,ul) were mixed with equal volumes of ammonium sulfate solutions of various molarity on a glass slide. The highest dilution of ammonium sulfate (final concentration) which gave visible aggregation in 2 min was scored as a numerical value for bacterial cell surface hydrophobicity (the SAT value). Partitioning in an aqueous two-phase system. An Aqueous polymer two-phase system containing PEG 6000 (Kebo) (7.13%, wt/wt) and Dx (molecular weight, 48,000; Sigma) (8.75%, wt/wt) in 0.015 M NaCl (pH 6.8) was prepared as a phase system (10). To determine particle negative charge, negatively charged Dx sulfate (Pharmacia, Uppsala, Sweden) at a concentration of 0.40% (wt/wt) was included in the phase system, replacing an equivalent amount of Dx. Similarly, positively charged DEAE-Dx at a concentration of 0.40% was included, replacing an equal amount of Dx in the phase system. Hydrophobic affinity partitioning was performed by including monosubstituted PEG-palmitate and Dx-palmitate, replacing an equal amount of PEG (for the PEG-palmitate) or Dx (for the Dx-palmitate) in the phase system. Partitioning was performed by adding 100 ,ul of coated latex particles (absorbance of 1.0 at 540 nm) to 0.9 ml of the phase systems
(previously homogenized by simple stirring), mixing by gently shaking, and allowing phase separation at 20°C for 1 h. The concentration of latex particles in the PEG-rich top phase and the Dx-rich bottom phase was then estimated turbidimetrically at 540 nm, and beads recovered in the top phase were expressed as a percentage of the original concentration of added latex beads. Differences in the hydrophobic and charge properties of the various coated latex beads are expressed as Alog G, which is defined by the equation:
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TABLE 1. Distribution of immobilized connective tissue and glycoprotein binding to Vibrio and Aeromonas strains by the PAA Latex beads coated with:
% Strains showing positive reactivity (no. of strains tested) Aeromonas sp.
Vibrio sp.
83 (147) NDa 69 (147) 78 (49)
58 (33) 88 (33) 90 (29) 44 (34)
58 (147) 55 (147)
93 (29) 70 (33)
53 69 59 40
(49) (49) (49) (20)
58 (36) 78 (32) 41 (32) 19 (27)
63 (49) 30 (49) 63 (49) 37 (49) 40 (20)
44 (35) 20 (25) 74 (35) 39 (31) 4 (28)
Ovalbumin (albumin, egg) BSA
53 (147) 55 (147)
6 (36) 7 (27)
Without protein
55 (147)
6 (36)
Connective tissue protein Cn-I Cn-II Cn-IV Fn Fg Gelatin
Glycoprotein Bovine submaxillary mucin Mucin (crude porcine stomach type II) Hog gastric mucin Asialomucin (bovine submaxillary gland) Orosomucoid (human plasma) Fetuin (calf serum, fraction V) Asialofetuin (calf serum, type I) Glycoprotein (bovine, fraction VI) Transferrin (rabbit)
a
ND, Not determined.
Alog G
=
log
G value of Dx-sulfate system (for Alog G charge) or G value of PEG-palmitate system (for Alog G hydrophobicity) G value of the PEG-Dx system
percent cells in top phase where G
=
percent cells in rest of system
12'I-labeled connective tissue protein binding assay. Samples of 50 ,ug of Fn, Cn-I, Cn-IV, and Fg were labeled with 0.2 mCi of 1251 by the method described in reference 19 with lodo-beads. Binding of 1251-connective tissue proteins to Aeromonas and Vibrio strains was quantitated as previously described (5). S. aureus Cowan 1 and Newman served as positive controls, and strain Wood 46 served as the negative control (21). RESULTS A selected number of Aeromonas and Vibrio strains were grown in and on various broth and agar media, respectively. Cell suspensions were then tested with a series of PAAs: Fn PAA, Fg PAA, Cn-I PAA, and Cn-IV PAA (Table 1). Identical experiments were conducted for latex particles coated with a series of glycoproteins (Table 1). BSA (0.1%) was added to the standard PAA buffer to prevent nonspecific
binding.
Cells of Vibrio strains generally bound to latex beads coated with Cn-II, Cn-IV, and Fg and, to a lesser degree, to beads coated with Cn-I, gelatin, and Fn (Table 1). Cells of these Vibrio strains did not agglutinate ovalbumin- or serum
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TABLE 2. Inhibition of binding of connective tissue protein-coated latex beads to Vibrio and Aeromonas strains by gelatin or BSA Reaction with the following PAA reagenta: SAT value Strain incubated with: Without BSA Ov Gel Fn Cn-IV Cn-I Fg protein
Vibrio cholerae 635-85 BSA (mg/ml) 10.0 5.0 2.5 1.25 Gelatin (mg/ml) 10.0 5.0 2.5 1.25 Control (without inhibitor) Aeromonas sobria AS-76 BSA (mg/ml) 10.0 5.0 2.5 1.25 Gelatin (mg/ml) 10.0 5.0 2.5 1.25 Control (without protein)
Aeromonas caviae E46985 BSA (mg/ml) 10.0 5.0 2.5 1.25 Gelatin (mg/ml) 10.0 5.0 2.5 1.25 Control (without protein)
1
0 0 1 1
2.0 2.0 2.0 2.0
0 0 1 1 2
0 0 0 0 2
0 0 0 0 1
1.0 1.0 1.0 1.0 2.0
1 1 2 3
2 3 3 3
2 3 3 3
2 3 3 3
1.0 1.0 1.0 1.0
0 1 1 1 3
0 1 1 2 3
0 0 1 1 3
0 0 0 1 3
0 0 1 2 3
1.0 1.0 1.0 1.0 1.0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
2.0 2.0 2.0 2.0
0 1 1 1 1
0 1 2 2 2
0 1 1 1 1
0 0 1 1 1
0 0 0 0 0
0 1 1 1 1
1.0 1.0 1.0 1.0 2.0
0 0 1 1
0 1 1 1
0 1 1 1
0 1 2 2
0 0 1 1
0 0 1 1
0 0 1
0 0 0 0 2
0 0 1 1 1
0 0 0 0 2
1 1 1 1 3
0 1 1 1 3
3 3 3 3
0 0 0 0
1 2 3 3
3 3 3 3
0 1 1 1 3
0 1 1 1 1
0 0 0 1 3
0 0 0 0
0 0 0 0
0 0 1 1 2
0 0 0 0 0
a See text for definition of reaction values. Gel, Gelatin; Ov, ovalbumin.
albumin (BSA)-coated latex beads or uncoated latex beads. In contrast, Aeromonas strains reacted equally well with all the proteins tested, with the highest affinity observed for Cn-I-coated latex beads (Table 1). Cells of various Vibrio and Aeromonas strains commonly agglutinated glycoprotein-coated latex beads (Table 1), with the exception of Vibrio strains and ovalbumin- or transferrincoated latex beads. Strains showing different reactions in a number of PAAs were investigated for surface hydrophobicity by the SAT and by phase partitioning. There was no correlation between bacterial cell surface hydrophobicity and/or charge and the PAA reactivity (unpublished data). Autoaggregating strains were grown in broth and on agar media to define conditions which inhibit expression of cell surface hydrophobicity without affecting the reactions in PAAs. However, no simple procedure to suppress nonspecific hydrophobic cell properties could be defined. Agglutination inhibition of latex beads coated with Cn-I, Cn-IV, Fg, gelatin, and Fn could be produced by preincubating Vibrio cells with gelatin (i.e., denatured Cn) before adding the PAA reagents and, to a lesser degree, by prein-
cubating the bacterial cells with BSA (concentration up to 5 mg/ml) (Table 2). Similarly, agglutination inhibition of latex beads coated with the various glycoproteins could be produced by preincubating bacterial cells with bovine serum glycoproteins or fetuin and, to a lesser degree, with orosomucoid, asialofetuin, and N-acetyl-D-galactosamine but could not be produced with bovine submaxillary mucin or porcine stomach mucin (Table 3). Differences in 251I-labeled Cn-I and Fg binding to V. cholerae 635-85 grown under various conditions were observed (Table 4). Similar results were obtained with Aeromonas strains of different species (data not shown). The highest 125I-labeled Cn-I binding to V. cholerae 635-85 was obtained with cells grown on blood agar incubated at 25°C. Bacteria grown in peptone water incubated at 25°C bound more 125I-labeled Fg. PAA reagents were tested for surface hydrophobicity and charge properties by partitioning in aqueous two-phase systems of Dx and PEG. In general, uncoated and coated latex beads possessed both hydrophobic and charged surface properties (Table 5).
TABLE 3. Inhibition of binding of glycoprotein-coated latex beads to cells of V. cholerae 635-85 by soluble glycoproteinsa Reaction with the following PAA reagentb:
Strain 635-85 incubated with:
Control (without inhibitor) Fetuin Asialomucin BSM Mucin (ps) HGM OM Gp (b) Ovalbumin N-Acetyl-D-galactosamine Tween 20 (0.5%)
Ft AsFt BSM Mucin
(Ps) 2
0
2
2
0 0 1 1 0 1 0 1 0
1 1 2 2 1 2 0 2 1
0 1 2 2 0 1 0 2 1
1 1 2 2 1 1 0 2 1
0
0
1
0
2
1
0 1 2 2 0 1 0 1
0 1 2 2 0 2 0 1
0 1 1 2 0 2 0 1
0
0
0
0
Glycoprotein Bovine submaxillary mucin Mucin (crude, porcine stomach, type II) Hog gastric mucin Asialomucin (bovine submaxillary gland) Orosomucoid (human plasma) Fetuin (calf serum) Asialofetuin (calf serum, type I) Glycoprotein (bovine fraction VI) Transferrin (rabbit) BSA Ovalbumin (egg albumin)
0.83 0.14 0.19 0.24 1.05 0.75 0.74 0.24 0.63 0.84 0.65
0.27 0.00 0.25 0.70 2.04 0.68 2.01 1.16 1.64 0.81 0.62
0
0
0
0
Connective tissue protein Cn-I Cn-IV
0.17 0.20
-0.08 0.37
Gelatin Fg (human) Without protein
0.52 0.52 0.15
1.09 0.62 0.27
OM
2
2
1
0 1 2 2 1 2 0 2
0 1 2 2 0 1 0 2
1
0
p
(b)
TABLE 4. Effect of culture medium and growth temperature on 1251I-labeled Cn-I and Fg binding to V. cholerae 635-85 % 125I-protein binding at the following growth temp:
a
Cn-I
Fg
250C
32°C
370C
250C
320C
370C
18.0 4.0 30.0 32.0 13.0 5.0 30.0 13.0
4.0 10.0 6.0 5.0 19.0 5.0 22.0 22.0
8.0 8.0 19.0 20.0 6.0 9.0 8.0 19.0
10.0 0.0 12.0 9.0 0.0 0.0 18.0 9.0
9.0 0.0 15.0 10.0 12.0 9.0 9.0 12.0
0.0 0.0 8.0 11.0 11.0 9.0 0.0 10.0
16.0 16.0
12.0 22.0
10.0 NDa
9.0 5.0
8.0 9.0
7.0 ND
ND, Not determined.
ALog G
rWithout protein
HGM
DISCUSSION Immobilizing various immunoglobulins on latex beads is a standard procedure in particle immunoassays for rapid detection of bacterial capsular and other surface antigens in various body fluids during infections (20). More recently, a number of PAAs have been developed, such as Fg-immunoglobulin latex tests, for rapid diagnosis of staphylococci with cell surface Fg receptors and protein A (i.e., S. aureus) and of other closely related species (3, 21). Accurate, reproducible, and rapid results obtained by using the PAA to screen staphylococci for Fg, Fn, and Cn-I and Cn-II cell surface receptors (21) encouraged us to evaluate the same assays for testing Vibrio and Aeromonas strains for similar cell surface receptors. Since Aeromonas and Vibrio species commonly produce a number of cell surface lectins, we expanded this list of PAA reagents by also coating latex beads with various glycoproteins, i.e., bovine and porcine mucins, orosomucoid, fetuin, and bovine serum glycoproteins (Table 1).
Growth medium
TABLE 5. Partitioning behavior of various PAA reagents in aqueous two-phase system of PEG-Dx Latex beads coated with:
a Abbreviations: AsFt, asialofetuin (calf serum, type I); BSM, mucin (bovine submaxillary gland); Mucin (ps), crude, porcine stomach type II mucin; HGM, hog gastric mucin; OM, orosomucoid (human plasma); Gp (b), glycoproteins (bovine serum, fraction VI). The concentration of inhibitor was 0.1% for glycoproteins and 0.1 M for carbohydrate. b See text for definition of reaction values.
CFA MacConkey agar Tryptic soy agar Blood agar 2216 agar TCBS agar Peptone water Brain heart infusion broth 2216 broth Tryptic soy broth
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Charge
Hydro-
phobicity
A total of 147 mesophilic Aeromonas strains and 30 Vibrio strains were tested. Vibrio strains showed affinity agglutination patterns with the various coated latex beads (with connective tissue and plasma proteins as well as glycoprotein PAA reagents, but not with ovalbumin PAA, BSA PAA, or uncoated latex beads). These results indicated the presence of specific cell surface receptors on various Vibrio strains for connective tissue proteins (Cn-I and Cn-IV, Fn, and Fg) and for sialic acid-rich glycoproteins (such as mucins and orosomucoid). Yamamoto et al. (30, 31) reported that hemagglutinating V. cholerae 01 strains and V. cholerae non-O1 strains adhere to the human intestinal mucosa. Krovacek et al. (14) reported that A. hydrophila and Vibrio anguillarum adhere to fish mucus-coated, glass slides. Aeromonas and Vibrio strains isolated from diseased fish agglutinated bovine submaxillary mucin- and porcine stomach mucin-coated latex beads (Table 1). Aeromonas strains have a complex cell surface composed of a mosaic of molecular structures which enable bacteria to bind to a range of biomolecules by lectinlike interactions between adhesins and specific glycoconjugates. Several reports have demonstrated how Aeromonas strains bind to various erythrocytes (1), mouse adrenal cells (R. B. Clark, et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, B-191, p. 50), fish cells, and cell surface glycoconjugates such as fish mucus (14). The Aeromonas strains used for the present study showed a high tendency to agglutinate latex beads, coated and uncoated, and, in the case of the former, regardless of the type of protein coating. It seems possible that nonspecific charge or hydrophobic interactions participate in the PAA agglutinating reactions. The following observations led us to this hypothesis. (i) A. hydrophila and related species (A. caviae and A. sobria) express high cell surface hydrophobicity and charge as revealed by partitioning in aqueous two-phase systems of Dx and PEG (unpublished data). (ii) The various coated latex beads used were also hydrophobic and negatively charged (Table 5). (iii) Inhibition of agglutination of latex beads coated with glycoproteins was obtained
1930
when the PAA was performed in the presence of 0.5% Tween 20 (Table 3). Moreover, our results suggest that Aeromonas strains exhibit surface structures capable of binding plasma glycoproteins such as Fg, albumin, lactoferrin, hemocyanin, and hemolymph (F. Ascencio, A. Ljungh, and T. Wadstrom; unpublished data). It is probable that these binding characteristics are mediated by protein receptors, as has been described for Streptococcus canis (15), and are the reason why bacteria are able to agglutinate various PAA reagents. To rule out the possibility that nonspecific aggregations contributed to PAA positivity, we also performed inhibition assays (i.e., particle agglutination inhibition assays) (Table 2). Agglutination reactions with Cn-I PAA and Fn PAA reagents were effectively blocked by incubating the bacterial suspensions (of both Vibrio and Aeromonas strains) in the presence of soluble gelatin. Further studies on inhibition of Vibrio and Aeromonas binding with natural and synthetic Cn and other proline-rich peptides (6) are now needed. Bacterial agglutination of various glycoprotein- and mucin-coated latex particles could be inhibited by specific glycoproteins or by monosaccharides (Table 3). Glycoproteins (bovine fraction VI) and fetuin were the best inhibitors. Hemagglutination inhibition with fetuin but not with single monosaccharides has been reported for a soluble hemagglutinin isolated from Aeromonas species (25); lack of such inhibition with single monosaccharides may still be due to specific interactions like that observed for the Escherichia coli fimbria adhesin binding of disaccharides with galactose-
a-1,4-galactose-type linkages (13). For Vibrio adhesins, the position of N-acetylneuraminic acid in the glycoprotein carbohydrate structures, as well as the galactose position, seems to be important for bacterial binding to such complex sequences of carbohydrates. In fact, 60% of the Vibrio strains tested bound to beads coated With various types of mucins which contain sialic acid in an a-2,6 linkage; 44% of Vibrio strains bound to beads coated with orosomucoid, which has sialic acid in an ot-1,4 linkage, and 5% of all Vibrio strains bound to beads coated with fetuin, which has sialic acid in an a-2,3 linkage (Table 1). The exact position of galactose in a glycoconjugate structure also seems to be important. Most (74%) of the Vibrio strains bound to beads coated with asialofetuin, which has a P-1,4 linkage; 19% of these strains bound to beads coated with desialylated bovine submaxillary mucin, which has N-
acetyl-D-galactosamine residues. Further studies are needed to elucidate the biochemical properties of the carbohydrate receptors of both Vibrio and Aeromonas species. We are now studying the use of mucin- and glycoprotein-coated latex beads as PAA reagents to rapidly screen for bacterial surface lectins. We found that many Vibrio strains bind '25I-labeled Fn (data not shown) as reported by Wiersma et al. (29). The number of Vibrio or Aeromonas strains that bind Fn PAA reagents corresponds to the strains that bind '251-labeled Fn, but the Fn PAA value does not correlate well with the amount of 125I-labeled protein binding. Similar test results
also obtained in Cn-I PAA and 125I-Cn-I binding assays (data not shown). Lack of quantitative correlation between PAA and 125I-labeled protein binding has been reported (21). Expression of bacterial cell surface properties commonly varies in relation to growth media and temperature (7, 24). Kabir and Ali (12) showed how the growth conditions influenced the expression of cell surface hydrophobicity and hemagglutination properties of V. cholerae. Statner et al. (24) have reported growth effects on A. hydrophila cell were
APPL. ENVIRON. MICROBIOL.
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surface proteins. Other studies have shown that growth conditions influence the 125I-labeled protein binding to cells of E. coli (A. Ljungh, L. Emody, H. Steinruch, B. Dahlback, P. Sullivan, E. Zetterberg, B. West, and T. Wadstrom, unpublished data) and various Salmonella strains (7). Protein binding to Vibrio cells was significantly influenced by growth conditions (Table 4). For 125I-labeled Cn-I, the best binding conditions were obtained with cells grown on blood agar at 25°C, while growth in peptone water at 25°C enhanced 1251I-labeled Fg binding. Studies on cell surface hydrophobicity and charge of both Vibrio and Aeromonas strains indicate that there is no correlation between agglutination of PAA reagents and cell surface hydrophobicity or charge (data not shown). The classical hemagglutination assay is still commonly used to screen for bacterial hemagglutinins or lectins (11, 25, 27). However, latex particles coated with a single glycoprotein or glycolipids with a-1,4 galactose-galactose disaccharide-type attachments (3) seem a more logical approach to screen for bacterial lectins, especially for organisms such as Vibrio and Aeromonas species able to produce a number of various hemagglutinins (1, 11), and also because of the long stability of the PAA reagents compared with erythrocytes when stored at 4°C (21). With the promising PAA results (except for autoaggregating strains), we are now in the process of preparing human small bowel and fish mucin PAA reagents to test for the presence of sugar-binding proteins (i.e., lectins) which may serve as colonization factor antigens (adhesins) for initial colonization of fish mucin and of the small bowel of humans
(30). ACKNOWLEDGMENTS This study was supported by grants from The Swedish Medical Research Council (MFR 16X 04723) and from The Swedish Board for Technical Development with a scholarship to F.A. LITERATURE CITED 1. Atkinson, H. M., and T. J. Trust. 1980. Hemagglutinating properties and adherence abilities of Aeromonas hydrophila. Infect. Immun. 27:938-946. 2. de Man, P., B. Cedergren, S. Enerback, A.-C. Larsson, H. Leffler, A.-L. Lundell, B. Nilsson, and C. Svanborg-Eden. 1987. Receptor-specific agglutination tests for detection of bacteria that bind globoseries glycolipids. J. Clin. Microbiol. 25:401-406. 3. Essers, L., and K. Radebold. 1980. Rapid and reliable identification of Staphylococcus aureus by latex agglutination test. J. Clin. Microbiol. 12:641-643. 4. Faris, A., M. Lindahl, and T. Wadstrom. 1982. High surface hydrophobicity of hemagglutinating Vibrio cholerae and other vibrios. Curr. Microbiol. 7:357-362. 5. Froman, G., L. M. Switalski, A. Faris, T. Wadstrom, and M. Hook. 1984. Binding of Escherichia coli to fibronectin. J. Biol. Chem. 23:14899-14905. 6. Gibbons, R. J., and D. I. Hay. 1988. Adsorbed salivary prolinerich proteins as bacterial receptors on apatitic surfaces, p. 143-163. In L. Switalski et al. (ed.), Molecular mechanisms of microbial adhesion. Springer-Verlag, New York. 7. Gonzalez, E. A., S. B. Baloda, J. Blanco, and T. Wadstrom. 1988. Growth conditions for the expression of fibronectin and collagen binding to Salmonella. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 269:437-446. 8. Hazen, T. C., G. W. Esch, R. V. Dimock, and A. Mansfield. 1982. Chemotaxis of Aeromonas hydrophila to the surface mucus of fish. Curr. Microbiol. 7:371-375. 9. Isaacs, R. D., S. D. Paviour, D. E. Bunker, and S. D. R. Lang. 1988. Wound infection with aerogenic Aeromonas strains: a review of twenty-seven cases. Eur. J. Clin. Microbiol. Infect. Dis. 7:355-360.
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10. Johansson, G. 1974. Partition of proteins and microorganisms in aqueous biphasic systems. Mol. Cell. Biochem. 30:169-180. 11. Jonson, G., J. Sanchez, and A. M. Svennerholm. 1988. Expression and detection of different biotype-associated cell-bound hemagglutinins of Vibrio cholerae 01. J. Gen. Microbiol. 135: 111-120. 12. Kabir, S., and S. Ali. 1983. Characterization of surface properties of Vibrio cholerae. Infect. Immun. 39:1048-1058. 13. Kallenius, G., R. Mollby, S. B. Svenson, H. B. Hellin, H. Hultberg, C. Cedergren, and J. Winberg. 1981. Occurrence of P-fimbriated Escherichia coli in urinary tract infections. Lancet 8260:1369-1372. 14. Krovacek, K., A. Faris, W. Ahne, and I. Mansson. 1987. Adhesion of Aeromonas hydrophila and Vibrio anguillarum to fish cells and to mucus-coated glass slides. FEMS Microbiol. Lett. 42:85-89. 15. Lammer, C., C. Frede, K. Gortork, A. Hildebrand, and H. Blobel. 1988. Binding activity of Streptococcus canis for albumin and other plasma proteins. J. Gen. Microbiol. 134:23172323. 16. Larsen, J. L., and S. Mellegard. 1984. Agglutination typing of Vibrio anguillarum isolated from diseased fish and from the environment. Appl. Environ. Microbiol. 47:1261-1265. 17. Ljungh, A. 1987. Aeromonas-toxins and other virulence factors. Experientia 43:367-368. 18. Ljungh, A., M. Osterlind, and T. Wadstrom. 1986. Cell surface hydrophobicity of group D viridans streptococci isolated from patients with septicemia. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 261:286-296. 19. Markwell, M. A. K. 1982. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labelling of antiserum. Anal. Biochem. 125:427-432. 20. Miller, C. A., W. L. Patterson, P. K. Johnson, C. T. Swartzell, F. Wogoman, J. P. Albarella, and R. J. Carrico. 1988. Detection of bacteria by hybridization of rRNA with DNA-latex and immunodetection of hybrids. J. Clin. Microbiol. 26:1271-1276. 21. Naidu, A. S., M. Paulsson, and T. Wadstrom. 1988. Particle agglutination assay for rapid detection of fibronectin, fibrinogen, and collagen receptors on Staphylococcus aureus. J. Clin.
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1931
Microbiol. 26:1549-1554. 22. Paula, S. J., P. S. Duffey, S. L. Abbott, R. P. Kokka, L. S. Oshiro, J. M. Janda, T. Shimada, and R. Sakazaki. 1988. Surface properties of autoagglutinating mesophilic aeromonads. Infect. Immun. 56:2658-2665. 23. Santos, Y., A. E. Toranzo, J. L. Barja, T. P. Nieto, and T. G. Villa. 1988. Virulence properties and enterotoxin production of Aeromonas strains isolated from fish. Infect. Immun. 56:32853293. 24. Statner, B., M. J. Jones, and W. L. George. 1988. Effect of incubation temperature on growth and soluble protein profiles of motile Aeromonas strains. J. Clin. Microbiol. 26:392-393. 25. Steward, G. A., C. S. Bundell, and V. Burke. 1986. Partial characterization of a soluble haemagglutinin from human diarrhoeal isolates of Aeromonas. J. Med. Microbiol. 21:319-324. 26. Stoebner, J. A., and S. M. Payne. 1988. Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae. Infect. Immun. 56:2891-2895. 27. Trust, T. J., I. D. Courtice, A. G. Khouri, J. H. Crosa, and M. H. Schiewe. 1981. Serum resistance and hemagglutination ability of marine vibrios pathogenic for fish. Infect. Immun. 34:702-707. 28. Trust, T. J., W. W. Kay, and E. E. Ishiguro. 1983. Cell surface hydrophobicity and macrophage association of Aeromonas salmonicida. Curr. Microbiol. 9:315-318. 29. Wiersma, E. J., G. Froman, S. Johansson, and T. Wadstrom. 1987. Carbohydrate specific binding of fibronectin to Vibrio cholerae cells. FEMS Microbiol. Lett. 44:365-369. 30. Yamamoto, T., and T. Yokota. 1988. Vibrio cholerae non-O1: production of cell-associated hemagglutinins and in vitro adherence to mucus coat and epithelial surfaces of the villi and lymphoid follicles of human small intestines treated with Formalin. J. Clin. Microbiol. 26:2018-2024. 31. Yamamoto, T., T. Kamano, M. Uchimura, M. Iwanaga, and T. Yokota. 1988. Vibrio cholerae 01 adherence to villi and lymphoid follicle epithelium: in vitro model using Formalin-treated human small intestine and correlation between adherence and cell-associated hemagglutinin levels. Infect. Immun. 56:32413250.
Vol. 56, No. 6
0099-2240/90/061926-06$02.00/0 Copyright © 1990, American Society for Microbiology
Particle Agglutination Assays To Identify Fibronectin and Collagen Cell Surface Receptors and Lectins in Aeromonas and Vibrio Species F. ASCENCIO,"2 P.
ALELJUNG,l AND T. WADSTROM1*
Department of Medical Microbiology, University of Lund, Solvegatan 23, S-223 62 Lund, Sweden,' and Department of Experimental Biology, Centro de Investigaciones Biologicas de Baja California Sur, La Paz, Baja California Sur, Mexico2 Received 30 November 1989/Accepted 25 March 1990
A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.
Aeromonas salmonicida is an important fish pathogen, while Aeromonas hydrophila and a number of Vibrio species are pathogens for both fish and other warm-blooded animals, including humans (8, 9, 22, 28). Most of these microorganisms produce a variety of hemolysins or cytolytic toxins and some (such as Vibrio cholerae) also produce cytotoxic enterotoxins such as choleragen (18, 24, 26). Studies by Atkinson and Trust (1) have shown that A. hydrophila produces a variety of surface hemagglutinins. Faris et al. (4) and others (11, 16, 27) showed that V. cholerae and other vibrios can produce several hemagglutinins. Moreover, Wiersma et al. (29) showed that V. cholerae binds specifically to one serum and connective tissue protein, i.e., fibronectin. We have recently developed a particle agglutination assay (PAA) for rapid screening of bacterial cell surface receptors with biospecific affinity to fibronectin (Fn), fibrinogen (Fg), and collagen (Cn) (21). We now report on the evaluation of such Fn, Fg, and types I, II, and IV Cn (Cn-I, Cn-II, Cn-IV) PAAs, as well as similar tests with various glycoprotein PAA reagents for rapid screening of cell surface receptors in strains of various Aeromonas and Vibrio species isolated from diseased fish, human stools, and the environment.
ferrin from rabbit plasma (T-6136), ovalbumin grade V (A-5503), bovine fraction VI glycoproteins (G-3259), crude mucin from porcine stomach (type II [M-2378]), asialomucin from bovine submaxillary gland (A-0789), fetuin, fraction IV (F-3004), and asialofetuin from fetal calf serum (type I [A-4781]) were purchased from Sigma Chemical Co., St. Louis, Mo. All other chemicals used were purchased from different commercial sources and were of analytical grade. Latex suspensions (control 764522) and the bacteriological media used were purchased from Difco Laboratories, Detroit, Mich. Bacterial strains and culture conditions. Strains used in this study were isolated from diseased fish, human infections, and the environment (a complete list of the bacteria strains used can be obtained from the corresponding author). Strains were grown on blood agar for 24 h for all binding assays. Bacterial colonies were suspended and washed once in 0.02 M potassium phosphate buffer (pH 6.8). Bacterial cells were resuspended in the same buffer to approximately 1010 cells per ml and immediately used in various binding assays. The same cell suspensions were used for the salt aggregation test (SAT). Preparation of standard latex reagents. Standard latex reagents were prepared and used as previously described (21). Briefly, 1 ml of latex particle suspensions (bead diameter of 0.8 ,um) was mixed with 3.0 ml of 0.17 M glycineNaOH buffer (pH 8.2) and centrifuged at 4,500 x g for 5 min, and the cell pellets were resuspended in 3.0 ml of the same buffer. Highly purified protein (100 ,ug) was added, and the mixtures were kept at 30°C for 12 h on a horizontal shaker at 50 rpm. The mixtures were centrifuged (9,200 x g for 5 min at 20°C), and the supernatants were discarded. Pellets were suspended in 2.0 ml of the glycine buffer containing 0.01% ovalbumin and 0.01% Merthiolate and kept at 4°C for 12 h. A gelatin reagent (gelatin PAA) was prepared as described above with a slight modification. Gelatin (200 ,ug) was added to 0.2 ml of glycine buffer, dissolved by warming at 45°C for
MATERIALS AND METHODS Chemicals. Porcine plasma Fn was a kind gift from Biolnvent International AB, Lund, Sweden. Highly purified human Fg (batch 61857) was kindly supplied by Kabi, Stockholm, Sweden. Orosomucoid (a-1-glycoprotein concentrate from human plasma, batch FVII 12) was a kind gift from the Scottish National Blood Transfusion Association Protein Fractionation Center, Edinburgh. Dextran (Dx)-palmitate, DEAE-Dx, and polyethylene glycol (PEG)-palmitate were kind gifts from Gote Johansson, Department of Biochemistry, University of Lund. Cn-IV from human placenta (C-7521; lot 52F-3848), gelatin (G-2500; lot 64C 0077), trans*
Corresponding author. 1926
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10 min in a water bath, and then added to latex suspensions. The reagents were thoroughly suspended before use. PAA. PAA was performed as previously described (21). Briefly, latex reagents (20 ,ul) were placed on a glass slide, and equal volumes of bacterial cell suspensions were added. The two drops were gently mixed, and the agglutination reaction was read after 2 min. The reactions were scored (PAA value) from strongly positive (3) to weakly positive (1) or negative (0) as previously described (21). Strains were tested for autoaggregation by mixing 1 drop of bacterial cell suspension with 1 drop of potassium phosphate buffer. Bacterial cell suspensions of Staphylococcus aureus Cowan 1 and Newman were used as positive controls and strain Wood 46 was used as a negative control for the Fn, Cn, Fg, and gelatin PAAs (21). Particle agglutination inhibition assay. PAA reactivity was blocked with soluble gelatin and bovine serum albumin (BSA) (at final concentrations of 0.12, 0.25, 0.5, and 1.0 mg/ml) or with glycoproteins and carbohydrates (0.1% fetuin, bovine serum glycoprotein, bovine and porcine mucin, orosomucoid, or 0.1 M N-acetyl-D-galactosamine). Briefly, 100 p,l of inhibitor was preincubated with an equal volume of a bacterial cell suspension as in the standard PAA for 30 min at 20°C and then mixed with the PAA reagents. Test reactions were scored as in the standard PAA test (21). SAT. SAT was performed as previously described (18). Bacterial cells were suspended in 0.001 M sodium phosphate buffer (pH 7.0), washed once in this buffer, and diluted to 1010 bacteria per ml. From a series of ammonium sulfate solutions of different molarities (0.02 to 4.0 M, pH 6.8), four concentrations, 0.02, 0.2, 2.0, and 4.0 M, were chosen as representing breakpoints for groups with different hydrophobicity. Bacterial suspensions (20 ,ul) were mixed with equal volumes of ammonium sulfate solutions of various molarity on a glass slide. The highest dilution of ammonium sulfate (final concentration) which gave visible aggregation in 2 min was scored as a numerical value for bacterial cell surface hydrophobicity (the SAT value). Partitioning in an aqueous two-phase system. An Aqueous polymer two-phase system containing PEG 6000 (Kebo) (7.13%, wt/wt) and Dx (molecular weight, 48,000; Sigma) (8.75%, wt/wt) in 0.015 M NaCl (pH 6.8) was prepared as a phase system (10). To determine particle negative charge, negatively charged Dx sulfate (Pharmacia, Uppsala, Sweden) at a concentration of 0.40% (wt/wt) was included in the phase system, replacing an equivalent amount of Dx. Similarly, positively charged DEAE-Dx at a concentration of 0.40% was included, replacing an equal amount of Dx in the phase system. Hydrophobic affinity partitioning was performed by including monosubstituted PEG-palmitate and Dx-palmitate, replacing an equal amount of PEG (for the PEG-palmitate) or Dx (for the Dx-palmitate) in the phase system. Partitioning was performed by adding 100 ,ul of coated latex particles (absorbance of 1.0 at 540 nm) to 0.9 ml of the phase systems
(previously homogenized by simple stirring), mixing by gently shaking, and allowing phase separation at 20°C for 1 h. The concentration of latex particles in the PEG-rich top phase and the Dx-rich bottom phase was then estimated turbidimetrically at 540 nm, and beads recovered in the top phase were expressed as a percentage of the original concentration of added latex beads. Differences in the hydrophobic and charge properties of the various coated latex beads are expressed as Alog G, which is defined by the equation:
1927
TABLE 1. Distribution of immobilized connective tissue and glycoprotein binding to Vibrio and Aeromonas strains by the PAA Latex beads coated with:
% Strains showing positive reactivity (no. of strains tested) Aeromonas sp.
Vibrio sp.
83 (147) NDa 69 (147) 78 (49)
58 (33) 88 (33) 90 (29) 44 (34)
58 (147) 55 (147)
93 (29) 70 (33)
53 69 59 40
(49) (49) (49) (20)
58 (36) 78 (32) 41 (32) 19 (27)
63 (49) 30 (49) 63 (49) 37 (49) 40 (20)
44 (35) 20 (25) 74 (35) 39 (31) 4 (28)
Ovalbumin (albumin, egg) BSA
53 (147) 55 (147)
6 (36) 7 (27)
Without protein
55 (147)
6 (36)
Connective tissue protein Cn-I Cn-II Cn-IV Fn Fg Gelatin
Glycoprotein Bovine submaxillary mucin Mucin (crude porcine stomach type II) Hog gastric mucin Asialomucin (bovine submaxillary gland) Orosomucoid (human plasma) Fetuin (calf serum, fraction V) Asialofetuin (calf serum, type I) Glycoprotein (bovine, fraction VI) Transferrin (rabbit)
a
ND, Not determined.
Alog G
=
log
G value of Dx-sulfate system (for Alog G charge) or G value of PEG-palmitate system (for Alog G hydrophobicity) G value of the PEG-Dx system
percent cells in top phase where G
=
percent cells in rest of system
12'I-labeled connective tissue protein binding assay. Samples of 50 ,ug of Fn, Cn-I, Cn-IV, and Fg were labeled with 0.2 mCi of 1251 by the method described in reference 19 with lodo-beads. Binding of 1251-connective tissue proteins to Aeromonas and Vibrio strains was quantitated as previously described (5). S. aureus Cowan 1 and Newman served as positive controls, and strain Wood 46 served as the negative control (21). RESULTS A selected number of Aeromonas and Vibrio strains were grown in and on various broth and agar media, respectively. Cell suspensions were then tested with a series of PAAs: Fn PAA, Fg PAA, Cn-I PAA, and Cn-IV PAA (Table 1). Identical experiments were conducted for latex particles coated with a series of glycoproteins (Table 1). BSA (0.1%) was added to the standard PAA buffer to prevent nonspecific
binding.
Cells of Vibrio strains generally bound to latex beads coated with Cn-II, Cn-IV, and Fg and, to a lesser degree, to beads coated with Cn-I, gelatin, and Fn (Table 1). Cells of these Vibrio strains did not agglutinate ovalbumin- or serum
1928
APPL. ENVIRON. MICROBIOL.
ASCENCIO ET AL.
TABLE 2. Inhibition of binding of connective tissue protein-coated latex beads to Vibrio and Aeromonas strains by gelatin or BSA Reaction with the following PAA reagenta: SAT value Strain incubated with: Without BSA Ov Gel Fn Cn-IV Cn-I Fg protein
Vibrio cholerae 635-85 BSA (mg/ml) 10.0 5.0 2.5 1.25 Gelatin (mg/ml) 10.0 5.0 2.5 1.25 Control (without inhibitor) Aeromonas sobria AS-76 BSA (mg/ml) 10.0 5.0 2.5 1.25 Gelatin (mg/ml) 10.0 5.0 2.5 1.25 Control (without protein)
Aeromonas caviae E46985 BSA (mg/ml) 10.0 5.0 2.5 1.25 Gelatin (mg/ml) 10.0 5.0 2.5 1.25 Control (without protein)
1
0 0 1 1
2.0 2.0 2.0 2.0
0 0 1 1 2
0 0 0 0 2
0 0 0 0 1
1.0 1.0 1.0 1.0 2.0
1 1 2 3
2 3 3 3
2 3 3 3
2 3 3 3
1.0 1.0 1.0 1.0
0 1 1 1 3
0 1 1 2 3
0 0 1 1 3
0 0 0 1 3
0 0 1 2 3
1.0 1.0 1.0 1.0 1.0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
2.0 2.0 2.0 2.0
0 1 1 1 1
0 1 2 2 2
0 1 1 1 1
0 0 1 1 1
0 0 0 0 0
0 1 1 1 1
1.0 1.0 1.0 1.0 2.0
0 0 1 1
0 1 1 1
0 1 1 1
0 1 2 2
0 0 1 1
0 0 1 1
0 0 1
0 0 0 0 2
0 0 1 1 1
0 0 0 0 2
1 1 1 1 3
0 1 1 1 3
3 3 3 3
0 0 0 0
1 2 3 3
3 3 3 3
0 1 1 1 3
0 1 1 1 1
0 0 0 1 3
0 0 0 0
0 0 0 0
0 0 1 1 2
0 0 0 0 0
a See text for definition of reaction values. Gel, Gelatin; Ov, ovalbumin.
albumin (BSA)-coated latex beads or uncoated latex beads. In contrast, Aeromonas strains reacted equally well with all the proteins tested, with the highest affinity observed for Cn-I-coated latex beads (Table 1). Cells of various Vibrio and Aeromonas strains commonly agglutinated glycoprotein-coated latex beads (Table 1), with the exception of Vibrio strains and ovalbumin- or transferrincoated latex beads. Strains showing different reactions in a number of PAAs were investigated for surface hydrophobicity by the SAT and by phase partitioning. There was no correlation between bacterial cell surface hydrophobicity and/or charge and the PAA reactivity (unpublished data). Autoaggregating strains were grown in broth and on agar media to define conditions which inhibit expression of cell surface hydrophobicity without affecting the reactions in PAAs. However, no simple procedure to suppress nonspecific hydrophobic cell properties could be defined. Agglutination inhibition of latex beads coated with Cn-I, Cn-IV, Fg, gelatin, and Fn could be produced by preincubating Vibrio cells with gelatin (i.e., denatured Cn) before adding the PAA reagents and, to a lesser degree, by prein-
cubating the bacterial cells with BSA (concentration up to 5 mg/ml) (Table 2). Similarly, agglutination inhibition of latex beads coated with the various glycoproteins could be produced by preincubating bacterial cells with bovine serum glycoproteins or fetuin and, to a lesser degree, with orosomucoid, asialofetuin, and N-acetyl-D-galactosamine but could not be produced with bovine submaxillary mucin or porcine stomach mucin (Table 3). Differences in 251I-labeled Cn-I and Fg binding to V. cholerae 635-85 grown under various conditions were observed (Table 4). Similar results were obtained with Aeromonas strains of different species (data not shown). The highest 125I-labeled Cn-I binding to V. cholerae 635-85 was obtained with cells grown on blood agar incubated at 25°C. Bacteria grown in peptone water incubated at 25°C bound more 125I-labeled Fg. PAA reagents were tested for surface hydrophobicity and charge properties by partitioning in aqueous two-phase systems of Dx and PEG. In general, uncoated and coated latex beads possessed both hydrophobic and charged surface properties (Table 5).
TABLE 3. Inhibition of binding of glycoprotein-coated latex beads to cells of V. cholerae 635-85 by soluble glycoproteinsa Reaction with the following PAA reagentb:
Strain 635-85 incubated with:
Control (without inhibitor) Fetuin Asialomucin BSM Mucin (ps) HGM OM Gp (b) Ovalbumin N-Acetyl-D-galactosamine Tween 20 (0.5%)
Ft AsFt BSM Mucin
(Ps) 2
0
2
2
0 0 1 1 0 1 0 1 0
1 1 2 2 1 2 0 2 1
0 1 2 2 0 1 0 2 1
1 1 2 2 1 1 0 2 1
0
0
1
0
2
1
0 1 2 2 0 1 0 1
0 1 2 2 0 2 0 1
0 1 1 2 0 2 0 1
0
0
0
0
Glycoprotein Bovine submaxillary mucin Mucin (crude, porcine stomach, type II) Hog gastric mucin Asialomucin (bovine submaxillary gland) Orosomucoid (human plasma) Fetuin (calf serum) Asialofetuin (calf serum, type I) Glycoprotein (bovine fraction VI) Transferrin (rabbit) BSA Ovalbumin (egg albumin)
0.83 0.14 0.19 0.24 1.05 0.75 0.74 0.24 0.63 0.84 0.65
0.27 0.00 0.25 0.70 2.04 0.68 2.01 1.16 1.64 0.81 0.62
0
0
0
0
Connective tissue protein Cn-I Cn-IV
0.17 0.20
-0.08 0.37
Gelatin Fg (human) Without protein
0.52 0.52 0.15
1.09 0.62 0.27
OM
2
2
1
0 1 2 2 1 2 0 2
0 1 2 2 0 1 0 2
1
0
p
(b)
TABLE 4. Effect of culture medium and growth temperature on 1251I-labeled Cn-I and Fg binding to V. cholerae 635-85 % 125I-protein binding at the following growth temp:
a
Cn-I
Fg
250C
32°C
370C
250C
320C
370C
18.0 4.0 30.0 32.0 13.0 5.0 30.0 13.0
4.0 10.0 6.0 5.0 19.0 5.0 22.0 22.0
8.0 8.0 19.0 20.0 6.0 9.0 8.0 19.0
10.0 0.0 12.0 9.0 0.0 0.0 18.0 9.0
9.0 0.0 15.0 10.0 12.0 9.0 9.0 12.0
0.0 0.0 8.0 11.0 11.0 9.0 0.0 10.0
16.0 16.0
12.0 22.0
10.0 NDa
9.0 5.0
8.0 9.0
7.0 ND
ND, Not determined.
ALog G
rWithout protein
HGM
DISCUSSION Immobilizing various immunoglobulins on latex beads is a standard procedure in particle immunoassays for rapid detection of bacterial capsular and other surface antigens in various body fluids during infections (20). More recently, a number of PAAs have been developed, such as Fg-immunoglobulin latex tests, for rapid diagnosis of staphylococci with cell surface Fg receptors and protein A (i.e., S. aureus) and of other closely related species (3, 21). Accurate, reproducible, and rapid results obtained by using the PAA to screen staphylococci for Fg, Fn, and Cn-I and Cn-II cell surface receptors (21) encouraged us to evaluate the same assays for testing Vibrio and Aeromonas strains for similar cell surface receptors. Since Aeromonas and Vibrio species commonly produce a number of cell surface lectins, we expanded this list of PAA reagents by also coating latex beads with various glycoproteins, i.e., bovine and porcine mucins, orosomucoid, fetuin, and bovine serum glycoproteins (Table 1).
Growth medium
TABLE 5. Partitioning behavior of various PAA reagents in aqueous two-phase system of PEG-Dx Latex beads coated with:
a Abbreviations: AsFt, asialofetuin (calf serum, type I); BSM, mucin (bovine submaxillary gland); Mucin (ps), crude, porcine stomach type II mucin; HGM, hog gastric mucin; OM, orosomucoid (human plasma); Gp (b), glycoproteins (bovine serum, fraction VI). The concentration of inhibitor was 0.1% for glycoproteins and 0.1 M for carbohydrate. b See text for definition of reaction values.
CFA MacConkey agar Tryptic soy agar Blood agar 2216 agar TCBS agar Peptone water Brain heart infusion broth 2216 broth Tryptic soy broth
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VOL. 56, 1990
Charge
Hydro-
phobicity
A total of 147 mesophilic Aeromonas strains and 30 Vibrio strains were tested. Vibrio strains showed affinity agglutination patterns with the various coated latex beads (with connective tissue and plasma proteins as well as glycoprotein PAA reagents, but not with ovalbumin PAA, BSA PAA, or uncoated latex beads). These results indicated the presence of specific cell surface receptors on various Vibrio strains for connective tissue proteins (Cn-I and Cn-IV, Fn, and Fg) and for sialic acid-rich glycoproteins (such as mucins and orosomucoid). Yamamoto et al. (30, 31) reported that hemagglutinating V. cholerae 01 strains and V. cholerae non-O1 strains adhere to the human intestinal mucosa. Krovacek et al. (14) reported that A. hydrophila and Vibrio anguillarum adhere to fish mucus-coated, glass slides. Aeromonas and Vibrio strains isolated from diseased fish agglutinated bovine submaxillary mucin- and porcine stomach mucin-coated latex beads (Table 1). Aeromonas strains have a complex cell surface composed of a mosaic of molecular structures which enable bacteria to bind to a range of biomolecules by lectinlike interactions between adhesins and specific glycoconjugates. Several reports have demonstrated how Aeromonas strains bind to various erythrocytes (1), mouse adrenal cells (R. B. Clark, et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, B-191, p. 50), fish cells, and cell surface glycoconjugates such as fish mucus (14). The Aeromonas strains used for the present study showed a high tendency to agglutinate latex beads, coated and uncoated, and, in the case of the former, regardless of the type of protein coating. It seems possible that nonspecific charge or hydrophobic interactions participate in the PAA agglutinating reactions. The following observations led us to this hypothesis. (i) A. hydrophila and related species (A. caviae and A. sobria) express high cell surface hydrophobicity and charge as revealed by partitioning in aqueous two-phase systems of Dx and PEG (unpublished data). (ii) The various coated latex beads used were also hydrophobic and negatively charged (Table 5). (iii) Inhibition of agglutination of latex beads coated with glycoproteins was obtained
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when the PAA was performed in the presence of 0.5% Tween 20 (Table 3). Moreover, our results suggest that Aeromonas strains exhibit surface structures capable of binding plasma glycoproteins such as Fg, albumin, lactoferrin, hemocyanin, and hemolymph (F. Ascencio, A. Ljungh, and T. Wadstrom; unpublished data). It is probable that these binding characteristics are mediated by protein receptors, as has been described for Streptococcus canis (15), and are the reason why bacteria are able to agglutinate various PAA reagents. To rule out the possibility that nonspecific aggregations contributed to PAA positivity, we also performed inhibition assays (i.e., particle agglutination inhibition assays) (Table 2). Agglutination reactions with Cn-I PAA and Fn PAA reagents were effectively blocked by incubating the bacterial suspensions (of both Vibrio and Aeromonas strains) in the presence of soluble gelatin. Further studies on inhibition of Vibrio and Aeromonas binding with natural and synthetic Cn and other proline-rich peptides (6) are now needed. Bacterial agglutination of various glycoprotein- and mucin-coated latex particles could be inhibited by specific glycoproteins or by monosaccharides (Table 3). Glycoproteins (bovine fraction VI) and fetuin were the best inhibitors. Hemagglutination inhibition with fetuin but not with single monosaccharides has been reported for a soluble hemagglutinin isolated from Aeromonas species (25); lack of such inhibition with single monosaccharides may still be due to specific interactions like that observed for the Escherichia coli fimbria adhesin binding of disaccharides with galactose-
a-1,4-galactose-type linkages (13). For Vibrio adhesins, the position of N-acetylneuraminic acid in the glycoprotein carbohydrate structures, as well as the galactose position, seems to be important for bacterial binding to such complex sequences of carbohydrates. In fact, 60% of the Vibrio strains tested bound to beads coated With various types of mucins which contain sialic acid in an a-2,6 linkage; 44% of Vibrio strains bound to beads coated with orosomucoid, which has sialic acid in an ot-1,4 linkage, and 5% of all Vibrio strains bound to beads coated with fetuin, which has sialic acid in an a-2,3 linkage (Table 1). The exact position of galactose in a glycoconjugate structure also seems to be important. Most (74%) of the Vibrio strains bound to beads coated with asialofetuin, which has a P-1,4 linkage; 19% of these strains bound to beads coated with desialylated bovine submaxillary mucin, which has N-
acetyl-D-galactosamine residues. Further studies are needed to elucidate the biochemical properties of the carbohydrate receptors of both Vibrio and Aeromonas species. We are now studying the use of mucin- and glycoprotein-coated latex beads as PAA reagents to rapidly screen for bacterial surface lectins. We found that many Vibrio strains bind '25I-labeled Fn (data not shown) as reported by Wiersma et al. (29). The number of Vibrio or Aeromonas strains that bind Fn PAA reagents corresponds to the strains that bind '251-labeled Fn, but the Fn PAA value does not correlate well with the amount of 125I-labeled protein binding. Similar test results
also obtained in Cn-I PAA and 125I-Cn-I binding assays (data not shown). Lack of quantitative correlation between PAA and 125I-labeled protein binding has been reported (21). Expression of bacterial cell surface properties commonly varies in relation to growth media and temperature (7, 24). Kabir and Ali (12) showed how the growth conditions influenced the expression of cell surface hydrophobicity and hemagglutination properties of V. cholerae. Statner et al. (24) have reported growth effects on A. hydrophila cell were
APPL. ENVIRON. MICROBIOL.
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surface proteins. Other studies have shown that growth conditions influence the 125I-labeled protein binding to cells of E. coli (A. Ljungh, L. Emody, H. Steinruch, B. Dahlback, P. Sullivan, E. Zetterberg, B. West, and T. Wadstrom, unpublished data) and various Salmonella strains (7). Protein binding to Vibrio cells was significantly influenced by growth conditions (Table 4). For 125I-labeled Cn-I, the best binding conditions were obtained with cells grown on blood agar at 25°C, while growth in peptone water at 25°C enhanced 1251I-labeled Fg binding. Studies on cell surface hydrophobicity and charge of both Vibrio and Aeromonas strains indicate that there is no correlation between agglutination of PAA reagents and cell surface hydrophobicity or charge (data not shown). The classical hemagglutination assay is still commonly used to screen for bacterial hemagglutinins or lectins (11, 25, 27). However, latex particles coated with a single glycoprotein or glycolipids with a-1,4 galactose-galactose disaccharide-type attachments (3) seem a more logical approach to screen for bacterial lectins, especially for organisms such as Vibrio and Aeromonas species able to produce a number of various hemagglutinins (1, 11), and also because of the long stability of the PAA reagents compared with erythrocytes when stored at 4°C (21). With the promising PAA results (except for autoaggregating strains), we are now in the process of preparing human small bowel and fish mucin PAA reagents to test for the presence of sugar-binding proteins (i.e., lectins) which may serve as colonization factor antigens (adhesins) for initial colonization of fish mucin and of the small bowel of humans
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