Background
Currently, infectious pathologies remain an important health problem, being among the 10 leading causes of death worldwide [
1]. In addition to the infectious diseases not yet eradicated, emerging and re-emerging infections may also appear. This is frequently due to multiple factors including environmental changes, lack of prevention measures, travel and global trade, alterations in host susceptibility and, particularly, adaptive genetic changes in the microorganisms themselves [
2]. New adaptations in causative agents provide them with a temporary evolutionary advantage against environmental factors, host defenses and antimicrobial drugs [
2,
3], as in the case of some
Staphylococcus aureus strains, which have acquired resistance to multiple antibiotics, resulting in it becoming the leading cause of chronic infections associated with indwelling medical devices [
4].
Of the myriad communicable pathologies currently affecting humankind, the World Health Organization has highlighted the threat from lower respiratory infections and tuberculosis, both of which continue to be among the global top ten causes of death [
1]. Although infections of the lower respiratory tract are caused by a variety of pathogens including viruses and fungi, bacteria are the main causative agents [
5].
The human body is largely exposed to different bacterial pathogens through the skin and mucous membranes, including the respiratory mucosa [
6]. After using a suitable portal of entry, the microorganisms must reach their target site in the body and accomplish the most critical step, the establishment of the focus of the infection. This crucial process implies that bacterial pathogens are capable of adhering to and remaining attached to the cell surface without being dislodged by host defenses [
7,
8]. Pathogenic microorganisms have developed diverse virulence factors, and these may cooperate to accomplish the establishment of a pathogen through mediation of the adhesion and colonization phases, through promoting tissue damage and through spreading the pathogen and overcoming the host immune system [
7,
8].
Bacterial adhesins need to recognize and interact specifically with host cell surface receptors in order to achieve adequate adherence and colonization [
6]. Eukaryotic receptors may also be involved in subsequent stages of the infectious process, including invasiveness, organotropism, and interference in host defense response [
7]. A variety of cell surface molecules can act as receptors for microorganisms, including proteins, carbohydrates, lipids, and various different combinations of these.
Proteoglycans (PGs) are a type of glycoconjugate that act as receptors for multiple microbial pathogens [
9]. These complex molecules are composed of long unbranched chains of polysaccharides called glycosaminoglycans (GAGs), which are covalently attached to a wide variety of core proteins [
10]. These molecules possess a high negative charge, and are formed by repeating units of uronic acid or galactose and an amino sugar, either N-acetyl glucosamine or N-acetylgalactosamine. There are four major classes of GAGs: heparin/heparan sulfate (HP/HS), chondroitin sulfate (CS), keratan sulfate, and hyaluronic acid, the latter being the only one not covalently bound to a core protein [
10]. GAGs display remarkable structural diversity, which is the result of interrelated enzymatic reactions, including N- and O- sulfations and epimerization, that occur heterogeneously along the chain [
11,
12]. Due to the diversity of core proteins, and especially to the diversity of composition patterns, length, epimerization and sulfation of saccharide chains, the PGs have great heterogeneity, which enables them to fulfil numerous functions. Modifications in GAG chains create specific binding motifs for many ligands, such as cytokines, chemokines, growth factors, enzymes and enzyme inhibitors, and extracellular matrix (ECM) proteins [
13‐
17]. These molecules are also involved in several physiological activities, including organization of the ECM, regulation of proliferation, differentiation and morphogenesis, cytoskeletal organization, tissue repair, inflammation and vascularization [
18‐
20]. In addition, a variety of roles have been ascribed to these molecules in pathological process, including non-infectious pathologies such as cancer [
21,
22] as well as infectious pathologies generated by diverse pathogens [
23,
24]. Different types of pathogen recognize GAGs as receptors, including a wide spectrum of viruses, bacteria and parasites [
24‐
26].
In addition to their remarkable heterogeneity, PGs are ubiquitous, and are widely distributed on cell surfaces, in pericellular locations and in cytoplasmic secretory vesicles [
10,
13]. The expression and composition of GAGs are variable, depending on the cell type and the physiological conditions [
25]. These features make PGs excellent candidates as host receptors for microorganisms, with microbial pathogens showing a preference for interacting with HS chains attached to cell surface proteoglycans (HSPGs), and principally with respect to two families: the transmembrane syndecans (SDCs) and the glycosylphosphoinositide-linked glypicans (GPCs), which have four and six members respectively [
26,
27].
GAGs play significant roles in many pathophysiological processes in the ECM of the lung: regulating hydration and water homeostasis, maintaining structure and function, modulating the inflammatory response, and influencing tissue repair and remodeling [
28]. Many studies have described the roles played by PGs in a wide range of pulmonary diseases, including malignant mesothelioma, pulmonary edema, fibrosis, asthma, emphysema, and bronchiectasis [
28‐
31].
The aim of this article is to investigate the involvement of PGs and their GAG chains as receptors for certain common respiratory bacterial pathogens. The study analyzes the role played by different molecular species of PGs in bacterial adherence, as well as the importance of specific molecular aspects of GAG chain structure on their interaction with microorganisms, particularly the influence of sulfation of saccharide chain residues. The analysis was carried out using both epithelial cells and fibroblasts of pulmonary origin since different profiles of cell surface GAGs are to be expected for the two cell types. Ultimately, understanding the complexity of the adhesion process would allow for the possibility of bacterial infections to be controlled via preventing the adhesion or invasion stages of bacterial pathogenesis.
Methods
Materials
The following materials were purchased from the manufacturers indicated: heparin, heparan sulfate, chondroitin sulfate A, B and C, heparinases I and III, chondroitinase ABC, fluorescein isothiocyanate (FITC), GenElute PCR clean-up kit, phospholipase C phosphatidylinositol-specific (PI-PLC) from Bacillus cereus, all from Sigma-Aldrich (St. Louis, MO, USA); 2-O, 6-O and N-desulfated heparins from Amsbio (Abingdon, UK); Dulbecco’s Modified Eagle’s minimal essential medium (DMEM) and Minimum Essential Medium (MEM), fetal bovine serum, penicillin-streptomycin, and PBS-phosphate-buffered saline from Gibco-Thermo Fischer Scientific (Waltham, MA, USA); Brain-Heart Infusion broth from Pronadisa (Madrid, Spain); RNeasy Kit and RNase-Free DNase from Qiagen (Hilden, Germany); High-Capacity cDNA Reverse Transcription Kit and PowerSYBR Green PCR Master Mix from Applied Biosystems (Foster City, CA, USA). Synthetic peptides were from Abyntek Biopharma (Derio, Spain); mouse monoclonal anti-syndecan 1 (CD138) from DakoCytomation (Carpinteria, CA, USA); and rabbit anti-syndecan 2, goat anti-syndecan 3 and rabbit anti-syndecan 4 polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were obtained from commercial sources and were of analytical grade.
Bacterial strains, cell lines and culture conditions
The species used in this study were the Gram-positive bacteria Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Enterococcus faecalis, and the Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae and Serratia marcescens, all of which were obtained from the Hospital Universitario Central de Asturias. All the bacteria were grown in Brain-Heart Infusion broth at 37 °C in a shaking incubator, except S. pneumoniae which was grown in a 5% (v/v) CO2 atmosphere without shaking.
The lung cell lines used in this study were lines A549 (epithelial, ATCC® CRL-11185 ™) and MRC5 (fibroblasts, ATCC® CRL-11171 ™). The two lines were grown in DMEM and MEM, respectively, with the culture broth being supplemented with 10% (w/v) fetal bovine serum and penicillin G/streptomycin (5000 IU/ml, 5000 μg/ml). Cultures were incubated at 37 °C in a 5% (v/v) atmosphere.
Fluorescein labeling
FITC labeling of bacteria was carried out using overnight cultures, which were washed four times with PBS and resuspended in a 0.1 mg/ml FITC solution to an A600 of 0.5; incubation in the dark at 37 °C under agitation proceeded for 1 h, and then the bacterial suspensions were centrifuged, washed 4 times with PBS to eliminate the FITC excess, and resuspended in PBS to an A600 of 0.5.
Adherence assays
Adhesion of the different pathogenic bacteria to A549 and MRC5 monolayers was performed in 24-well plates grown to 70–90% confluence. The media were aspirated, and the cells washed twice with PBS and then blocked with 10% fetal bovine serum in PBS for 2 h at 37 °C in a 5% CO2 atmosphere. After further washing with PBS, 100 μl of FITC-labeled bacteria in 400 μl of DMEM was added and the mixture incubated for 1 h at 37 °C and 5% CO2. Thereafter, the wells were rinsed (4 x) with 500 μl PBS to remove unbound bacteria. At the end of the experiment, lung cells were disaggregated with 1% SDS, and the fluorescence of the pathogens attached to them was quantified in a Perkin Elmer LS55 fluorometer set at 488 nm (excitation) and 560 nm (emission). Data for the different experiments were normalized using the adhesion values without any additive or treatment, which was given the arbitrary value of 100%. Assays were performed at least in triplicate and the data are expressed as the mean ± SD.
Inhibition of glycosaminoglycan synthesis
Cell cultures in 24 well plates at approximately 70% confluence were incubated in medium containing rhodamine B 50 μg/ml or genistein 30 μM (final concentrations) overnight at 37 °C. The cultures were washed twice with PBS, and subjected to adherence assays as described in the previous paragraph.
Enzymatic digestion of lung cell-surface GAGs
Digestion of HS from cell cultures was achieved by incubation at 37 °C for 3 h in a 5% CO2 atmosphere in minimal culture medium with a mix of 500 mU/ml (final concentration) each of heparinase I and III. Digestion of CS chains was carried out by 3 h of incubation under the same conditions, but using 250 mU/ml (final concentration) of chondroitinase ABC. The digestion of both GAGs was performed through simultaneous incubation with heparinases I and III and chondroitinase ABC in the same conditions. The reactions were stopped with 2 washes in PBS buffer and the cell cultures were immediately submerged in the appropriate supplemented culture medium, and subjected to adherence assays with pathogens as described in the previous paragraph.
Adherence inhibition assays
The effect of GAGs on adherence interference experiments was performed through the addition of either HS, CS-A, CS-B, CS-C, or a mixture of all four, at concentrations ranging between 0.01 and 400 nM to the labeled bacteria before their addition to the monolayers.
The effect of peptides which included the consensus HP binding sequences, QKKFKN and FKKKYGKS on adherence interference experiments was analyzed at a final concentration of 20 nM of each peptide. The peptides were added to the monolayers before the addition of the labeled bacteria.
The effect of the location of sulfations in HS chains on bacterial adherence was studied using native HP as control, and 2-O, 6-O and N-desulfated HPs at a final concentration of 20 nM as competitors in the adherence. The HPs were added to the labeled bacteria before their addition to the monolayers.
RNA isolation, cDNA synthesis and qRT-PCR reactions
RNA was isolated using the RNeasy kit following the manufacturer’s specifications. Samples were subjected to treatment with RNase-free DNase during the purification process itself. The concentration of RNA obtained was determined spectrophotometrically by measuring absorbance using a Picodrop Microliter UV/Vis pectrophotometer (Picodrop Limited, UK).
cDNA synthesis was carried out using the High Capacity cDNA Transcription Kit following the manufacturer’s specifications. The reaction products were cleaned using the PCR Clean-Up GenElute kit as per the manufacturer’s instructions.
qRT-PCR reactions were carried out as previously described [
17], and actin was used as a control gene to compare run variation and to normalize individual gene expression. The expression values of genes were calculated as 2
–ΔCt (relative to actin as the housekeeping gene).
Antibody inhibition assays
Antibody inhibition assays were carried out in 24-well plates grown to 80% confluence. The media was aspirated, and the cells washed twice with PBS and then blocked with 10% fetal bovine serum in PBS for 2 h at 37 °C in a 5% CO2 atmosphere. After further washing with PBS, a mixture of anti SDC-1, anti SCD-2, anti SDC-3, and SDC-4 were diluted 1:100 in PBS, except anti-SDC2, which was diluted 1:250, and incubated for 1 h. After the treatment, adherence assays were performed as indicated above.
Enzymatic removal of glypicans using phospholipase-C
Glypicans from cell surfaces were removed enzymatically using PI-PLC. Cells were grown in 24-well plates to 80% confluence. After washing with PBS, the cells were incubated in the absence (control) or presence of 80 mU/ml PI-PLC for 40 min at 37 °C in a 5% CO2 atmosphere. After the treatment, adherence assays were performed, as previously described.
Statistical analysis
All experiments were carried out at least three times, with at least three replicates being used on each occassion. All analyses were performed using the Statistics for Windows program (Statsoft Inc.; Tulsa, OK). Mean values were compared between two samples by the Mann-Whitney U test and between multiple samples using the Kruskal-Walis test. The p value accepted as significant was p < 0.05. All data are presented as means ± standard errors of the means.
Discussion
PGs are involved in many infectious processes, especially by their GAG moieties, which act as receptors for the adherence and attachment of many pathogenic microorganisms, including some which affect the respiratory tract [
24,
26]. Lung epithelial cells are in contact with the environment, creating a barrier against pathogens, along with the other passive and active defense mechanisms. Lung fibroblasts are found in the lower layers, and are thus less exposed to environmental factors and microorganisms. It would therefore be expected that both cell types present a distinct profile of PGs, including GAGs, with different structures, which would affect their interaction with pulmonary pathogens. The characterization of GAG-pathogen interactions could allow new and more efficient infection avoidance strategies to be developed via preventing adhesion.
To determine the involvement of PGs, and especially their GAG moieties, in bacterial attachment to A549 and MRC5 cell lines, their levels were reduced by the use of two specific inhibitors of their biosynthesis: rhodamine B or genistein, and the effect on pathogen adherence analyzed. These treatments resulted in a significant decrease in bacterial adherence in all cases, indicating that the lung pathogens use, at least in part, GAGs as receptors for adhesion. We have recently published a study of bacterial adherence to corneal epithelial cells, where it was noted that treatment with rhodamine B or genistein had a different effect on bacterial adherence depending on the Gram nature of the microorganism in question. Specifically we found that in Gram-positive bacteria binding was most affected by rhodamine, while genistein more strongly reduced Gram-negative adhesion [
36]. Interestingly, the current study was unable to determine the existence of such patterns in the adherence of microorganisms to lung cell lines, being the behavior observed dependent, for each specific cell line, of the bacterial species analyzed.
The results observed may be due to the fact that these molecules affect the biosynthesis of GAGs at different levels. Rhodamine B is thought to inhibit chain elongation, acting as a nonspecific inhibitor that reduces GAG synthesis in a range of cells, and produces reduced lysosomal GAG storage in some types of mucopolysaccharidosis [
32,
33]. The isoflavone genistein inhibits the kinase activity of epidermal growth factor receptor, which is required for the full expression of genes coding for enzymes involved in GAG production [
34], although it has been described that the effect of this molecule on the biosynthesis of GAGs is strongly dependent on their type and localization [
35]. Each bacterium has different types of adhesins to bind to the chains of GAGs of lung cells, and each one of these molecules has a variable binding specificity, which can be determined by various factors, such as the length of the chains, which would be affected by rhodamine B, or by the structure of the GAG chains, which can be altered by treatment with genistein.
The involvement of GAG species in bacterial adherence to lung cells was analyzed by reducing the levels of GAG chains by means of enzymatic degradation using bacterial lyases. The results show that both types of GAG are involved in the binding of bacteria to lung cells. In A549 cells, heparinase treatment significantly decreased adherence relative to chondroitinase treatment, but not compared with simultaneous digestion of HS and CS chains, suggesting that HS is the main species involved in bacterial adherence, and that there is no additive effect in the presence of both molecules. In MRC5 cells, HS also seems to be the principal molecule involved in adherence, but in this case, chondroitinase treatment produced a similar decreases as HS with respect to certain bacteria, suggesting that in this cell line CS has a greater involvement in bacterial adhesion. The differences observed in chondroitinase treatment for both cell lines were statistically significant, which could be a reflection of the existence of differences between A549 and MRC5 cell lines as regards PG composition and structure of GAG chains, considering that the expression pattern of GAGs varies depending on cell type and their physiological state.
The participation of the predominant GAG species present on the cell surface in the adhesion of pathogens was also studied by analyzing their ability to compete in adherence interference experiments. HS showed the highest inhibitory ability, suggesting that it constitutes the predominant cell surface receptor for all the bacteria analyzed, although in the case of binding of S. marcescens to fibroblasts, CS-B showed an effect comparable to HS. There was no clear pattern in the inhibition produced by the diverse types of CS tested in both epithelial cells and fibroblasts depending on the bacteria tested. When the inhibitory effect of an equimolar mixture of all the GAG species was analyzed, in most cases results were similar to HS used alone, suggesting, again, that this species is the main cell surface receptor. An interesting exception to the previous conclusion was the binding of S. aureus to fibroblasts, where the effect of the mixture was considerably higher than that produced by HS alone, suggesting that in this cell type different molecular species cooperate in the binding of the pathogen.
All the results obtained in this work suggest HS to be the main mediator in bacterial cell adhesion, which fits with the fact that it is the most widespread GAG on cell surfaces [
12,
13]. A large number of bacteria use this molecule as a receptor for adhesion to different tissues, including
Helicobacter pylori,
E. faecalis,
Neisseria meningitidis,
Pseudomonas aeruginosa, and
S. aureus, among others [
24]. Other GAGs are also able to act as receptors for microorganisms, such as
Borrelia burgdorferi, which uses a variety of these molecules as receptors depending on the host cells; HS to bind to endothelial cells and CS-B and HS to glial cells [
37]. HS and CS-B chains also mediate in
Chlamydia trachomatis, S. pyogenes binding [
24]. Both HS and CS-B share a feature in their chemical structure, variable proportions of the C-5 epimer of glucuronic acid, the iduronic acid, providing the molecule with more flexibility [
38]. Some of the most common pathogenic bacteria in the respiratory tract also use GAGs, mainly HS, for attachment, such as
S. pneumoniae [
39], 75% of nontypeable
Haemophilus influenzae [
40],
Chlamydia pneumoniae, and
P. aeruginosa on basolateral epithelial surfaces [
24].
HS has a complex domain structure, consisting of highly sulfated NS-domains interspaced with poorly sulfated NA-domains. Most interactions between proteins and HS occurs mainly through NS-domains, either by electrostatic interactions or by the specific recognition of sulfated sequences in saccharide chains. Most heparin-binding proteins have clusters of basic amino acids alternating with hydropathic residues that have been shown to be required for binding [
41]. In accordance with these consensus sequences, two peptides, FKKKYGKS and QKKFKN, were designed in order to figure out their role as competitors for bacterial attachment and, consequently, their ability to mimic different binding motives present on the bacterial adhesins. Both peptides partially inhibited adhesion in both cell lines, and in most cases the longer peptide was more efficient. However, the pattern observed changed depending on the bacteria and, particularly, on the pulmonary cell type, suggesting that different bacterial adhesins with different HP binding sequences are mediating in interaction with lung cells [
8,
42,
43].
Generally, HS chains occur as HSPGs, and two gene families, SDCs and GPCs, account for most cell surface HSPGs. Some previous studies have related specific species of cell surface HSPGs with the adhesion of and colonization by certain pathogens [
7,
14,
42]. Since HSPGs are expressed in varying amounts depending on the tissue, we quantified their transcript levels in both lines of pulmonary cell. In the MRC5 line, transcripts for the four SDCs could be detected at similar levels, but in the A549 line isoforms 1 and 4 were much more abundant. With respect to GPCs, the six isoforms showed large differences in their transcript levels, dependent on the cell type, although isoform 1 was the most abundant in both cases. The enzymatic removal of GPCs from the cell surface did not show any great effect, while blocking experiments with a combination of anti-SDC specific antibodies resulted in a decrease in adherence in both cell lines, suggesting that SDCs are involved in bacterial attachment to lung cells.
Similar results were found in a study of bacterial adhesion to corneal epithelial cells, where all the isoforms of SDCs participate cooperatively in the attachment of bacteria, indicating that they play a far more important role in this process than GPCs [
36]. The involvement of SDCs in bacterial adherence has been described for different pathogens such as
Streptococcus agalactiae, Listeria monocytogenes and
S. aureus, which interacts with syndecan-1 for adherence to cells; and
Neisseria gonorrhoeae, whose adhesin OpaA uses SDC-1 and -4 as receptors [
44‐
46]. There is little information on the involvement of GPCs in infectious processes, an interesting exception being the case of the intracellular pathogen
Chlamydophila pneumonia, which is able to use a variety of cell-type specific binding mechanisms, e.g. it uses GAG chains to bind to epithelial cells, but in Jurkat lymphoid cells, which only express GPC-1, it uses a GAG-independent mechanism to bind. However, it is known that GPCs are involved in other non-infectious diseases, including tumoral processes, neurological syndromes and disorders, and prion diseases [
22,
47‐
50].
In some pathogens, it has been described that specific patterns of sulfation are required for adherence; this is the case for
C. trachomatis, whose binding to HeLa 229 cells is inhibited by 2-O desulfated HP [
51], certain viruses such as the hepatitis E virus, which interacts with 6-O-sulfated HS [
52], and baculovirus, whose entrance is promoted by 6-O-and N-sulfated HS [
53]. To investigate the importance of specific sulfated positions of the HS disaccharide unit on the binding of bacteria to lung cells, the effect of native HP compared to 2-O, 6-O and N-desulfated HPs in competition adherence assays was analyzed.
All desulfated HPs showed less inhibitory capacity than the native HP, which points to the importance of sulfation in adherence. However, the effect was slightly different between the two cell lines; while in the epithelial cells results varied widely depending on the pathogen analyzed, and no significant differences between the different desulfated heparins were observed, in fibroblasts results were more homogeneous, and sulfation in C2 of uronic acid showed a greater effect on competition sulfation in C6 of the glucosamine residue. These data suggest that binding involves different sulfated sequences in each cell type, which could also relate to the use of different bacterial adhesins in each case.