2.2. A. Salmonicida Autoinducer-2 Production Starts before the Log Phase of Growth and Its Kinetics Differs Depending on Culture Media

The timecourse of extracellular AI-2 level changes differed depending on if A. salmonicida was cultured in CM9 (Figure 2A) or DM (Figure 2B). The common trait was that the majority of the signals were produced before the log phase started. The AI-2 increase in CM9 was slow, reaching maximum levels after 9 h (Figure 2A) compared to 2 h in DM medium (Figure 2B). In CM9, the largest increase occurred before the log phase and the increase continued at a slower rate (Figure 2A). In DM, the level of AI-2 reached a plateau in the lag phase and their level remained similar throughout the time course (Figure 2B).

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Figure 2

Autoinducer-2 production in A. salmonicida cultures. Time course of AI-2 production by A. salmonicida cultured in CM9 (A) and DM (B).

2.3. Exogenous DPD Enhances A. Salmonicida Growth

A. salmonicida responded to DPD treatment with increased growth in both media (Figure 3A,B). In CM9, OD₅₆₀ of the cultures increased linearly with increasing DPD concentration (ΔOD₅₆₀ in log phase vs. DPD conc.: r² = 0.91; p = 0.0002; n = 4, Figure 3A). In DM, the increase in OD₅₆₀ in response to DPD treatment was less pronounced (ΔOD₅₆₀ in log phase vs. DPD conc.: r² = 0.66; p = 0.0078, Figure 3B; n = 4). CFU/mL vs. DPD growth curves of A. salmonicida calculated using CFU/mL-OD₅₆₀ standard curves in the logarithmic range of growth (Figure 3C,D) indicate a stronger effect of DPD on growth in CM9 compared to DM. A. salmonicida doubling times calculated from the logarithmic growth phase inversely correlated with DPD concentrations in both media (r² = 0.82; p = 0.002, n = 4 in CM9, Figure 3E and r² = 0.58; p = 0.0172, n = 4 in DM, Figure 3F).

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Figure 3

Effect of exogenous DPD on A. salmonicida growth. The effect of DPD on A. salmonicida growth was monitored over time (A,B) and correlated against A. salmonicida doubling time (E) ( p = 0.0002; r² = 0.82 in CM9 and (F); p = 0.017; r² = 0.58 in in DM). A. salmonicida growth curves in the log phase of growth expressed as CFU/ml in CM9 (C) and DM (D) media. The horizontal dotted lines in panels (E,F) denote the doubling time of the negative control (no added DPD). Data points are expressed as Mean ± Standard Error of the Mean (n = 4). Statistical test: Pearson correlation test. Abbreviations: DPD = (S)-4,5-Dihydroxy-2,3-pentandione. The results are based on three independent experiments.

2.4. Atlantic Salmon Mucins Reduce A. Salmonicida AI-2 Secretion

Atlantic salmon mucins from skin, pyloric ceca, proximal and distal intestine reduced AI-2 signals from A. salmonicida cultures (p = 0.0001, p = 0.0049, p = 0.0001 and p = 0.0008, respectively, n = 5, Figure 4A). Skin mucins reduced AI-2 secretion by 81%, proximal intestinal mucins by 72%, distal intestinal mucins by 56% and pyloric cecal mucins by 48%. The effect of mucins on the AI-2 production and A. salmonicida growth did not correlate (r² = 0.05, p = n.s., n = 19; Figure 4B), which together with the results in Figure 2 suggest that mucin effects on A. salmonicida growth are not the main cause for the effects on A. salmonicida AI-2 production.

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Figure 4

Effect of salmon mucins on A. salmonicida AI-2 levels in the culture medium. Extracellular AI-2 levels were measured after culturing A. salmonicida to the late log phase in the presence of 100 µg/mL Atlantic salmon mucins. These experiments were performed in DM medium to avoid interference of saccharides present in the CM9 medium. Mucins from all organ sites decreased the AI-2 levels in the A. salmonicida supernatant (skin, pyloric ceca, proximal intestine and distal intestine p = 0.0001, p = 0.0049, p = 0.0001 and p = 0.0008, respectively; (A). Data points correspond to biological replicates of individual fish and are expressed as Mean ± Standard Error of the Mean (n = 5). Statistical test: One-way ANOVA with Dunnett’s post hoc test. ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001. AI-2 production and growth-modulating effect of mucins did not correlate (r² = 0.05, n = 19, p = n.s.); (B). Statistical test: two-tailed Pearson correlation test. Abbreviations: Pyloric = Pyloric ceca; Proximal = proximal intestine and Distal = distal intestine. The results were reproduced three times with similar results and the assay was performed within its linear range.

2.5. Among the Saccharides Abundant on Mucins, Fucose, N-Acetylneuraminic Acid and GlcNAcβ1-3Gal Inhibit AI-2 Secretion of A. Salmonicida

The disaccharide GlcNAcβ1-3Gal, which is a common epitope on Atlantic salmon mucins, reduced the AI-2 levels in the A. salmonicida supernatant by 31% (n = 6; p = 0.0297; Figure 5). Fucose decreased AI-2 levels by 44% (n = 6; p = 0.0008; Figure 5) while NeuAc decreased them by 35% (n = 6; p = 0.0101; Figure 5). Galactose, GalNAc and the galactose-containing saccharides lactose and GalNAcβ1-3Gal also had a tendency to reduce AI-2 production in A. salmonicida cultures in DM (n = 6; p = n.s.; Figure 5), whereas no such tendency was present for N-glycolylneuraminic acid and N-acetylglucosamine.

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Figure 5

Effect of saccharides on A. salmonicida AI-2 levels. Mono- and disaccharides in 100 µg/mL concentration were added to A. salmonicida cultures in DM medium, after which extracellular AI-2 levels were measured from the late log phase. Fucose, NeuAc and GlcNAcβ1,3Gal decreased the AI-2 secretion of A. salmonicida in DM medium (p = 0.0008; p = 0.0101 and p = 0.0297, respectively). Data points are expressed as Mean ± Standard Error of the Mean (n = 6) Statistical test: One-way ANOVA with Dunnett´s post hoc test. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001. The results are based on two independent experiments and the graph includes all data points from the two experiments. The data points are normalized for A. salmonicida growth, and the assay was performed within its linear range. Abbreviations: Glc = glucose; GlcNAc = N-acetylglucosamine; Gal = galactose; GalNAc = N-acetylgalactosamine; Fuc = fucose; NeuAc = N-acetylneuraminic acid; NeuGc = N-glycolylneuraminic acid.

2.6. Removal of N-Acetylneuraminic Acid from Atlantic Salmon Mucins Attenuates the AI-2 Reducing Effects of Mucins on A. Salmonicida

Among the terminal monosaccharides located on salmon mucins that reduced A. salmonicida AI-2 production (Figure 5), N-acetylneuraminic acid is the most prevalent [8]. Enzymatic removal of N-acetylneuraminic acid from salmon mucins increased the AI-2 production of A. salmonicida (skin, pyloric ceca, proximal intestine, and distal intestine p = 0.0041, p = 0.0021, p = 0416 and p = 0.0035, respectively, Figure 6). N-acetylneuraminic acid removal from both skin and pyloric cecal mucins increased the AI-2 levels by 32% compared to mock-treated mucins (Figure 6A,B), whereas removal from proximal and distal intestinal mucins increased AI-2 levels by 22% and 25%, respectively (Figure 6C,D).

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Figure 6

Effect of sialidase treatment on the effect of mucins on A. salmonicida AI-2 secretion. Extracellular AI-2 was measured after A. salmonicida culture with Atlantic salmon mucins treated with Sialidase A or with the same treatment in the absence of the enzyme (mock). All modified mucins from the four groups increased the AI-2 quantity relative to their mock-treated counterparts (A: skin, B: pyloric ceca, C: proximal intestine and D: distal intestine p = 0.0041, p = 0.0021, p = 0416 and p = 0.0035, respectively; n = 6). Statistical test: Student´s t-test. * = p ≤ 0.05, ** = p ≤ 0.01. The data points are normalized for A. salmonicida growth. Abbreviations: Mock = enzyme buffer-treated negative-control mucins; Sial A = sialidase A-treated mucins; pyloric = pyloric cecal mucins.

2.7. Identification of the luxS Gene in the Genome of A. Salmonicida Ssp. Salmonicida Strain VI-88/09/03175

The genome sequence of the A. salmonicida ssp. salmonicida strain VI-88/09/03175 was determined, resulting in a draft genome of 4,738,132 bp over 123 contigs, with an average coverage of 52-fold coverage. From the annotation, a luxS homolog was identified. The luxS gene sequence of A salmonicida ssp. salmonicida strain VI-88/09/03175 (510 bp) showed a 100% homology with its homolog in the A. salmonicida subsp. salmonicida A449 reference genome (accession number CP000644.1). The draft genome was submitted to GenBank under BioProject number PRJNA698804.

2.8. Construction of ΔluxS Gene-Deletion Mutant Strain

To investigate the relationship between luxS gene and AI-2 activity in A. salmonicida we first deleted the luxS gene, creating a mutant strain by homologous recombination. As shown in Figure 7, the deletion was then verified by PCR using genomic DNA (gDNA) as a template with two approaches: (1) Amplification of the flanking regions of luxS gene with luxS-up-F and luxS-down-R primers produced bands with 980 bp for the wild type (WT) while the ΔluxS mutant clones produced 470 bp bands, in accordance with the theoretical size (Figure 7B); (2) Amplification mapping inside the luxS gene with LuxS-F and LuxS-R primers produced a product only in the WT strain (Figure 7C).

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Figure 7

Construction of A. salmonicida ssp. salmonicida strain VI-88/09/03175 ΔluxS mutant. (A) Deletion of the luxS gene by overlapping PCR using Gibson Assembly (NEB Inc., Ipswich, MA, the USA). (B,C) The deletion was verified by PCR using genomic DNA as template with two approaches: (B). Gel electrophoresis for PCR with primers amplifying flanking regions of luxS gene; the WT shows a band with 980 bp while the ΔluxS mutant clones (2,3,4) show bands with a size of 470 bp, in accordance with the theoretical size. (C). Gel electrophoresis confirming deletion of the gene using primers mapping inside the luxS gene; the WT shows a band with 106 bp while the ΔluxS mutant clones (2,3,4) shows no band, indicating deletion of the gene. A 1 kb plus DNA ladder (Thermo Scientific, Whaltham, MA, USA) was used as molecular-weight marker.

2.9. Growth Characteristics of A. Salmonicida WT and ΔluxS

To investigate the influence of luxS on A. salmonicida growth, growth rates from the ΔluxS mutant and WT were compared (Figure 8). The growth for the first few hours after starting a liquid culture in fresh DM was similar for the WT and ΔluxS mutant. At later time points, the ΔluxS mutant growth lagged behind that of the WT, and by 8 h the ΔluxS mutant growth was approximately 2-fold less than that of the WT (Figure 8).

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Figure 8

Growth comparison of the A. salmonicida ssp. salmonicida strain VI-88/09/03175 WT strain and its ΔluxS mutant. A. salmonicida WT and ΔluxS were cultured in DM with a starting optical density at 600 nm of 0.1. The assay was carried out at 20 °C and 120 rpm and monitored both by fluorescent detection of AlamarBlue reduction (A: n = 6) and OD₅₆₀ (B: n = 4) readings every hour. Data points are expressed as Mean ± Standard Error of the Mean.

4.2. Culture Media

The DM medium contained 0.1 g/L Ca(NO₃)₂·4H₂O, 0.4 g/L KCl, 0.1 g/L MgSO₄·7H₂O, 6 g/L NaCl, 0.8 g/L Na₂HPO₄, 2 g/L NaHCO₃, 2 mg/L FeSO₄, 5 g/L BSA, 50 mg/L adenine, 3 mg/L lipoic acid. For amino acids, DM medium contained 44.5 mg/L alanine, 632 mg/L arginine, 75 mg/L asparagine, 66.5 mg/L aspartic acid, 120 mg/L cysteine, 73.5 mg/L glutamic acid, 300 mg/L glutamine, 37.5 mg/L glycine, 110 mg/L histidine, 262.5 mg/L isoleucine, 262 mg/L leucine, 362.5 mg/L lysine, 75.5 mg/L methionine, 165 mg/L phenylalanine, 57.5 mg/L proline, 52.5 mg/L serine, 238 mg/L threonine, 51 mg/L tryptophan, 180 mg/L tyrosine, 234 mg/L valine. The vitamin content of DM medium was as follows: 0.2 mg/L D-biotin, 3 mg/L choline chloride, 1 mg/L folic acid, 35 mg/L myo-inositol, 1 mg/L niacinamide, 1 mg/L p-aminobenzoicacid, 1.25 mg/L D-pantothenicacid, 1 mg/L pyridoxinehydrochloride, 0.2 mg/L riboflavin, 1 mg/L thiamine hydrochloride, 5 µg/L vitamin B12 (cyanocobalamin). The CM9 medium contained 3 g/L KH₂PO₄, 12.8 g/L Na₂HPO₄, 0.5 g/L NaCl, 1.0 g/L NH₄Cl, 0.24 g/L MgSO₄, 11.1 mg/L CaCl₂ and 40 g/L Bacto^TM Casamino acids (BD sciences). See Table 2 for a side by side comparison of the media. The Bacto^TM Casamino acids derive from hydrolysis of casein further supplemented with an undisclosed amount of growth factors, cystine, maltose, iron and inorganic salts, therefore making CM9 medium a nondefined medium. In addition, due to the high amount of carbohydrates, CM9 medium was not suitable for our assay to investigate the effect of mucins and saccharides on A. salmonicida extracellular AI-2 levels.

4.3. DNA Manipulation

Bacterial DNA-extraction manipulations were performed routinely, as described by Sambrook et al. [32]. All restriction enzymes were used according to the manufacturers’ instructions (New England Biolabs, Ipswich, MA, USA). PCR were carried out in an Applied Biosystems 2720 Thermal Cycler (Thermo Scientific) using Platinum™ II Hot-Start Green PCR Master Mix (2x) (Invitrogen). The reaction mixture (50μL) contained 22μL ddH₂O, 25.0μL Platinum™ II Hot-Start Green PCR Master Mix, 1.0μL PCR forward primer (10mM), 1.0μL PCR reverse primer (10mM), 1.0μL DNA sample, using the following PCR program: initial denaturation for 2min at 94 °C; 30 cycles each for denaturation for 15 s at 94 °C, annealing for 15 s at 60 °C; and extension for 1.5min at 68 °C. DNA fragments for cloning purposes were extracted and purified from agarose gel using Qiagen Gel Extraction kit (Qiagen, Inc., Hilden, Germany). The homologous A and B fragments and pRE112 plasmid were connected by using Gibson Assembly^® Master Mix-Assembly (NEB Inc., Ipswich, MA, USA). A. salmonicida ssp. Salmonicida strain VI-88/09/03175 whole-genome sequencing was performed using the Illumina MiSeq platform v3 chemistry, 2 × 300 bp read length. Data QC, de novo assembly and annotation were performed using the BACTpipe pipeline [50]. From the annotated coding sequences, a S-ribosylhomocysteine lyase (luxS) homolog was identified, which was compared to its A. salmonicida ssp. salmonicida A449 reference strain equivalent using psi-BLAST (National Center for Biotechnology Information, NCBI).

4.4. Construction of ΔluxS Mutant

The luxS gene was knocked out with the suicide vector pRE112 (Table 3) by homologous recombination, according to the method described by Meng et al. [12]. Flanking regions of the luxS gene (A- upstream and B- downstream) were amplified from the A. salmonicida ssp. salmonicida strain VI-88/09/03175 gDNA template, using A-F/R and B-F/R primers and the plasmid pRE112 was digested by KpnI enzyme (NEB Inc., Ipswich, MA, USA) These two fragments were connected to the plasmid by using Gibson Assembly^® Master Mix-Assembly (NEB Inc., Ipswich, MA, USA). Subsequently, the plasmid ΔluxS_pRE112 was chemically transformed into E. coli S17-1 competent cells, containing pir and sacB gene, and chloramphenicol selection was performed for further bacterial conjugation with A. salmonicida. A sucrose selection (15%) for pRE112 absence was performed and confirmation of the ΔluxS genotype was made by PCR (Figure 7).

All the primers used in this study are shown in Table 3.

4.5. Quorum-Sensing Assay

For harvesting culture supernatant for AI-2 detection, A. salmonicida was cultured with 100 µg/mL mucin samples or with the same concentration of mono- and disaccharides, as described previously [8], in the denoted medium until cultures reached the stationary phase of growth. This is a time point where AI-2 production of A. salmonicida had reached maximum levels (Figure 2). Cultures were collected from wells and replicates were pooled and centrifuged at 4000× g for 5 min at RT. Supernatants were sterile-filtered (0.22 µm pore size, cellulose acetate filter, VWR), pipetted into white opaque 96-well plates and stored frozen at −20 °C ready for analysis. Freeze–thaw cycles and storage above −20 °C were avoided. Fresh, filtered A. salmonicida culture media (CM9 or DM) were used as negative controls in the bioreporter assay. To verify that the higher nutrient content in the negative control had no effect on the assay, we made a serial dilution from the negative control and subjected it to the bioreporter assay. No effect on nutrient dilution was detected in the DM media. Although a small effect could be detected in the CM9 media, the effect was less than 1% of the signal of the assays and therefore was considered negligible.

The measurement of autoinducer production was carried out with a quorum-sensing reporter bacterium: Vibrio harveyi transposon insertion mutant with the LuxN- phenotype (strain BB170 (ATCC^® BAA-1117™), courtesy of Bassler BL). V. harveyi strain BB170 was cultured overnight in Bioluminescence Autoinducer medium (ATCC medium: 2746) at 30 °C with continuous shaking at 100 rpm. The culture of the reporter strain having OD₆₀₀ = 1.0 was diluted 1:5000 in fresh BA medium. 50 µL from the diluted V. harveyi BB170 suspension was added to 50 µL sterile-filtered A. salmonicida supernatant in each well of a white opaque 96-well cell-culture plate (Corning Inc., Corning, NY, USA). The mono- and disaccharides used in the study were tested separately and found to not interfere with the detection of AI-2 signals of the A. salmonicida supernatants. The plate was incubated at 30 °C with continuous shaking at 100 rpm and light production was measured in 10 min intervals for 12 h in a Clariostar plate reader. To evaluate the quantity of quorum-sensing signal molecules, one timepoint (corresponding to the induction phase) was used, where the decrease in light production turned into an increase. This corresponds to the lowest luminescence value of the “U”-shaped luminescence curve (Figure 10). The higher the concentration of AI-2 added, the lower the reduction in luminescence before the luminescence-autoinduction phase of V. harveyi BB170 began.

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Figure 10

Light-production time course of V. harveyi BB170 reporter. This graph is an example showing the timepoint selection for evaluation of luminescent signals for standard curves and subsequent calculation of DPD equivalent AI-2 values. V. harveyi BB170 luminescence was measured in the QS-reporter assay in response to CM9 medium (negative control), DPD dissolved in CM9 medium and V. harveyi own supernatant (positive control). The dashed vertical line shows the time point corresponding to the start of autoinduction in V. harveyi coinciding with the lowest luminescence count of the negative control. Data points in the intersection of curves and the dashed line were used to plot “DPD concentration-relative luminescence” standard curves. Data points are expressed as Means (n = 3). Abbreviations: DPD = 4,5-dihydroxy-2,3-pentanedione.

4.6. Assay Sensitivity Testing

The sensitivity of the V. harveyi BB170 bioluminescent assay was tested with 4,5-dihydroxy-2,3-pentanedione (DPD, Sigma Aldrich). The sensitivity testing was carried out as described above, except that instead of A. salmonicida supernatants, DPD diluted to the desired concentration in 50 µL DM and CM9 media was used.

4.7. Growth Assay

A. salmonicida cells, at a concentration equivalent to an optical density at 600 nm (OD₆₀₀) of 0.1, were cultured in DM and CM9 media to test the linear range of detection of AI-2 signals and to analyze the effect of DPD on A. salmonicida. DM medium was used to test the effect of Atlantic salmon mucins and mucin saccharides on AI-2 production of A. salmonicida.

4.8. Growth Assay with DPD

DPD was diluted in 20 µL PBS and added to 80 µL A. salmonicida culture in 4–8 replicates of a Nunc-Delta surface flat-bottom plate (Nunc A/S, Roskilde, Denmark) to reach the specified final concentrations. 20 µL PBS added to 80 µL culture was used as a negative control. The assay was carried out at 23 °C and 120 rpm and monitored both by OD₅₆₀ readings and fluorescent detection of AlamarBlue (Invitrogen) reduction. Changes in growth were calculated in the logarithmic phase of growth and were expressed relative to the negative control.

4.9. Growth Assay with Mucins and Saccharides

Purified mucin samples in 4 M GuHCl were dialyzed eight times against PBS and diluted in sterile PBS to a concentration of 100 µg/mL. The saccharides were used in the same concentration. The GuHCl-containing isolation buffer was dialyzed and diluted in parallel and used as a reference of normal growth (dialysis control [DC]). PBS was used as a reference for growth in the presence of monosaccharides and oligosaccharides. Monosaccharides were purchased from Sigma-Aldrich. GD1a-oligosaccharide, GlcNAcβ1,3Gal, and GalNacβ1,3Gal were purchased from Elicityl, France. Core 1 glycan (Galβ1,3GalNAc) was purchased from Dextra Laboratories, UK. Bacteria were cultured in 96-well Nunc-Delta surface flat-bottom plates (Nunc A/S, Roskilde, Denmark) in 4 to 8 replicates at 23 °C and 120 rpm in the presence of alamarBlue (Invitrogen) to monitor reduction of the dye. AlamarBlue reduction corresponds to the metabolism of the culture and correlates well with CFU/mL [8].

4.10. Fish and Sampling Procedure

Atlantic salmon parr (Långhult lax, Långhult, Sweden) were transported to the Department of Biology and Environmental Science and kept in 500 L tanks. The fish were held in recirculating 10 °C fresh water (FW), supplemented with 10% salt water (SW) (yielding a salinity of 2–3 ‰), at a flow rate of 8.5 l/min. The fish were exposed to a simulated natural photoperiod and hand-fed ad libitum once daily with a commercial dry pellet (Nutra Olympic 3 mm; Skretting Averøy, Ldt., Stavanger, Norway).

Five fish (31.06 ± 0.49 cm bl and 280.70 ± 12.78 g bw; mean ± Standard Error of the Mean) were randomly netted, anesthetized in metomidate (12.5 mg/L) and killed with a sharp blow to the head. Mucus from the skin was sampled by gently scraping the skin off, including mainly the epidermis and secreted mucus of the entire fish, using two microscopy glass slides. The fish was then opened longitudinally and the intestine, from the last pyloric ceca to the anus, was quickly dissected out. The intestine was cut open along the mesenteric border and the proximal region was separated from the distal at the ileorectal valve and the mucus and mucosa was scraped off using microscopy slides. The pyloric ceca were dissected out using small scissors and were pulverized in liquid nitrogen. All samples were placed in 10 mM sodium di-hydrogen phosphate containing 0.1 mM phenylmethanesulphonyl fluoride (PMSF), pH 6.5 (sampling buffer), to inhibit proteolytic cleavage. The experiment on fish in the present study was approved by the Ethical Committee for Animal Experiments in Gothenburg, Sweden under licence #46/2009. All methods involving fish were performed in accordance with the relevant guidelines and regulations.

4.11. Isolation and Purification of Mucins

The scrapings and pulverized tissues in sampling buffer were placed into five sample volumes of extraction buffer (6 M GuHCl, 5 mM EDTA, 10 mM sodium phosphate buffer, pH 6.5, containing 0.1 M PMSF), dispersed with Dounce homogenizer (four strokes with a loose pestle) and stirred slowly at 4 °C, overnight. The insoluble material was removed by centrifugation at 23,000× g for 50 min at 4 °C (Beckman JA-30 rotor) and the pellet was re-extracted twice with 10 mL extraction buffer. The supernatants from these three extractions were pooled and contained the GuHCl soluble mucins, hereafter used in our assays. The samples were filled up to 26 mL with extraction buffer. CsCl was added to the samples by gentle stirring and the samples were transferred to Quick Seal ultracentrifuge tubes (Beckman Coulter). The tubes were filled with 10 mM NaH₂PO₄ to give a starting density of 1.35 g/mL and samples were subjected to density-gradient centrifugation at 40,000× g for 90 h at 15 °C. The fractions were collected from the bottom of the tubes with a fraction collector equipped with a drop counter. Density-gradient fractions of purified mucin samples were analyzed for carbohydrates as periodate-oxidizable structures in a microtiter-based assay: Fractions diluted 1:100, 1:500 and 1:1000 in 4 M GuHCl were coated into 96-well plates (PolySorpTM, NUNC A/S, Roskilde, Denmark) and incubated overnight at 4 °C. The rest of the assay was carried out at 23–24 °C. After washing three times with washing solution (5 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween 20, 0.02% NaN₃, pH 7.75), the carbohydrates were oxidized by adding 25 mM sodium metaperiodate in 0.1 M sodium acetate buffer, pH 5.5 for 20 min. The plates were washed again and the wells were blocked with DELFIA blocking solution (50 mM Tris-HCl, 0.15 M NaCl, 90 mM CaCl₂, 4 mM EDTA, 0.02% NaN₃, 0.1% BSA, pH 7.75) for 1 h. After further washing steps, the samples were incubated for 1 h with 2.5 mM biotin hydrazide in 0.1 M sodium acetate buffer, pH 5.5, followed by another washing step. Europium-labeled streptavidin was diluted 1:1000 in Delfia assay buffer (50 mM Tris-HCl, 0.15 M NaCl, 20 mM DTPA, 0.01% Tween 20, 0.02% NaN₃, 1.5% BSA, pH 7.75) and was added to the wells. After 1 h incubation, the plates were washed six times and then incubated with Delfia enhancement solution (0.05 M NaOH, 0.1 M ftalat, 0.1% Triton X-100, 50 mM TOPO, 15 mM b-NTA) for 5 min on an orbital shaker. Fluorescence (λ_excitation = 340 and λ_emission = 615) was measured using a Wallac 1420 VICTOR₂ plate reader with the Europium label protocol (PerkinElmer, Waltham, MA, USA). Density measurements were performed using a Carlsberg pipette as a pycnometer: 300 µL of sample was aspirated into the pipette and weighed, and density was calculated as g/mL. DNA content was measured by UV light absorbance at 260 nm.

4.12. Preparation of Mucin Samples

Gradient fractions containing mucins were pooled together to obtain one sample for each gradient. Mucin concentrations in pooled samples were determined by serial dilution of the samples, as well as a standard curve of a fusion protein of the mucin MUC1, 16TR and IgG2a Fc starting at a concentration of 20 mg/mL and using seven 1:2 serial dilutions and detection of carbohydrate as periodate-oxidizable structures in a microtiter-based assay, as described above. The mucin concentrations were calculated from the standard curve. This method of concentration determination was chosen as not all mucins come into solution after freeze drying, and determining concentration by freeze drying can therefore contain large errors or remove mucin species selectively. Although this is not an exact measure of concentration, it can be used to ensure that the mucins are at the same concentration for comparative assays, and since bacteria–mucin interactions largely occur via the mucin glycans [6], setting the concentration based on the glycan content appears most appropriate.