Precio

The CW extracts (resuspended in M199) from strains AGR1485 and AGR1487, (described in Section 4.3.2.2) were further fractionated using SE-HPLC to separate out the proteinaceous components present in the CW extract. SE-HPLC is reported to be the most suitable method for separation of macromolecules based on their molecular weights [325]. This analytical technique may be used to provide the “absolute”

molecular weights and characterisation of polymers and biopolymers, in a non- destructive process from which the recovered samples can be used for subsequent studies [326].

4.3.3.1. Size-exclusion high performance liquid chromatography

For both bacterial CW extracts, SE-HPLC was carried out using a Biologic Duo Flow HPLC system (Bio-Rad, USA) with detection at 280 nm and 214 nm, using a Biologic Quad Tec UV-Vis detector and an automated fraction collector. The column used was a GE Healthcare Tricorn 10/600 high performance column packed with

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Superdex 200 matrix (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The Superdex 200 column (dextran-agarose gel, bed height 600 mm, bed volume of approximately 45 mL and an internal diameter of 10 mm) has a separation range of 10 to 600 kDa for globular proteins. Standard proteins that result in peaks of known molecular weights, comprising of cytochrome c (12.4 kDa, Sigma-Aldrich, New Zealand), BSA (66 kDa, Sigma-Aldrich, New Zealand), and β-amylase (200 kDa, Sigma-Aldrich, New Zealand) were run and the elution times of the resulting peaks were then used to generate a standard curve. The equation from the standard curve was used to determine the approximate molecular weight range of the protein peaks that resulted from SE-HPLC of the AGR1485 and AGR1487 CW extracts. Each standard protein (1 mg) dissolved in 150 μL of M199. The three standard proteins were combined, filter sterilised using a 0.22 μm filter (Ultrafree Centrifugal filters, Durapore, Millipore) and applied to the Superdex 200 column using a 1 mL loop. The column was eluted with M199 (no supplements added) at a flow rate of 0.6 mL/min. A standard curve of elution time versus log molecular weight was plotted and used to determine the molecular weights of peaks resulting from SE-HPLC of the AGR1485 and AGR1487 CW extracts. Elution time was used as a parameter to calculate the corresponding molecular weights of the peaks.

The bacterial CW extracts were obtained from sonication and ultracentrifugation of bacterial culture (described in Section 4.3.2). Each strain was grown to late stationary phase (2.5 OD600) and 40 mL of each bacterial culture was used to yield a bacterial CW pellet, which was resuspended in 500 μL of M199 and subjected to SE-HPLC fractionation. As sonication and ultracentrifugation were carried out in a non-sterile

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environment, the CW extract from each strain was filtered sterilised using a 0.22 μm filter (Ultrafree Centrifugal filters, Durapore, Millipore) and applied to the Superdex 200 column. The Durapore microporous filter membranes display the lowest protein binding of any of the filter membranes and therefore a minimum loss of proteins due to filtration was assumed. Fractions were collected over the entire range of the elution volume, irrespective of a peak in absorbance, to ensure collection of non-protein (non- absorbing) compounds such as lipids and glycans. For both strains AGR1485 and AGR1487, the first fraction (Fraction 1) collected was determined by elution time rather than peak size, because this fraction was likely to contain non-proteinaceous bacterial CW components. The other three fractions were collected based on the generated peaks. The fractions collected were filter sterilised using 0.22 μm syringe filters (Millipore), concentrated using Vivaspin 20 concentrators (10 kDa MWCO for fractions 1 through 3, and 3 kDa MWCO for fraction 4) to a final volume of 2.5 to 3 mL for each fraction. The pH of each concentrated fraction was measured using a Beckman 340 pH Meter. The concentrated fractions collected from SE-HPLC, were filter sterilised using 0.22

μm syringe filters before being applied as treatments in TEER assays.

4.3.3.2. Protein concentration determination of bacterial cell wall fractions Protein concentrations of the bacterial CW fractions resulting from SE-HPLC were determined by using the Bradford assay [324] as previously described in Section 4.3.4. To test the assumption that CW proteins present in normal concentrations can affect Caco-2 cell TEER, the CW protein concentration of live AGR1487 was determined. Live AGR1487 re-suspended in M199 containing 1% NEAA to an optical density of 0.9 OD600 was applied (500 μL/Transwell) to Caco-2 cell monolayers and

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incubated for 12 hours at 37ºC in 5% CO2. After 12 hours, bacteria and Caco-2 cells were harvested and subjected to sonication as described in Section 4.3.2.2. The protein concentration of the resulting CW extract of AGR1487 was determined using the Bradford assay as described in Section 4.3.4. The protein concentration of the Caco-2 cells from the control Transwells (without bacteria), harvested after the 12 hour incubation period, was used as background.

The proteins present in AGR1487 CW fraction 4 was calculated as a percentage of total protein present in all CW fractions. The latter was determined using the AGR1487 SE-HPLC chromatograms and by calculating the area under all the peaks (A=280 nm) resulting from the runs. The area under peaks 3 and 4 that were combined to prepare fraction 4 was then used to calculate the percentage of total CW proteins that were present in fraction 4. The calculations were done using R with function “auc” in

package “MESS” to compute the area under the curves (AUC). The concentration of proteins of fraction 4 and the percentage of proteins present in fraction 4 compared to total proteins present in all CW fractions was used to calculate the concentration of proteins (fraction 4 proteins) that would be present in AGR1487 CW (0.9 OD600) which has been shown to affect TEER of Caco-2 cell monolayers.

4.3.4. Transepithelial electrical resistance assay with crude bacterial