Pseudomonas aeruginosa binds to neoglycoconjugates bearing mucin carbohydrate determinants and predominantly to sialyl-Lewis x conjugates

Andrée Scharfman, Sophie Degroote, Julie Beau, Geneviève Lamblin, Philippe Roussel2 and Joël Mazurier1

Unité INSERM no. 377 and Université de Lille 2, Place de Verdun, 59045 Lille Cedex, France and 1UMR CNRS no. 111, USTL, 59655 Villeneuve d'Ascq Cedex, France

Received on July 14, 1998; revised on December 23, 1998; accepted on January 29, 1999

Pseudomonas aeruginosa plays an important role in the colonization of the airways of patients suffering from cystic fibrosis. It binds to the carbohydrate part of respiratory and salivary mucins and its binding to cystic fibrosis mucins is even higher, suggesting that qualitative or/and quantitative modifications of the carbohydrate chains may be involved in this process. In order to find out the best carbohydrate receptors for P.aeruginosa, a flow cytometry technique using a panel of polyacrylamide based glycoconjugates labeled with fluorescein was developed. The neoglycoconjugates contained neutral, sialylated or sulfated chains analogous to carbohydrate determinants found at the periphery of respiratory mucins (Lea, Ley, Lex, sialyl- and 3[prime]-sulfo-Lex, and blood group A determinants). We used also neoglycoconjugates containing Gal([alpha]1-2)Gal[beta] and sialyl-N-acetyllactosamine determinants. The interaction of these glycoconjugates with the nonpiliated strain of P.aeruginosa, 1244-NP, was saturable except for the glycoconjugates containing blood group A or sialyl-N-acetyllactosamine epitopes. The measure of Kd indicated that strain 1244-NP had a higher affinity for the glycoconjugate bearing the sialyl-Lex determinant than for all the other glycoconjugates studied. The role of sialic acid was confirmed by competition assay using mainly sialylated mucin glycopeptides. In order to find out if this behavior was the same for pathological strains as for the 1244-NP mutant, four mucoid strains of P.aeruginosa isolated from cystic fibrosis patients were analyzed with the Lex neoglycoconjugate, its sialylated and its sulfated derivatives. Individual variations in the binding of these strains to the three glycoconjugates were observed. However, three strains out of four had a higher affinity for the sialyl-Lex than for the 3[prime]-sulfo-Lex derivative.

Key words: binding/mucin-carbohydrates/Pseudomonas aeruginosa/sialyl-Lewis x

Introduction

Adhesion of P.aeruginosa, an opportunistic pathogen, to respiratory mucins and/or to epithelial glycoconjugates has attracted much attention because of its role as the major airway colonizer in immunocompromised hosts and patients with cystic fibrosis (Høiby, 1982).

Cystic fibrosis (CF), a general exocrinopathy, is the most common severe genetic disease among Caucasians. In its most typical form, the severity of the disease is due to mucus hypersecretion and to chronic lung infection characterized by the predominance of three bacteria, Hemophilus influenzae and Staphylococcus aureus in early life, and thereafter P.aeruginosa, often in a mucoid form, a pathogen which is responsible for most of the morbidity and mortality of the disease. CF is caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel of low conductance activated by protein-kinase A (Riordan, 1993) which has probably other functions (Barasch et al., 1991; Stutts et al., 1995; Devidas and Guggino, 1997; Pasyk and Foskett, 1997). The relation between the CFTR defect and the specificity of airways infection by P.aeruginosa is not yet elucidated.

The binding of P.aeruginosa to the human airway mucosa may involve several host receptors and bacterial adhesins. Pili present on the surface of P.aeruginosa recognize the sequence GalNAc([beta]1,4)Gal of asialo-GM1 and -GM2 receptors and may contribute to the binding of bacteria to epithelial cells (Lee et al., 1994), especially in a regenerating respiratory epithelium following damage (De Bentzman et al., 1996).

However, human airway mucins, a population of heavily glycosylated glycoproteins synthesized by goblet cells and bronchial glands of the human airway mucosa, are part of the mucociliary escalator which normally removes inhaled particles or microorganisms. Their very diverse carbohydrate chains act as recognition sites for bacteria or viruses. Indeed, several reports have demonstrated that various strains of P.aeruginosa adhere to mucins (Vishwanath and Ramphal, 1984; Ramphal et al., 1989; Sajjan et al., 1992; Carnoy et al., 1993; Devaraj et al., 1994) and that the adhesion process involves several adhesins localized on the outer membrane of P.aeruginosa (Carnoy et al., 1994). More recently, the role of flagella in the binding of the bacteria to mucins (Ramphal et al., 1996; Arora et al., 1998) and to cell surface glycolipids (Feldman et al., 1998) has been emphasized and the flagellar cap protein, Fli D, has been shown to be responsible for the adhesion of another nonpiliated strain of P.aeruginosa, PAK-NP, to human bronchial mucins (Arora et al., 1998).

In the airways of CF patients suffering from cystic fibrosis colonized by P.aeruginosa, most bacteria are embedded in the airway mucus and their interactions with respiratory mucins probably represent a major step in the development of the colonization process. Bronchial mucins secreted by patients suffering from cystic fibrosis (Roussel et al., 1975; Boat et al., 1976; Lamblin et al., 1977; Chace et al., 1985), by CF cells (Frates et al., 1983; Chen et al., 1989) or by xenograft models of CF cells (Zhang et al., 1995) are oversulfated, and it has been suggested that the [Delta]F508 deletion, the most frequent mutation in CF, leads to a misfolding of CFTR which is retained in the endoplasmic reticulum and degraded (Pind et al., 1994). Barasch et al., (1991) have suggested that this mislocation might affect the pH of the trans-Golgi network and modify the activity of various glycosyl- and sulfo-transferases. However, these data are controversial and it has been recently suggested that the concentration of PAPS in the Golgi lumen, the sulfate donor, is directly regulated by CFTR, which therefore may influence the sulfation processes (Pasky and Foskett, 1997).


Figure 1. Influence of the concentration of fluorescent glycoconjugate on the binding to P.aeruginosa 1244-NP. Bacterial suspension was incubated with increasing concentrations (from 6.25 to 250 nM) of Lex-PAA-flu (A), sialyl-Lex- (B) and sialyl-N-acetyllactosamine- (C) epitopes. After 30 min in the dark, a flow cytometry analysis was performed.

Previous reports have demonstrated an increased affinity of P.aeruginosa for respiratory (Devaraj et al., 1994) and salivary CF mucins (Carnoy et al., 1993), suggesting that the qualitative or/and quantitative modifications of the mucin carbohydrate chains may be involved in this process. Using different approaches, several neutral carbohydrate determinants have been shown to be potential receptors for different strains of P.aeruginosa (Ramphal et al., 1991a; Rosenstein et al., 1992). However, recent data indicate that, in addition to oversulfation, airway mucins from patients severely infected by P.aeruginosa may undergo increased sialylation as well as increased expression of sialyl-Lex epitopes (Davril et al., 1999).

In an attempt to find out the better carbohydrate receptors for P.aeruginosa, we have developed a flow cytometry technique using a panel of neoglycoconjugates and different strains of P.aeruginosa, (1) strain 1244-NP which is known to adhere to respiratory and salivary mucins carbohydrate chains (Ramphal et al., 1991b; Carnoy et al., 1993, 1994) and (2) four strains isolated from CF patients. The neoglycoconjugates contained neutral, sialylated or sulfated carbohydrate chains mostly analogous to carbohydrate determinants commonly found at the periphery of human respiratory mucins (Roussel and Lamblin, 1996), multivalently bound to a polyacrylamide carrier labeled with fluorescein (Bovin et al., 1993; Veerman et al., 1997). In most strains, the sialyl-Lex determinant was found to be a better receptor than its 3[prime] sulfated analog.

Results

Comparative binding of the different Gly-PAA-flu derivatives to P.aeruginosa 1244-NP

The different fluorescent neoglycoconjugates (Gly-PAA-flu) used to evaluate the binding to the non-mucoid and nonpiliated strain 1244-NP are listed in Table I. The binding of these neoglycoconjugates to Pseudomonas strains was analyzed by flow cytometry and was shown to be time dependent, reaching a plateau at 30 min (data not shown). This period was retained for subsequent experiments. The binding of the fluorescent derivatives containing Lea, Ley, Lex, sialyl-Lex, 3[prime]-sulfo-Lex or Gal([alpha]1-2)Gal[beta] was dose dependent and was saturable at a concentration varying from 62.5 nM up to 250 nM as was shown for Lex and sialyl-Lex in Figure 1A,B. In contrast, the binding to neoglycoconjugates containing the sialyl-N-acetyllactosamine and blood group A Gly-PAA-flu determinants was not saturable at concentrations up to 250 nM (Figure 1C). In the range of concentration used in the study (6.25-250 nM) the different fluorescent conjugates did not aggregate bacteria as shown by the dot plot analysis of forward scatter versus side scatter (data not shown).

Table I. Synthetic neoglycoconjugates used in the study
Carbohydrate Neoglycoconjugate
Lea Gal([beta]1-3)[Fuc([alpha]1-4)]GlcNAc[beta]-PAA-flu
Ley Fuc([alpha]1-2)Gal([beta]1-4)[Fuc([alpha]1-3)]GlcNAc[beta]-PAA-flu
Lex Gal([beta]1-4)[Fuc([alpha]1-3)]GlcNAc[beta]-PAA-flu
Blood group A determinant GalNAc([alpha]1-3)[Fuc([alpha]1-2)]Gal[beta]-PAA-flu
Gal([alpha]1-2)Gal[beta] Gal([alpha]1-2)Gal[beta]-PAA-flu
Sialyl-Lex Neu5Ac([alpha]2-3)Gal[beta]1-4[Fuc([alpha]1-3)]GlcNAc[beta]-PAA-flu
3[prime]-Sulfo-Lex HSO3-3Gal[beta]1-4[Fuc([alpha]1-3)]GlcNAc[beta]-PAA-flu
Sialyl-N-acetyllactosamine Neu5Ac([alpha]2-3)Gal([beta]1-4)GlcNAc[beta]-PAA-flu
PAA-flu, Polyacrylamide carrier labeled with fluorescein.


Figure 2. Scatchard analysis of the binding of Lex (A), sialyl-Lex (B) and sialyl-N-acetyllactosamine (C) fluorescent derivatives to P.aeruginosa 1244-NP. After incubation of bacteria with increasing concentrations of the fluorescent glycoconjugates, flow cytometry analysis was performed.

Scatchard analysis of the data (Figure 2, Table II) showed that there was differences in the binding of the different fluorescent derivatives to P.aeruginosa 1244-NP. The binding of derivatives containing Lea, Ley, Lex, 3[prime]-sulfo-Lex or Gal([alpha]1-2)Gal[beta] epitopes was monophasic, suggesting that there was only one single class of receptors (Figure 2A); the dissociation constant (Kd) ranged from 87 nM for Gal([alpha]1-2)Gal[beta] to ~50 nM for the other derivatives (Table II). The number of binding sites varied from 1569 ± 199 for the Ley-fluorescent derivative to 4500 ± 262 for the sulfo-Lex derivative. In contrast, the binding of P.aeruginosa 1244-NP to sialyl-Lex-PAA-flu was biphasic (Figure 2B) suggesting that there were two classes of binding sites for this determinant, one with high affinity (Kd =12 nM ± 4.2, number of binding sites = 1106 ± 345) and another one with low affinity (data not shown). The data obtained for the binding of blood group A and sialyl-N-acetyllactosamine fluorescent derivatives demonstrated that the binding of these two derivatives was non specific as shown for sialyl-N-acetyllactosamine in Figure 2C.

Table II. Comparative binding of the different Gly-PAA-flu to P.aeruginosa 1244-NP
Carbohydrate epitope Number of binding sites Dissociation constant (Kd nM)
Sialyl-Lex a 1106 ± 345 12 ± 4.2d
Sulfo-Lex a 4500 ± 262 49 ± 11
Lex a 4264 ± 159 60 ± 5.3
Lea a 2236 ± 273 51 ± 13
Ley b 1569 ± 199 50 ± 12
Gal([alpha]1-2)Gal[beta]c 3600 ± 332 87 ± 23
Results are expressed as mean ± SD.
aNumber of data point fitted for Scatchard analysis = 36.
bNumber of data point fitted in Scatchard analysis = 18.
cNumber of data point fitted in Scatchard analysis = 12.
dSignificantly different when compared to Kd obtained with the other Gly-PAA-flu (p < 0.001).

Altogether the values obtained for Kd and number of binding sites showed that the affinity of fluorescent derivatives containing Lea, Ley, Lex, 3[prime]-sulfo-Lex or Gal([alpha]1-2)Gal[beta] epitopes for P.aeruginosa 1244-NP was in the same order of magnitude. The value of Kd obtained for the sialyl-Lex derivative indicated that P.aeruginosa 1244-NP had an higher affinity for this neoglycoconjugate than for all the other glycoconjugates studied so far (p < 0.001).

Specificity of the binding of the Gly-PAA-flu to P.aeruginosa 1244-NP

To check the specificity of the interaction between fluorescent derivatives and P.aeruginosa 1244-NP, competition binding assays were performed for three of them, Lex-, Gal([alpha]1-2)Gal[beta]-, and sialyl-Lex-PAA-flu.

Incubation of Lex-PAA-flu and bacteria in presence of 100-fold molar excess of the corresponding unlabeled Lex derivative, resulted in a decrease of the logarithm of fluorescence intensity, when compared to control as shown in Figure 3 (4250 equivalent bound particles but 8250 in the absence of unlabeled Lex polyacrylate derivative). Reduction of intensity of fluorescence was evidenced by the Kolmogorov-Smirnov two-sample test. On the contrary, the fluorescence intensity of the Lex-PAA-flu did not change when incubation was performed in presence of unlabeled Gal([alpha]1-2)Gal[beta] polyacrylate derivative. Moreover, Kd value obtained for the binding of Lex-PAA-flu was not modified (p > 0.05) when incubation was performed in presence of unlabeled Gal([alpha]1-2)Gal[beta] derivative.


Figure 3. Specificity of the binding of Lex-PAA-flu to P.aeruginosa 1244-NP. Bacterial suspension was incubated with PBS alone (A),with a 62.5 nM solution of Lex-PAA-flu in presence (B) or in absence (C) of a 100 molar excess of unlabeled Lex polyacrylate derivative. After 30 min in the dark, flow cytometry analysis was performed. Representative histograms: x-axis, log of the fluorescence intensity; y-axis number of fluorescent cells.

Competitive binding between Gal([alpha]1-2)Gal[beta]-PAA-flu and the corresponding unlabeled polyacrylate derivative resulted in a decrease of the fluorescence intensity (5400 equivalent bound particles but 8150 in absence of unlabeled derivative). Reduction of intensity was evidenced by the Kolmogorov-Smirnov two-sample test. Fluorescence intensity was not changed when incubation with Gal([alpha]1-2)Gal[beta]-PAA-flu was performed in presence of unlabeled Lex polyacrylate derivative.

Competition binding assays were also performed with sialyl-Lex-PAA-flu and unlabeled glycoconjugates bearing either sialyl-Lex, Lex or Gal([alpha]1-2)Gal[beta] epitopes. The presence of an excess of unlabeled sialyl-Lex derivative in the incubation mixture led to an aggregation of the bacteria as visualized by an increase of the forward scatter (data not shown). The addition of Lex- and Gal([alpha]1-2)Gal[beta]-PAA to incubation mixtures did not modify the binding of sialyl-Lex-PAA-flu to P.aeruginosa 1244-NP. In addition, Kd value obtained for the binding of sialyl-Lex was not changed in presence of an excess of the unlabeled Gal([alpha]1-2)Gal[beta] derivative (p > 0.05), confirming the absence of cross-binding of these two derivatives to P.aeruginosa. Additional experiments also demonstrated that the Kd values of the binding of sialyl-Lex-PAA-flu was not modified by addition of free stachyose in the incubation mixture. Altogether, these results demonstrated that there was no cross-reactivity between Lex, Gal([alpha]1-2)Gal[beta] epitopes and sialyl-Lex-PAA-flu to P.aeruginosa 1244-NP.

Competitive binding of Gly-PAA-flu and airways mucins glycopeptides to P.aeruginosa 1244-NP

Binding assays were also performed in presence of an excess of different airway mucin glycopeptides, neutral, mainly sialylated, or mainly sulfated glycopeptides (Ramphal et al., 1989). As demonstrated by the number of equivalent bound particles, the binding of Gly-PAA containing Lex, sialyl-Lex or 3[prime]-sulfo-Lex to P.aeruginosa was partly inhibited by mucin glycopeptides (Table III). The binding of Lex-PAA-flu was equally inhibited by the three classes of glycopeptides, while the binding of 3[prime]-sulfo-Lex-PAA-flu was better inhibited by mainly sulfated glycopeptides and the binding of sialyl-Lex-PAA-flu better inhibited by mainly sialylated glycopeptides (Table III). Inhibition experiments were also performed using purified airway mucins from CF and non-CF patients showing that the binding to the sialyl-Lex derivatives was better inhibited by CF mucins (23% of inhibition) than by non-CF mucins (7%).

Table III. Inhibition of binding of Lex, sialyl-Lex, 3[prime]-sulfo-Lex-PAA-flu to P.aeruginosa 1244-NP after competition with airway mucin glycopeptides
Gly-PAA-flu % of inhibition by glycopeptidesa
Neutral Mainly sialylated Mainly sulfated
Lex 17 15 11
Sialyl-Lex 20 35 13
3[prime]-Sulfo-Lex 10 15 25
aThe percentage of inhibition was calculated from the mean values of fluorescence intensity obtained after incubation with or without mucin glycopeptides.

Binding of Lex, sialyl-, and 3[prime]-sulfo Lex-PAA-flu derivatives to clinical strains of P.aeruginosa

In order to determine whether pathological strains recognize also preferentially sialyl-Lex determinants, the binding of Lex-, sialyl-Lex-, or 3[prime]-sulfo-Lex-PAA-flu to four mucoid strains isolated from CF patients was compared by using flow cytometry and Scatchard analysis. All these neoglycoconjugates bound to the four pathological strains. However, there were individual variations in the binding (Table IV). Strain 6118 bound equally to the three derivatives. In contrast, the three other strains (690, 6190, and 130308) bound better to the sialyl-Lex derivative than to the 3[prime]-sulfo-Lex derivative. For two of these strains (690 and 6190), there was no significant differences between their binding to the Lex and to the sialyl-Lex derivatives.

Table IV. Binding of Lex-, sialyl-Lex- and 3[prime]sulfo-Lex-neoglycoconjugates to four strains of P.aeruginosa isolated from CF patients
Mucoid strains Kd p
LPF Sialyl-LPF 3[prime]Sulfo-LPF LPF vs sialyl-LPF LPF vs 3[prime]sulfo-LPF Sialyl-LPF vs sulfo-LPF
690a 80 ± 22 46 ± 6 88 ± 7 N.S. N.S. < 0.005
6118b 70 ± 13 43 ± 34 98 ± 46 N.S. N.S. N.S
6190b 36 ± 14 27 ± 3 93 ± 18 N.S. < 0.05 < 0.05
130,308b 100 ± 25 43 ± 6 129 ± 23 < 0.05 N.S. < 0.0125
LPF, Lex-PAA-flu; N.S., not significantly.
aNumber of data fitted in Scatchard analysis = 26.
bNumber of data fitted in Scatchard analysis = 16.

Discussion

The aim of the present work was to set up a flow cytometry assay using polyacrylamide-based glycoconjugates labeled with fluorescein (Gly-PAA-flu) (Bovin et al., 1993), in order to compare the binding of P.aeruginosa to a panel of conjugates containing carbohydrate determinants analogous to the peripheral part of many carbohydrate chains present in human airway mucins (Roussel and Lamblin, 1996). Several proteins of the outer membrane or the flagella of P.aeruginosa have already been shown to be recognized by various human mucins (Carnoy et al., 1994; Ramphal et al., 1996; Arora et al., 1998), and several carbohydrate determinants have been identified as possible sites of attachment of this bacteria. In contrast to previous techniques used to identify carbohydrate receptors recognized by P.aeruginosa, such as chromatography of glyco- or neoglycolipids followed by bacteria overlay (Ramphal et al., 1991a; Rosenstein et al., 1992), flow cytometry allows the interactions bacteria/receptors to occur in a liquid phase. With this technique, using polyacrylamide based glycoconjugates labeled with fluorescein, carbohydrate receptors of P.aeruginosa surface lectins can be identified. The type of binding of the different glycoconjugates (Table I) to P.aeruginosa 1244-NP was not identical and was dependent on the carbohydrate epitope. The binding of blood group A and sialyl-N-acetyllactosamine fluorescent glycoconjugates was not saturable. It was saturable and monophasic for polyacrylate derivatives bearing Lea, Ley, Lex, 3-sulfo-Lex and Gal([alpha]1-2)Gal[beta] epitopes while there was two binding sites, for the polyacrylate derivative containing the sialyl-Lex epitope, one of them with high affinity.

To assess the specificity of the binding, different competition experiments were performed using three different fluorescent conjugates. Competition experiments demonstrated that stachyose which does not exist in respiratory mucins was not recognized by P.aeruginosa 1244-NP. The binding of Lex and Gal([alpha]1-2)Gal[beta]-PAA-flu to P.aeruginosa 1244-NP showed a decrease of 50% in the presence of an excess of the corresponding unlabeled polyacrylate derivative. However, it was not possible to obtain a total inhibition: this may be due to a nonspecific fixation of the fluorescent groups on the bacteria (Babiuk and Paul, 1970; Chatelier et al., 1995). Similarly, competing molecules, such as mucin glycopeptides, are not able to inhibit more than 50% of the binding of the fluorescent glycoconjugates. Incubation of sialyl-Lex with an excess of the unlabeled derivative led to an aggregation of the bacterial population; Komiyama and associates (Komiyama et al., 1987) have suggested a role of sialic acid in the aggregation of P.aeruginosa by saliva.

In the present study, Lea, Lex, and Ley determinants were identified as receptors for P.aeruginosa 1244-NP and the Kd values of the binding of these three neoglyconjugates were not significantly different. On another hand, the binding of this bacteria to the blood group A and to the sialyl-N-acetyllactosamine neoglycoconjugates was found to be not saturable, attesting a nonspecific binding of these two neoglycoconjugates to P.aeruginosa 1244-NP which again may be due to the fluorescent groups.

Since P.aeruginosa synthesizes an internal lectin, PA-IL, which binds to d-galactose in [alpha] or [beta] anomeric configuration (Chen et al., 1998) and since its best ligands have a terminal Gal[alpha], we also assayed the Gal([alpha]1,2)Gal[beta]-PAA-flu, although human mucins are not supposed to contain terminal Gal[alpha], except in mucins with blood group B and mucins which contain a few Gal([alpha]1,3)GalNAc chains (van Halbeek et al., 1994). The binding of P.aeruginosa to this determinant was in the same order of magnitude as the binding to Lex or Lea determinants which have a terminal Gal[beta]. Competition experiments revealed the absence of cross-binding between fluorescent polyacrylate derivatives carrying Lex and Gal([alpha]1-2)Gal[beta] derivatives, suggesting that these two epitopes are recognized by different lectins. Glick and Garber, (1983) have reported that, even if most PA-IL is internal, small quantities of the lectin may exist on the outer membranes and therefore may be involved in the recognition of the Gal([alpha]1-2)Gal[beta] epitope. Since the pattern of the outer membrane proteins that act as bacterial adhesins for airway mucins varies from one strain of P.aeruginosa to the other (Carnoy et al., 1994), it will be necessary, in the future, to determine the different surface adhesins involved in the recognition of the different neutral carbohydrate chains.

Neoglycoconjugates containing acidic determinants, sialyl-Lex or 3[prime]-sulfo-Lex, were also recognized by P.aeruginosa 1244-NP. The present study indicates that these neoglyconjugates were much better receptors than the neoglyconjugate containing sialyl-N-acetyllactosamine. As far as the binding of the sialyl-Lex derivative is concerned, strain 1244-NP had a better affinity for this derivative than for all the other glycoconjugates studied. The dissociation constant obtained for the binding of 1244-NP to 3[prime]-sulfo-Lex containing glycoconjugates suggested that the preferential affinity of 1244-NP strain for the sialyl-Lex epitope was not due to the substitution of Lex by any acidic residue but indeed by the presence of sialic acid residue. The role of sialic acid was confirmed by competition binding assays showing that the binding of P.aeruginosa to the sialyl-Lex epitope was better inhibited by mainly sialylated mucin glycopeptides than by neutral or mainly sulfated glycopeptides, and also better by CF mucins than by non-CF mucins. CF mucins indeed contain more sialic acid than non-CF mucins (Davril et al., 1999). Competitive experiments demonstrated the absence of cross binding between sialyl-Lex-PAA-flu and polyacrylate bearing neutral epitopes such as Lex and Gal([alpha]1-2)Galb, suggesting that the receptor for sialyl-Lex glycoconjugate had a restricted specificity. Therefore, for most strains analyzed in the present study, the sialyl-Lex polyacrylate derivative is a major receptor.

The role of sialic acid in the binding of P.aeruginosa has already been suggested by several reports (Baker et al., 1990; Ramphal et al., 1991a). Moreover, desialylation of human salivary mucin glycopeptides has also been shown to reduce their binding to the piliated strain 1244-NP (Carnoy et al., 1993).

Finally, the preferential inhibition of the binding of strain 1244-NP to the sialyl-Lex or the sulfated Lex-neoglycoconjugates by the corresponding sialylated or sulfated mucin glycopeptides is in favor of at least two adhesins having different affinities to these two neoglycoconjugates.

There are differences in the binding of P.aeruginosa PAK and 1244 strains, as well as their nonpiliated derivatives, to different glycoconjugates and in the pattern of outer membrane proteins recognized by airway mucins (Ramphal et al., 1991b; Carnoy et al., 1994). In order to find out whether the behavior of P.aeruginosa is the same for pathological strains as for the 1244-NP strain, four mucoid strains isolated from CF patients were also analyzed with the Lex neoglyconjugate and its sulfated and sialylated derivatives. Three strains out of four bound better to the sialyl-Lex than to the 3[prime]-sulfo-Lex derivatives. It should be noticed that the results obtained with these clinical isolates showed some variations in the binding to the three neoglyconjugates from one experiment to another, as indicated by standard deviations. A similar problem has been encountered by Devaraj et al. in studying the binding of P.aeruginosa to CF and non-CF mucins (Devaraj et al., 1994). Moreover, Govan et al. have pointed out the instability of mucoid strains during in vitro cultures leading to reversion of the mucoid to nonmucoid forms (Govan et al., 1979).

These findings concerning sialylated or sulfated neoglycoconjugates are of special interest when considering the abnormalities of salivary and respiratory mucins secreted by CF patients. CF salivary mucins have been found to be more sulfated and more sialylated than mucins from controls (Carnoy et al., 1993) and, moreover, they are better recognized by various strains of P.aeruginosa. An overexpression of sialyl-Lex determinants has been observed in various inflammatory conditions, for instance among the acute phase glycoproteins synthesized in the liver under cytokine stimulation (Van Dijk et al., 1998). Recently, we have observed an increased sialylation and an increased expression of sialyl-Lex epitopes in the airway mucins from patients suffering from CF, in addition to increased sulfation (Davril et al., 1999). This increased sialylation was not specific for cystic fibrosis since it was also observed in the airway mucins secreted by severely infected patients suffering from chronic bronchitis. This observation may suggest that overexpression of sialyl-Lex determinants on glycoproteins synthesized by the airway mucosa may be viewed as the signature of local inflammation, in the same way as acute phase proteins synthesized by the liver which contain more sialyl-Lex epitopes during inflammation (Van Dijk et al., 1998). Several reports currently suggest that, in CF airways, the inflammatory response to aggression is abnormal (Birrer et al., 1994; Khan et al., 1995; Bonfield et al., 1997) and that inflammation might pave the way to the initial lung colonization by P.aeruginosa (Heeckeren et al., 1997). Therefore, the attachment of P.aeruginosa to the sialyl-Lex epitope may be especially important in the development of lung infection in CF.

In conclusion, P.aeruginosa recognizes a whole set of neutral and acidic carbohydrate determinants, and especially the sialyl-Lex epitope. In the future, it will be important to find out (1) how many bacterial adhesins from P.aeruginosa may be involved in the airway colonization of CF patients, (2) which one is specific for the sialyl-Lex epitope, and (3) whether it is expressed in all strains colonizing CF patients.

Material and methods

Bacteria strains and culture conditions

Strain 1244-NP (provided by S.Lory from University of Washington) is an isogenic nonpiliated, nonmucoid strain (Ramphal et al., 1991b) which has already been shown to adhere to mucin carbohydrate chains (Carnoy et al., 1993) and to neoglycolipids (Ramphal et al., 1991a). Other strains were isolated from CF patients as mucoid isolates. All strains were grown in tryptic soy broth (TSB medium, Difco, Detroit) for 18 h at 37°C. After centrifugation of the cultures at 4000 × g for 30 min, the cell pellets were washed twice by a filtered physiologic saline containing 5% (v/v) TSB and then resuspended in the same solution. Optical density measurements were used to obtain a bacterial suspension of ~107 CFU/ml. The exact number of bacteria was determined by dilution and plating of the suspension.

Preparation of human respiratory mucins and mucins glycopeptides

High molecular weight mucins were prepared from sputum of patients (blood group O) suffering either from chronic bronchitis or from CF, by two steps of cesium bromide density-gradient ultracentrifugation as already described (Houdret et al., 1986). Mucins obtained after the first step of ultracentrifugation were treated by a cocktail of enzymes (Lo Guidice et al., 1994) to get rid of nucleic acids and proteoglycans, often associated with mucins of patients suffering from cystic fibrosis (Rahmoune et al., 1991), and then submitted to the second step of ultracentrifugation.

Glycopeptides were obtained by pronase digestion of mucins followed by fractionation by ion exchange chromatography allowing to obtain neutral, mainly sialylated and mainly sulfated glycopeptides (Ramphal et al., 1989).

Binding assay

Analysis of the binding of the different glycoconjugates to P.aeruginosa was performed by flow cytometry using fluorescent polymeric neoglycoconjugates obtained from Syntesome (Munich, Germany) (Bovin et al., 1993). In these compounds (Gly-PAA-flu), a fluorescent-labeled polyacrylamide matrix is N-substituted every fifth amide group by a carbohydrate determinant on a spacer arm (-(CH2)3-). The carbohydrate/fluorescein molar ratio is 20:1. Unsubstituted sides group of the polymer were converted into -CONHCH2CH2OH. The different Gly-PAA-flu used in the present study are listed in Table I.

Bacteria cells were resuspended at a concentration of 2 × 106 CFU/ml in phosphate-buffered saline (PBS) containing 1% (w/v) bovine serum albumin (BSA), and 0.5 ml aliquots were incubated for 30 min in the dark with increasing amounts of the different Gly-PAA-flu (6.25-250 nM). Controls were obtained by omitting glycoconjugates in the incubation mixture (Figure 3A). The mixtures were analyzed on a FACScan cytofluorimeter (Becton-Dickinson) using FACScan and LYSIS software for acquisition and analysis respectively. The light scatters channels were set on a linear gain scale, the flow rate was kept at approximately 200 events per second and 5000 events were collected. Acquisition was done on gated populations (by forward and side scatters) to exclude cell debris and cell aggregates. The green fluorescence was set on a logarithmic scale and the mean value of logarithm of fluorescence was converted in equivalent bound particles using calibrated beads to which a known number of fluorescent particles has been covalently bound (Immuno-Britt, Coulter Counter). Results were expressed as equivalent bound particles after deduction of the value of control. Experimental data were expressed as mean value ± standard variations. Binding capacity and dissociation constants were calculated according to Scatchard using the nonlinear progression data analysis program Enzfitter (Cambridge, UK). For statistical analysis, the Student's test was used. To determine t, the following equation was used, t = [kd1(1) - kd1(2)]/[sqrt(SE(1)^2 + SE(2)^2)] for(n1 + n2 - 4) degrees of freedom, where kd1(1) and kd1(2) are fitted values for the parameters for data sets 1 and 2, SE(1) and SE(2) are the standard errors for the fitted values, and n1 and n2 are the numbers of datum points in data sets 1 and 2.

Inhibition experiments

Competition assays were performed for the binding of fluorescent glycoconjugates bearing Lex, Gal([alpha]1-2)Gal[beta] and sialyl-Lex epitopes. The bacterial suspension was incubated with a 62.5 nM solution of each of the fluorescent glycoconjugates in presence of a 100 molar fold excess of unlabeled polyacrylate derivatives. Bacteria incubated under the same experimental conditions, without any potential inhibitors, are used as controls. After flow cytometry analysis, the value of mean of fluorescence intensity (expressed as equivalent bound particles) were compared. The Kolmogorov-Smirnov two-sample test was used to calculate the probability that two histograms were different.

Incubation with Lex-PAA-flu was performed in presence or in absence of unlabeled polyacrylate derivatives bearing Lex or Gal([alpha]1-2)Gal[beta] epitopes. The same experiments were performed with Gal([alpha]1-2)Gal[beta]-PAA-flu, using in this case, unlabeled conjugates with Lex or Gal([alpha]1-2)Gal[beta] epitopes.

Competition experiments of the binding of sialyl Lex-PAA-flu were performed in presence of the corresponding unlabeled polyacrylate derivative and of unlabeled polyacrylate derivatives bearing Lex or Gal([alpha]1-2)Gal[beta] epitopes.

In some experiments increasing concentration of Lex or sialyl-Lex (6.25 to 250 nM) were incubated with bacterial suspension in presence of an 40 molar excess of unlabeled Gal([alpha]1-2)Gal[beta] polyacrylate derivative and of free stachyose. After flow cytometry analysis, binding capacities and dissociation constants were calculated as described above.

Competition binding of Gly-PAA-flu and mucins glycopeptides to P.aeruginosa 1244-NP

Assays were performed in the presence of a 100-fold excess of mucin glycopeptides (neutral, mainly sialylated or mainly sulfated).

The bacterial suspension (0.5 ml) was incubated with a 62.5 nM solution of Gly-PAA-flu in presence or absence of mucin glycopeptides (100 µg/ml). After flow cytometry analysis, the percentage of inhibition was calculated from the mean values of fluorescence intensity (expressed as equivalent bound particles), obtained after incubation with or without mucin glycopeptides. In some experiments, incubation of sialyl-Lex-Gly-PAA-flu with P.aeruginosa was performed in presence of CF or non-CF high molecular weight mucins (50 µg/ml).

Analytical procedure

Sugar analysis was carried out by gas-liquid chromatography of trimethylsilyl derivatives of methyl-glycosides (Lamblin et al., 1984). N-acetylneuraminic acid was measured by the thiobarbituric acid assay of Aminoff, (1961) after hydrolysis with 0.1 M H2SO4 for 30 min at 80°C. Sulfate content was determined by HPAEC after hydrolysis with 1 M HCl as described previously (Lo-Guidice et al., 1994).

Acknowledgments

We thank Claude Galabert (Hopital Renée Sabran, Giens 83400 Hyères, France) and Christelle Neut (Laboratoire de Microbiologie, Faculté de Pharmacie, Lille, France) for providing us with strains from CF patients. This investigation was supported by the Association Française de Lutte contre la Mucoviscidose and the Réseau Régional d'Études des Interactions Hôtes-Microorganismes (Centre Hospitalier et Université de Lille-France).

Abbreviations

CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; asialo-GM1, gangliotetraosyl ceramide; asialo GM2, gangliotriaosyl ceramide; PAPS, adenosine 3[prime]-phosphate 5[prime]-phosphosulfate; NP, nonpiliated; Gly-PAA-flu, polyacrylamide-based glycoconjugates labeled with fluorescein; Le, Lewis; Kd, dissociation constant; PA-IL, Pseudomonas aeruginosa (PA-I) lectin; TSB, tryptic soy broth; CFU, colony forming unit; Gal, galactose; Fuc, fucose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Neu5Ac, N-acetylneuraminic acid.

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2To whom correspondence should be addressed at: Unité INSERM no. 377, Place de Verdun, 59045 Lille Cedex, France


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