Inhibition of cathelicidin activity by bacterial exopolysaccharides

Summary

The interaction of bacterial exopolysaccharides, produced by opportunistic lung pathogens, with antimicrobial peptides of the innate primate immune system was investigated. The exopolysaccharides were produced by Pseudomonas aeruginosa, Inquilinus limosus and clinical isolates of the Burkholderia cepacia complex, bacteria that are all involved in lung infections of cystic fibrosis patients. The effects of the biological activities of three orthologous cathelicidins from Homo sapiens sapiens, Pongo pygmaeus (orangutan) and Presbitys obscurus (dusky leaf monkey) were examined. Inhibition of the antimicrobial activity of peptides was assessed using minimum inhibitory concentration assays on a reference Escherichia coli strain in the presence and absence of exopolysaccharides, whereas complex formation between peptides and exopolysaccharides was investigated by means of circular dichroism, fluorescence spectroscopy and atomic force microscopy. Biological assays revealed that the higher the negative charge of exopolysaccharides the stronger was their inhibiting effect. Spectroscopic studies indicated the formation of molecular complexes of varying stability between peptides and exopolysaccharides, explaining the inhibition. Atomic force microscopy provided a direct visualization of the molecular complexes. A model is proposed where peptides with an α-helical conformation interact with exopolysaccharides through electrostatic and other non-covalent interactions.

Introduction

Bacteria produce a number of polysaccharidic molecules that are exposed to the surrounding medium. Polysaccharides can be part of the external membrane (e.g. lipopolysaccharides of Gram-negative bacteria), can form a capsule around the microbe (capsular polysaccharides) or can be released into the extracellular medium (exopolysaccharides, EPS). In all cases, saccharidic macromolecules constitute an important interface between bacteria and their environment, a milieu essential for their own survival. This paper focuses on bacterial EPS.

Considering pathogenic bacteria, polysaccharides are involved in a large number of biological functions either promoted by their common structural properties (i.e. abundance of hydroxyl groups, hydrophilicity, etc.) or driven by specific chemical and conformational motifs of each EPS. As far as their common properties are concerned, EPS help constitute an environmental niche around bacteria or colonies serving to maintain the proper hydration, acting as a physical barrier against external threats and trapping useful micronutrients (Ophir and Gutnick, 1994). Together with other biomacromolecules, EPS form the skeleton of biofilms where bacterial colonies grow in organized communities, regulated by the switching on and off of specific genes. As a matter of fact, microorganisms able to produce large amount of EPS (mucoid strains) can be found after the onset of lung infection in cystic fibrosis patients, as reported for Pseudomonas aeruginosa (Deretic et al., 1990; Govan and Deretic, 1996) and for some species of the Burkholderia cepacia complex (Chung et al., 2003; Zlosnik et al., 2008).

More specific biological functions exhibited by bacterial polysaccharides have been investigated in recent studies. Pier et al. reported that acetylated alginate produced by P. aeruginosa prevented activation of the alternative pathway of the complement system and scavenged hypochlorite produced by activated phagocytes (Pier et al., 2001). O-acetyl substitution on EPS is believed to be used by bacteria to mask hydroxyl groups that are the binding site for opsonins C3b and C4b. Reckseidler-Zenteno et al. (2005) demonstrated a drastically reduced persistence in human serum of a mutant strain of Burkholderia pseudomallei unable to produce the capsular polysaccharide. Using immunofluorescence microscopy in the presence of antibodies for the human complement factor C3b, they demonstrated that the capsule contributes to resistance to phagocytosis by reducing C3b deposition on the surface of the bacterium. Finally, Brown and Gordon (2001) recognized the presence of a new dectin-like receptor on macrophages capable of binding β-(1→3) glucans. This shed new light on innate immune recognition of β-glucans, which exert potent effects on the immune system by stimulating anti-tumour and antimicrobial activity.

In order to further increase the understanding of the connections between bacteria and the innate immune system, this paper presents data on the interaction between EPS produced by opportunistic bacteria and antimicrobial peptides (AMP) from the cathelicidin family, which are secreted onto mucosal surfaces and stored in the granules of phagocytes (Ganz and Lehrer, 1998). The interaction of AMP with bacterial membranes leads to their disruption and eventually to cell death. The precise mechanism of action of cathelicidins has not yet been completely elucidated, although a model based on a ‘carpet’ interaction between AMP and bacterial membrane, possibly also involving the formation of ‘wormhole’ or toroidal pores, has been proposed (Dürr et al., 2006; Porcelli et al., 2008). Regardless the actual mechanism, AMP must in any case be free to interact with the cytoplasmic membrane. Interactions with (macro)molecules in the bacterial cell environment will modulate, and could jeopardize their biological action. Preliminary results on this topic have already been reported (Herasimenka et al., 2005) and a possible model of the molecular interaction of human and sheep cathelicidins with EPS, based on AMP association and complexation with the polysaccharide chain, was proposed. A more systematic study is now presented where human (hssLL-37), Pongo pygmaeus (ppyLL-37) and Presbytis obscurus (pobRL-37) orthologous of the α-helix forming cathelicidin LL-37, are investigated in the presence of EPS produced by bacteria involved in lung infections of cystic fibrosis patients. These orthologues vary in charge (from +4 to +10) and property for assuming a helical conformation in aqueous buffer (Zelezetsky et al., 2006). The primary structure, net positive charge and amphipatic properties of the AMP used are reported in Table 1. HssLL-37 and ppyLL-37 exhibit a moderate and low net positive charge (q) respectively, and possess a higher mean residue hydrophobicity (〈H〉) than the more highly cationic pobRL-37, whereas the amphipathicities (〈μH_rel〉) are similar.

Table 1.  Primary structure, net positive charge and amphypathic properties of cathelicidins used in this study

Cathelicidin		+q	〈H〉	〈μHrel〉
+q, net positive charge; 〈H〉, mean residue hydrophobicity (the negative value indicates a prevalence of the hydrophilic character); 〈μHrel〉, relative amphipathicity.
Homo sapiens (hssLL-37)	LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES	6	−1.83	0.59
Pongo pygmaeus (ppyLL-37)	LLGDFFRKAREKIGEEFKRIVQRIKDFLRNLVPRTES	4	−1.70	0.59
Presbitys obscurus (pobRL-37)	RLGNEFRKAKKKIGRGLKKIGQKIKDFLGNLVPRTES	10	−2.12	0.62

Different EPS have been considered: (i) alginate produced by P. aeruginosa strain PAO1; (ii) cepacian produced by Burkholderia pyrrocinia strain BTS7 (Lagatolla et al., 2002); (iii) a mixture composed by a mannan and a glucan, both fully pyruvilated, produced by Inquilinus limosus, hereafter referred to as IL (Herasimenka et al., 2007); and (iv) a mixture of cepacian, PS-I and dextran, hereafter referred to as C9343, produced by Burkholderia cenocepacia strain C9343 (Conway et al., 2004). Cepacian is the EPS produced by the majority of the B. cepacia complex clinical isolates, either as the only EPS, or in a mixture with other different EPS, so that it can be considered as representative of this bacterial group. The primary structures of these EPS are reported in Fig. 1. Mixtures of different EPS cosynthesized by bacteria were also included in this study to try to asses how this characteristic influences the biological activity of EPS. It is worth mentioning that the above EPS are able to form polymer aggregates that contribute to the biofilm architecture, where acetyl groups play a stabilizing role (Nivens et al., 2001; Herasimenka et al., 2008).

Figure 1. Chemical structures of the EPS investigated.

In the present investigation, besides microbiological assays and physico-chemical studies, microscopy experiments, based on the powerful atomic force technique, were performed for a direct visualization of AMP–EPS interactions.

Results and discussion

EPS and AMP structures

All EPS investigated are negatively charged due to the presence of carboxylate moieties, except for dextran, which is part of the C9343 mixture. Due to the high number of charged groups, and to the polydispersity of the molecular masses, the distribution of electric charges present on a polyelectrolyte is often indicated as the charge density, which is proportional to the number of charges in the polymer repeating unit divided by its contour length (Table S1). Alginate and EPS from I. limosus exhibit a high charge density, as every sugar unit bears a carboxylate group, while cepacian contains only one glucuronic acid per repeating unit (see Fig. 1). The charge density of the mixture C9343 is less easy to evaluate. The mixture is composed of cepacian (38.5%, w/w), PS-I (23.0%), which bears one negative charge every two residues, and the neutral dextran (38.5%). Therefore, considering the mean overall charge density of the three polymers, the mixture C9343 should exhibit a value similar to or slightly higher than that of cepacian. Both alginate and cepacian sugar residues are also modified by the presence of acetyl groups, with a variable degree of substitution: 60% of the monomers in alginate bear acetyl groups (Theilacker et al., 2003), while cepacian repeating units contain three acetyl groups, as shown by NMR spectra.

All three cathelicidins considered have the tendency to form an α-helical structure that is the conformation that promotes both peptide aggregation in aqueous buffer and interaction with bacterial membranes (Johansson et al., 1998; Zelezetsky et al., 2006; Morgera et al., 2008). This is in part due to an asymmetric distribution of hydrophilic and hydrophobic amino acid residues onto the cylindrical α-helical surface, which is quantified as the relative amphipaticity (〈μH_rel〉, Table 1). In the case of hssLL-37 and ppyLL-37, the formation of intramolecular salt-bridging stabilizes helix formation in aqueous solution in the presence of physiological ions, whereas for pobRL-37 helix formation is normally observed only in the presence of a membrane-like environment (Zelezetsky et al., 2006). A schematic representation of the amphipaticity of these peptides is provided by their α-helical wheel projection, as shown for hssLL-37 in Fig. 2.

Figure 2. Helical wheel projection of human cathelicidin hssLL-37. Residues from 1 to 18 are reported in the internal ring; residues from 19 to 36 are reported in the external ring. Black squares indicate hydrophobic residues whereas white circles indicate hydrophilic residues. Amino acid residues are labelled as one-letter code.

Minimum inhibitory concentration assays

The evaluation of the minimum amount of AMP required to inhibit the growth of Escherichia coli strain ML-35 in the presence of bacterial EPS gave the data shown in Fig. 3. The four EPS investigated had different effects on the antibacterial activity of a specific cathelicidin, but the three cathelicidins showed a similar behaviour in the presence of a given EPS. In particular, alginate afforded the highest peptide inhibition activity, while cepacian gave the lowest. IL had an activity similar to alginate, while C9343 exhibited an effect similar to cepacian. The addition of C9343 to AMP solutions caused some precipitation, which on one hand might induce artefacts in the experimental data, but on the other may indicate a strong capacity to segregate AMP.

Figure 3. E. coli strain ML-35 minimum inhibiting concentration experiments for hssLL-37; pobRL-37 and ppyLL-37 in the presence of 0.5 mg ml⁻¹ of alginate, cepacian, IL and C9343.

The influence of EPS on AMP activity correlated well with the amount of negative charge carried by the different EPS. In fact, alginate and IL bear the highest number of charges per chemical repeating unit, cepacian exhibits the lowest charge density and the mixture C9343 should exhibit a value similar or slightly higher than that of cepacian, as already discussed. It is worth mentioning that positively charged polysaccharides synthesized by bacteria as important biofilm component also protect against AMP activity. As reported by Vuong et al. (2004a,b), poly N-acetylglucosamine (PNAG) produced by Staphylococcus epidermidis protects against human β-defensin and LL-37. As it has been proposed that PNAG adheres to the negatively charged bacterial surface via electrostatic interactions, the increased positive potential around bacteria might provide an electrostatic barrier against positively charged peptides, thus explaining the PNAG protecting role.

The different behaviour of ppyLL-37, in the presence of alginate and IL, suggested that, besides the charge density, the specific conformational features of a given EPS and/or AMP play an additional role in modulating the AMP activity. In fact, alginate has an extended polymer chain conformation due to the 1→4 glycosidic linkages, while the two EPS produced by I. limosus exhibit a more folded chain conformation due to the presence of 1→2 and 1→3 glycosidic bond respectively (Herasimenka et al., 2007). The different behaviour of ppyLL-37 in the presence of EPS was also revealed by spectroscopic studies.

Circular dichroism experiments

Circular dichroism (CD) spectroscopy was used to investigate the ability of EPS to induce the α-helical conformation in AMP. Ionic strength, provided by ions and polyions present in the medium as well as by the outer layer of the bacterial membrane, induce some cathelicidins to assume this secondary structure, which is in any case required for the interaction with the bacterial cell membrane eventually leading to cell lysis. The data obtained by means of CD spectroscopy of EPS–AMP systems are reported in Fig. 4, where the molar ellipticity was transformed into per cent of α-helix conformation in AMP by using the formula reported in (Juban et al., 1997). Although the addition of C9343 caused some precipitation in AMP solutions, its CD data were also included in the discussion as some structural information could still be obtained. PpyLL-37 and, to a lesser extent, hssLL-37, exhibited a low but significant amount of α-helix formation in water, even in the absence of EPS (about 17% for ppyLL-37 and 7% for hssLL-37), as they are known to adopt ordered structures in bulk solution in a salt-dependent manner (Johansson et al., 1998; Zelezetsky et al., 2006).

Figure 4. Circular dichroism data, expressed in per cent of α-helix calculated at 222 nm, of AMP interactions with EPS. A. hssLL-37. B. pobRL-37. C. ppyLL-37. The abscissa refers to the ratio between EPS and AMP concentrations.

Alginate was the EPS with the greatest effect on the structure of all the AMP examined causing the maximum α-helical formation upon its addition. PpyLL-37 showed the highest percentage of α-helix conformation (about 70%), whereas both pobRL-37 and hssLL-37 reached a value of about 40% at most. Although the different EPS had diverse effects on AMP, the maximum content of α-helix was consistently obtained at an EPS/AMP concentration ratio between 5 and 10. Considering ppyLL-37 and hssLL-37, it is worth noting that the per cent α-helix content decreased upon further addition of some EPS, a feature that will be better clarified when discussing data from fluorescence spectroscopy, but can be traced to a different type of EPS/AMP interaction.

The CD data for pobRL-37 and hssLL-37 paralleled the minimum inhibitory concentration (MIC) findings. Alginate and IL had the strongest effect on AMP conformation, while cepacian and C9343 exhibited a low influence on hssLL-37 and very little or no influence in the case of pobRL-37. However, experiments conducted with ppyLL-37 showed that cepacian caused an α-helix induction as high as that observed in the presence of alginate, although its influence on ppyLL-37 activity was relatively low, as evidenced by the MIC data (Fig. 3). This behaviour might be explained considering the physico-chemical properties of ppyLL-37 (see Table 1); it has the lowest net positive charge (q) and the highest mean hydrophobic character among the three peptides, as indicated by the least negative value of 〈H〉. As a matter of fact, the rather low q value may facilitate the induction of helical conformation by an EPS with low negative charge density because electrostatic repulsions between positive charges are weaker. Moreover, the effect of IL, which is highly charged, on ppyLL-37 was lower than that of cepacian, thus confirming the hypothesis that, besides electrostatic effects, peculiar structural characteristics of both EPS and AMP influence the transition to the ordered α-helix conformation. Conformational and/or chemical features of EPS chains might either result in the formation of domains with different hydrophilic/hydrophobic characteristics, or affect negative charge distances on the polysaccharide backbone that may induce a local increase in charge density. Both characteristics might influence binding with AMP. In order to further investigate the interaction between AMP and EPS, and correlate the MIC and CD data with a molecular interaction between these two species, fluorescence experiments were carried out.

Fluorescence spectroscopy

When the aromatic side-chain of Phe residues is masked from solvent interactions by complex formation, its fluorescence yield increases. This property is often used to detect binding within interactive species. The binding of EPS with the three investigated AMP could thus be estimated by looking at the emission spectrum around 282 nm, as hssLL-37 and ppyLL-37 both contain four phenylalanine residues, and pobRL-37 contains three. As found for CD spectroscopy data, the fluorescence results (Fig. 5) paralleled those obtained with the MIC assays. In fact, alginate and IL gave larger fluorescence increases than cepacian or C9343. An oscillating behaviour observed for hssLL-37 and ppyLL-37 in the presence of C9343 might be due to partial precipitation, as already described, due to the fact that light scattering effects in the presence of relatively large particles in solution are more visible in fluorescence than in CD spectroscopy.

Figure 5. Fluorescence data of the interactions between AMP and EPS. A. hssLL-37. B. pobRL-37. C. ppyLL-37. The abscissa refers to the ratio between EPS and AMP concentration.

The large increase of fluorescence yield of all investigated AMP in the presence of alginate and IL clearly supported the formation of complexes between the two interacting species, while cepacian and C9343 seemed to complex to a lesser extent. It is worth stressing that ppyLL-37 showed the highest degree of complex formation with all the EPS considered, again confirming its peculiar behaviour. Moreover, fluorescence data showed that the interaction between ppyLL-37 and cepacian was stronger than that of cepacian with hssLL-37 and pobLL-37 (higher observed fluorescence intensity), thus supporting the CD data, but not as strong as the interactions with alginate and IL, in agreement with MIC data.

The maximum fluorescence emission was achieved at an EPS/AMP concentration ratio between 4 and 10, the same value range that induced the most α-helix formation, and then decreased in agreement with CD data. This effect can be explained by the amphipathic nature of the cathelicidin α-helix, which exhibits two chemically different surfaces on the helix: one polar, and positively charged, and the other apolar and rich in phenylalanine residues. As complexes between negatively charged EPS and positively charged AMP were likely to form throughout interaction of the hydrophilic surface of the α-helix, upon complex formation the aromatic Phe residues were still exposed to the solvent being on the opposite surface of the α-helical structure with respect to that interacting with the EPS chain. The interaction of polar solvent molecules with the non-polar surface of the α-helix leads to an energetically unfavourable configuration, and would promote complexation with a second peptide molecule, which assumes a helical conformation. The hydrophobic surfaces of the peptides would interact and mask Phe residues from the aqueous medium. In this way, a single negatively charged stretch of the EPS could complex a peptide dimer or multimer, which in turn would expose only its polar residues to the solvent, as depicted in Fig. 6. This configuration is more likely to occur at low EPS/AMP concentration ratios, when the number of AMP molecules was roughly equal to that of EPS repeating units, thus producing an excess of AMP molecules with respect to the binding potential of EPS chains. When the concentration of EPS increases (high EPS/AMP ratio), the AMP molecules redistribute along the EPS chain, an entropy-favoured process, resulting in a partial dissociation of the AMP complexes and of AMP dimers. This process would lead to solvent exposed Phe residues and, consequently, to a decrease of the fluorescence yield values (Fig. 5). The effect of the ionic strength was tested at different phosphate buffer concentration. Passing from water to 5 mM buffer the fluorescence intensity increased due to an increase of the α-helical content in AMP. However, on further increasing the buffer concentration to 20 mM a net decrease in fluorescence intensity was detected, as expected considering the lowering of electrostatic interactions due to the increase of the solution dielectric constant (data not shown).

Figure 6. Cross-section of the model of the complex formation between one EPS chain and two AMP molecules as inferred from CD and fluorescence spectroscopy data at low EPS/AMP concentration ratio.

In order to have an independent and more direct visualization of complex formation between EPS and AMP, atomic force microscopy (AFM) experiments were carried out.

AFM investigation

The AFM technique is based on the detection of the attractive and/or repulsive forces acting between a small, sharp tip and a sample deposited on a flat surface. The tip hovers over the sample at a very short distance (a few dozen nm) while oscillating, and the variation in oscillation amplitude produced by interactions between the tip and the surface is transformed into images revealing the topology of the investigated surface. Macromolecular species deposited onto the supporting surface can be visualized, and in addition their lateral dimensions can be measured. AFM experiments were carried out only on alginate and cepacian as the other two EPS are composed of mixtures, rendering data interpretation more complex.

The AFM images of alginate and cepacian deposited onto a freshly cleaved mica surface using the spray-drying procedure are shown in Fig. 7A and B. The concentration used for alginate (15 μg ml⁻¹) resulted in the formation of a clearly detectable EPS extended network, and this might function as a polymeric scaffold similar to that used by bacteria to build biofilms. The lower concentration used for cepacian (5 μg ml⁻¹) produced images where single chains or small polymeric aggregates were detectable. In addition, Fig. 7B shows that cepacian polymer chains are topologically very elongated, suggesting a rather high conformational rigidity, as discussed in a previous paper (Herasimenka et al., 2008). By measuring the distance between the tip and the mica, which varied as a function of the dimensions of the object perpendicularly to the mica surface, the mean diameters of the polymer chains were obtained by averaging a sufficiently large number of EPS chain images; they were found to be 0.68 ± 0.08 nm for alginate and 1.03 ± 0.31 nm for cepacian (Herasimenka et al., 2008).

Figure 7. AFM images of EPS and EPS interacting with AMP. Concentrations refer to solutions prior to deposition and drying on the mica surface. A. Alginate from P. aeruginosa at a polymer concentration of 15 μg ml⁻¹. B. Cepacian from B. pyrrocinia at a polymer concentration of 5 μg ml⁻¹. C. Alginate interacting with hssLL-37: [EPS] = 5 μg ml⁻¹, [AMP] = 5 μg ml⁻¹. D. Cepacian interacting with hssLL-37: [EPS] = 5 μg ml⁻¹, [AMP] = 5 μg ml⁻¹. E. Cepacian interacting with hssLL-37: [EPS] = 5 μg ml⁻¹, [AMP] = 2.5 μg ml⁻¹.

When alginate and cepacian solutions were spray-dried on the mica surface in the presence of hssLL-37, the images recorded were dramatically different. Figure 7C shows a typical image of alginate chain obtained with EPS/AMP concentration ratio equal to 5, a value corresponding to the highest fluorescence intensity (Fig. 5) and hence to complex formation between AMP multimers and EPS segments. Blobs along polymer chains were clearly detected and might be explained as hssLL-37 peptides interacting with the polysaccharide. The lateral dimensions of alginate chains increased dramatically with respect to those detected in solutions of pure alginate. Values taken in different sections of the image gave 4.68 ± 0.64, 3.01 ± 0.42 and 1.74 ± 0.23 nm, thus confirming the presence of stable AMP/EPS complexes. Considering that the mean diameter of an α-helix cylinder is about 1.2 nm, the diameter of a complex composed of an EPS chain and two AMP molecules can be estimated about 3.5 nm, in reasonable agreement with AFM data. Figure 7D and E show the images obtained with cepacian at two different EPS/AMP concentration ratios: 5 and 10. The morphology of the complexes was different from that detected in the presence of alginate, a result that in turn can be correlated to the diverse fluorescence intensity detected at those EPS/AMP ratios. At a concentration ratio of 5, the presence of complexes between cepacian and hssLL-37 was clearly detected as large blobs of 3.22 ± 1.12 nm average thickness, indicated by the arrows in Fig. 7D. Upon increasing the concentration ratio to 10, the average dimension of the blobs appeared to decrease to 1.44 ± 0.50 nm. These findings were in good agreement with the CD and fluorescence spectroscopy data and indicated that on increasing the EPS/AMP concentration ratio, more EPS chain stretches were available for interacting with hssLL-37 molecules, thus allowing a redistribution of hssLL-37 onto the polysaccharidic chain, with a possible consequent AMP monomerization and a decrease in the dimension of the EPS–AMP complexes.

Conclusions

The MIC experiments, together with a physico-chemical characterization of the interaction between AMP and EPS, produced by opportunistic bacteria, demonstrated that bacteria can use EPS to bind and segregate AMP, thus lowering the efficiency of the primary innate host defences.

Bacterial polysaccharides are often negatively charged, which certainly promotes their complexation with positively charged peptides. In fact, MIC and fluorescence experiments showed that alginate and the mixture of EPS produced by I. limosus, having one negative net charge per monosaccharide residue, lowered the biological activity of AMP and generated stable complexes in a wide range of EPS concentration. On the contrary, the charge of the peptide seemed not to have a relevant influence on the stability of the complexes. As a matter of fact, both ppyLL-37 (+4) and pobRL-37 (+10) peptides, which are quite different in terms of cationicity, exhibited similar fluorescence behaviour with respect to the EPS tested. In order to complex with EPS, the three investigated AMP need to assume an α-helical conformation, at least for a relatively long stretch of their primary structure. The electrolytic nature of EPS certainly helps peptides to undergo the coil-to-helix conformational transition by increasing the ionic strength and thus lowering repulsions between homologous electric charges. However, CD experiments indicated that specific stereochemical motifs may also play a role in the interaction between EPS and AMP. Although both alginate and IL have a linear polymer structure with one negative charge per saccharidic unit, they behaved differently in the presence of different peptides. Alginate and IL were equally efficient in segregating pobRL-37, and exhibited almost the same efficiency with hssLL-37, but showed a very different behaviour in the presence of ppyLL-37. Here, at high EPS concentration, IL induced almost the same α-helical content as that in the absence of EPS, while alginate supported the ppyLL-37 helical structure. Some considerations on the differences in the conformations of alginate and IL polymers might help to explain this different behaviour. The 1→4 glycosidic linkages in alginate lead to an elongated conformation that can be classified as ‘cellulosic type’. Contrary to this, the 1→2 and 1→3 glycosidic bonds present in the EPS produced by I. limosus force the polymer chain to assume a more ‘helicoidal’ conformation.

Finally, AFM revealed itself to be a potent tool to visualize the interaction of EPS with AMP. Although the experimental conditions generally used for AFM are rather different from those present in vivo or used in vitro for different experiments, the comparison between images of EPS obtained in the absence and in the presence of AMP were very indicative of the presence of supramolecular aggregates, which may conveniently explain some details of the structure–function relationships governing the biological activities of bacterial EPS.

Experimental procedures

Materials

The water used in all experiments was purified using an Elix system (Millipore). P. aeruginosa strain PAO1 was a kind gift of Professor J. Govan (UK), while I. limosus strain LMG 20952^T was donated by Professor P. Vandamme (Belgium). B. pyrrocinia strain BTS7 was isolated from a cystic fibrosis patient in care at the Regional Centre for Cystic Fibrosis located in Trieste, Italy (Lagatolla et al., 2002). The mixture of three different EPS [cepacian, PS-I (Cérantola et al., 1996) and dextran] produced by B. cenocepacia strain C9343 (Conway et al., 2004) was a kind gift of Professor D. Speert (Canada). The reference E. coli strain ML-35 was used for the biological assays.

Growth of microorganisms for EPS production

Burkholderia pyrrocinia strain BTS7 and I. limosus strain LMG 20952^T were grown overnight in LB culture medium enriched with 0.2% glucose, diluted 1000 times in NaCl 0.9% and 100 μl were then plated on Yeast-Extract-Mannitol solid medium. B. pyrrocinia was incubated at 30°C, while I. limosus at 37°C, both for 3 days followed by 2 days at room temperature. P. aeruginosa strain PAO1 was grown directly on Pseudomonas Isolation Agar plates at 37°C for one night followed by 2 days of incubation at room temperature. The mucoid cultures were recovered by scraping the plates with 0.9% NaCl, the viscous material was centrifuged at 17 000 r.p.m., at 4°C for 30 min to remove the cells. The supernatant was precipitated in 4 vols of isopropanol at 4°C, the solid material was dissolved in water, dialysed first against 0.1 M NaCl and then against water. Finally, the pH was adjusted to neutrality and the solution was heated at 100°C for 10 min, followed by cooling and filtration. UV, CD and NMR spectra were recorded to check the presence of protein and/or nucleic acid contaminants. The lyophilized polymers, in the form of sodium salt, were stored at 4°C.

Synthesis of cathelicidins

The cathelicidins from man (Homo sapiens sapiens), orangutan (P. pygmaeus) and dusky leaf monkey (P. obscurus) were obtained by solid phase synthesis using the Fmoc-chemistry starting from the gene sequences (Zelezetsky et al., 2006); they were named hssLL-37, ppyLL-37 and pobRL-37 respectively.

MIC assays

Escherichia coli strain ML-35 was grown overnight at 37°C in Tryptic Soy Broth. The cell density was determined by measuring the turbidity at 600 nm and referred to previously determined standards. MIC for hssLL-37, ppyLL-37 and pobRL-37, in the presence or absence of selected EPS at 0.5 mg ml⁻¹ concentration, were determined by the microdilution susceptibility test in sterile polystyrene 96-well plates (Sarstedt). The assays were performed using 2.5 × 10⁴ cfu per well of E. coli ML-35 in 40% Tryptic Soy Broth, 10 mM phosphate buffer, according to the NCCLS guidelines, as previously described (Gennaro et al., 1989; Ganz and Lehrer, 1998). The plates were incubated overnight at 37°C, and MIC evaluation corresponded to the lowest peptide concentration capable of inhibiting bacterial growth upon visual inspection.

CD experiments

The CD experiments were performed on a Jasco J-600 instrument, using 10 mm quartz cells and recording the spectra at room temperature in the 200–250 nm wavelength region. Spectra were the result of the accumulation of at least two scans. CD spectra were recorded at room temperature on 10 μM peptide solution in water, followed by the addition of aliquots of EPS aqueous solution to give a saccharide final concentration ranging from 0 to 300 μM. The concentration of AMP was expressed in moles, while the concentration of the EPS was expressed in moles of EPS repeating unit. For the EPS mixture produced by the C9343 B. cenocepacia strain, the mass of the repeating unit was calculated taking into account the mass of the repeating unit of each component normalized by its weight percentage in the mixture. The percentage of α-helix content in AMP was determined as previously described (Juban et al., 1997; Ganz and Lehrer, 1998).

Fluorescence spectroscopy

Fluorescence measurements were performed using a Perkin-Elmer LS50B luminescence spectrometer, using 10 mm quartz cells at room temperature. The emission at 282 nm was measured using an excitation wavelength of 257 nm and a slit width of 7 nm. The fluorescence intensity of phenylalanine residues in AMP was recorded using 10 μM water solution of peptides, followed by the addition of polysaccharides to a final saccharide concentration in the range of 0–300 μM. The concentrations of AMP and EPS were expressed as reported in the previous paragraph.

Atomic force microscopy

The AFM images were obtained with a Multimode Scanning Probe (Veeco) coupled with a Nanoscope IIIa control device. The tips (Veeco) were made of phosphorus-doped silicon. Aqueous solutions of EPS having the desired concentrations were filtered using 0.22 μm membranes (Millipore Millex GP), subsequently spray-dried onto a freshly cleaved mica surface 15 h prior AFM imaging. Solutions with (EPS)/(AMP) concentration ratios equal to 5 and 10 were used to detect polymer–peptide interactions.

Acknowledgements

Professor D.P. Speert (Department of Pediatrics, University of British Columbia, Vancouver, Canada) is kindly acknowledged for the EPS mixture produced by B. cenocepacia strain C9343. We are grateful to Professor J. Govan (Centre for Infectious Diseases, University of Edinburgh, Scotland) and Professor P. Vandamme (Laboratory of Microbiology, University of Gent, Belgium) for the strains P. aeruginosa PAO1 and I. limosus LMG 20952^T respectively. Professor M. Prato (Department of Pharmaceutical Sciences, University of Trieste, Italy) is kindly acknowledged for the use of the AFM instrumentation. This work was carried out with the financial support of the Regione Friuli Venezia Giulia (projects # L.R.11/2003 and L.R.26/2005), of the Italian Ministry of the University and Research (PRIN 2007) and of the Italian Cystic Fibrosis Foundation (project #11/2006, with the contribution of CF delegation of Belluno, Italy).