Contribution of Hydrophobicity to Hemagglutination Reactionsof ...

INFECTION AND IMMUNITY, Oct. 1985, p. 336-337 0019-9567/85/100336-02$02.00/0 Copyright C 1985, American Society for Microbiology

Vol. 50, No. 1

Contribution of Hydrophobicity to Hemagglutination Reactions of Pseudomonas aeruginosa N. GARBER,' N. SHARON,2 D. SHOHET,' J. S. LAM,3 AND R. J. DOYLE4* Division of Biological Sciences, Bar-Ilan University, Ramat-Gan,' and Department of Biophysics, The Weizmann Institute of Science, Rehovoth 76100,2 Israel; Department of Microbiology, University of Guelph, Ontario NJG 2WI, Canada3; and

Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, Kentucky 402924 Received 25 March 1985/Accepted 11 July 1985

Several strains of Pseudomonas aeruginosa exhibited the ability to hemagglutinate erythrocytes. Hydrophobic bond-breaking agents, but not sugars and saccharides, were effective inhibitors of hemagglutination. The results suggest the involvement of hydrophobic bonds in hemagglutination reactions of P. aeruginosa.

For many gram-negative bacteria, lectin-mediated interactions appear to be involved in adherence to animal cells. For example, fimbrial lectins of Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium interact with mannose-containing oligosaccharides on erythrocytes, resulting in hemagglutination (2, 3, 7; I. Ofek and N. Sharon, in D. Mirelman, ed., Microbial Lectins and Agglutinins, in press). For Pseudomonas aeruginosa, the determinants of adherence to animal cells have not been clearly established (8). Ramphal and Pyle (9) observed that P. aeruginosa binds more readily to acid-injured epithelial cells than to normal cells and that the binding may involve sialic acid. Other work by Woods et al. (13) suggested that removal of fibronectin by trypsin enhanced the binding of P. aeruginosa to buccal cells. Recently, Shohet et al. (Isr. J. Med. Sci. 21:183, 1985) and Garber et al. (Pseudomonas Newsl., vol. 9, 1984) found that a fimbriae-deficient strain of P. aeruginosa agglutinated papain-treated human erythrocytes as well as erythrocytes from rabbits, chickens, dogs, and rats. Moreover, sugars were incapable of hemagglutination inhibition, but a partial inhibition of hemagglutination was observed with fetuin and normal human serum. We now show that several strains of P. aeruginosa have the ability to hemagglutinate and that the hemagglutination depends on hydrophobic interactions, and probably not on lectin-carbohydrate complexes. P. aeruginosa strains GEL (gelatinous), MCD (mucoid), DWF (dwarf), CLS (classic), and RGH (rough) were characterized in a previous study (1). P. aeruginosa G-6 was a strain deficient in fimbriae and in the internal mannose- and galactose-specific lectins (4, 5). A clinical isolate of a P. aeruginosa strain from a patient with cystic fibrosis was obtained from D. Katznelson, Tel-Hashomer Hospital, Ramat-Gan, Israel. The bacteria were maintained on MacConkey agar slants at room temperature. The bacteria were cultured in brain heart infusion broth for 16 h at 37°C in 15-ml volumes without shaking and were then washed twice in distilled deionized water and suspended in 50 mM sodium phosphate-150 mM sodium chloride at pH 7.2 (PBS). For strain GEL, after resuspension in PBS, small aggregates of insoluble material were removed by centrifugation at 1,000 x g for 2 min. Human type 0 erythrocytes were from dated citrated whole blood (Magen-David, Jaffa, Israel). Animal erythrocytes were freshly drawn. Treatment of erythrocytes with papain was done as described by Gilboa-Garber (4). *

Briefly, the erythrocytes were incubated with a 1 mg/ml concentration of papain and a 0.1 mg/ml concentration of cysteine-hydrochloride in PBS at 37°C for 30 min. The cells were then washed three times in PBS. Treatment of cells with chymotrypsin and trypsin (50 jig/ml, final concentrations) and with Vibrio cholerae sialidase (0.001 U/ml, final concentration) (enzyme was from Behring-werke AG, Marburg, Federal Republic of Germany) was in PBS containing 1.0 mM Ca2' at 37°C for 1 h. All cells were then washed three times in PBS. Table 1 shows that all of the strains promoted hemagglutination reactions. Digestion of human or rabbit erythrocytes with papain resulted in a marked increase in hemagglutination titer. For human erythrocytes, strain GEL generally gave the highest titer. The V. cholerae sialidase, an enzyme which removes terminal sialic acid residues from glycoproteins, did not change the interaction between the erythrocytes and the bacteria. Moreover, when this enzyme was used before or after treatment of the erythrocytes with papain, no change in titer was found. Agglutination of erythrocytes may be mediated by lectins, antibodies, sialic acid-binding proteins from certain viruses, certain polyelectrolytes, and lipids (12). The agglutination of TABLE 1. Hemagglutination reactions of P. aeruginosaa Hemagglutination reaction with the following mammalian

erythrocytes Strain Human type 0

GEL MCD CLS RGH DWF G-6 Clinical isolate

1:4 0 0 0 1:2 1:2 0

Papain-

Papain-

treated human type

Rabbit

treated

Goat

Mouse

Sheep

1:64 0 0 1:8 1:16 1:16 1:32

1:8 0 1:8 1:16 1:16 1:4 1:8

1:64 1:64 1:128 1:128 1:128 1:16 1:64

0 0 0 0 1:16 0

0 0 0 0 1:16 0 ND

1:8 0 0 0 1:8 1:2 ND

rabbitr

NDb

a Hemagglutination reactions were conducted with a final erythrocyte density of 1%. Bacterial densities were adjusted to a Klett reading of 300 (ca. 109 cells per ml) before making dilutions. Values shown represent the greatest bacterial dilution capable of promoting agglutination. Treatment of human erythrocytes with V. cholerae sialidase, trypsin, or chymotrypsin did not result in a change in agglutination by the bacteria. bND, Not determined.

Corresponding author. 336

VOL. 50, 1985

NOTES

TABLE 2. Inhibition of hemagglutinationa Inhibitor

L-Alanine .................................

L-Aspartic acid ............................ Asialofetuin .............................. Fetuin .................................... L-Leucine ............................... N-Methyl-L-leucine ........... ............. 4-Methylumbelliferone ......... ............ 4-Methylumbelliferyl-a-D-mannoside ........ p-Nitrophenyl-(3-D-galactoside ...... ........ p-Nitrophenyl-a-D-glucoside ...... ..........

Minimum concentration required for inhibitionb

800 (8.98) >1,000 (>7.51) 100

100 200 (1.52) 200 (1.33) 50 (0.26) 300 (0.89) 250 (0.82) 500 (1.66) 250 (0.83) p-Nitrophenyl-a-D-mannoside ............... p-Nitrophenol ............................. 100 (0.55) Ovalbumin ............................... 200 L-Tryptophan ............................. 200 (0.98) Yeast mannan ............................ 300 a P. aeruginosa GEL was used. Inhibition was assayed with papain-treated human type 0 cells. b Concentrations are in micrograms per milliliter. Numbers in parentheses refer to millimolar concentrations. Noninhibitors included dextran, D-glucose, D-mannose, D-galactose, L-fucose, sialic acid, bovine serum albumin, ao-acid glycoprotein, D-fructose, lactose, stachyose, melibiose, D-galactosamine, lactosamine, raffinose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, hyaluronic acid, and methyl-a-D-mannoside (each at 3,000 ;Lg/ml, final concentration). When other strains of P. aeruginosa were employed, similar results to those described above were obtained. Results shown represent the minimal concentrations giving complete inhibition of hemagglutination.

erythrocytes by bacteria frequently occurs because of the presence of lectins on the latter. Attempts to demonstrate a lectin-mediated hemagglutination by P. aeruginosa were therefore made. In these experiments, sugars or sugarcontaining polymers were used in an effort to inhibit hemagglutination. Simple sugars were found to be without effect on the hemagglutination reactions (Table 2). In contrast, compounds such as 4-methylumbelliferone, 4-methylumbelliferyl-a-D-mannoside, L-leucine, N-methyl-L-leucine, tryptophan, p-nitrophenol, and p-nitrophenyl-containing saccharides were capable of hemagglutination inhibition. In addition, yeast mannan, fetuin, asialofetuin, and ovalbumin were observed to be inhibitors. The most effective inhibitors of hemagglutination were 4-methylumbelliferone, p-nitrophenol, fetuin, and asialofetuin. When 4-methylumbelliferone (1 mg/ml, final concentration) was incubated with erythrocytes for 1 h at 37°C and then cells were washed twice with PBS, there was no change in titer with the bacteria. The similar incubation of P. aeruginosa with 4-methylumbelliferone, however, resulted in a loss of titer from 1:64 to 1:8, suggesting that 4-methylumbelliferone was binding to the bacterial surface. The ability of the yeast mannan to inhibit hemagglutination may be related to the fact that mannan preparations have contaminating proteins. Our yeast mannan preparation contained 10.4% (wt/wt) protein, based on the standard Lowry assay. It is thought that fetuin, asialofetuin, and ovalbumin have hydrophobic domains which can bind to hydrophobic sites on the bacteria, erythrocytes, or both. We can find no basis for believing that lectin-like interactions are involved in the hemagglutination of erythrocytes by the P. aeruginosa strains examined by us. This does not, of course, exclude the possibility that other strains possess surface

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lectins (11) or that even our strains may express lectins under the proper conditions. Hydrophobic forces alone may account for the hemagglutination reactions. The inhibition of adherence of P. aeruginosa to buccal cells by fibronectin may be a result of the interaction of hydrophobic sites (10) between the epithelia and the protein. Fibronectin may possess hydrophobic domains (14) capable of binding to hydrophobic residues on buccal cells, thereby masking receptor sites for bacteria. Hydrophobic bond formation has also been implicated in the adhesion of Streptococcus sanguis to saliva-coated hydroxylapatite (6). When the washed P. aeruginosa strains were subjected to partitioning by hexadecane (10), there was no appreciable adherence to the bacteria to the nonaqueous phase (data not shown). This does not, however, rule out the presence of hydrophobic areas that could bind to complementary sites on erythrocytes. The results of this work suggest that, when studying the adherence and colonization of P. aeruginosa, interactions based on hydrophobic bonds must be considered. R.J.D. was a Fulbright Fellow to Israel when this research was

performed. We thank I. Ofek and N. Firon for discussions. LITERATURE CITED 1. Chan, R., J. S. Lam, K. Lam, and J. W. Costerton. 1984. Influence of culture conditions on expression of the mucoid mode of growth of Pseudomonas aeruginosa. J. Clin. Microbiol. 19:8-16. 2. Duguid, J. P., and D. C. Old. 1980. Adhesive properties of enterobacteriacae, p. 186-218. In E. H. Beachey (ed.), Bacterial adherence, receptors and recognition. Chapman & Hall, London. 3. Firon, N., I. Ofek, and N. Sharon. 1984. Carbohydrate-binding sites of the mannose-specific fimbrial lectins of enterobacteria. Infect. Immun. 43:1088-1090. 4. Gilboa-Garber, N. 1982. Pseudomonas aeruginosa lectins. Methods Enzymol. 83:378-385. 5. Glick, J., and N. Garber. 1983. The intracellular localization of Pseudomonas aeruginosa lectins. J. Gen. Microbiol. 129:30853090. 6. Nesbitt, W. E., R. J. Doyle, and K. G. Taylor. 1982. Hydrophobic interactions and the adherence of Streptococcus sanguis to hydroxylapatite. Infect. Immun. 38:637-644. 7. Ofek, I., E. H. Beachey, and N. Sharon. 1978. Surface sugars of animal cells as determinants of recognition in bacterial adherence. Trends Biochem. Sci. 3:159-160. 8. Pollack, M. 1984. The virulence of Pseudomonas aeruginosa. Rev. Infect. Dis. 6(Suppl. 3):617-626. 9. Ramphal, R., and M. Pyle. 1983. Evidence for mucins and sialic acid as receptors for Pseudomonas aeruginosa in the lower respiratory tract. Infect. Immun. 41:339-344. 10. Rosenberg, M., D. Gutnick, and E. Rosenberg. 1980. Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol. Lett. 9:29-33. 11. Speert, D. P., F. Eftekhar, and M. L. Puterman. 1984. Nonopsonic phagocytosis of strains of Pseudomonas aeruginosa from cystic fibrosis patients. Infect. Immun. 43:1006-1011. 12. Tsivion, Y., and N. Sharon. 1981. Lipid-mediated hemagglutination and its relevance to lectin-mediated agglutination. Biochim. Biophys. Acta 642:336-344. 13. Woods, D. E., D. C. Strauss, W. G. Johanson, Jr., and J. A. Bass. 1981. Role of fibronectin in the prevention of adherence of Pseudomonas aeruginosa to buccal cells. J. Infect. Dis. 143:784-790. 14. Yamada, K. M. 1983. Cell surface interactions with extracellular materials. Annu. Rev. Biochem. 52:761-799.