Absorbance of CBBR-250 Dye Vs LTA Concn (in PBS)
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 1 10 100 1000 10000 LTA-PO4 Concentration (μM) A b s at 596 nm
Figure 18: Determination of Critical Micelle Concentration of LTA from HN001 Using CBBR-250
Results of a typical CMC determination experiment, carried out in PBS buffer. The increase in absorbance at 596 nm of CBBR-250 dye in solution with LTA indicates the formation of micelles. The point at which the absorbance begins to rapidly increase is defined as the CMC. At concentrations below the CMC the LTA is monomeric, and above the CMC the LTA is incorporated into micelles, as indicated by the schematic diagrams in the figure.
Lipoteichoic acid is an amphipathic molecule, and has the potential to form micelles at sufficiently high concentrations. The micellar form of LTA may not interact with PRRs on immune cells in the same way as monomeric LTA, due to the differences in size and shape. The clustering of MAMP-PRR complexes on immune cells may also be affected by the presentation of LTA as micelles or monomers. After binding of LTA to a PRR such as TLR2, the interaction of the LTA-PRR complex with other receptors on immune cells is likely to be altered. The potential for recognition of the lipid part of LTA would certainly be diminished by micelle formation, as the location of the hydrophobic moiety would be buried within the micelles, making it unavailable for interaction with PRRs. For these reasons, LTA micelles might induce a different immune response than would monomeric LTA. It is therefore important to know the concentration at which micelles begin to form, i.e. the critical micelle concentration (CMC) for each LTA. The CMCs of the purified LTA from all three strains in this study
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were determined using Coomassie Brilliant Blue R-250 dye (CBBR-250). The green form of this dye is incorporated preferentially into micelles, causing an equilibrium driven increase in the concentration of the blue form, which absorbs at 596 nm. The absorbance of a fixed concentration of CBBR-250 was measured in the presence of a range of LTA concentrations, and the CMC was determined from the point at which the colour begins to change, as shown in Figure 18.
The CMC of LTA from HN001 was initially determined in PBS (Figure 18). Note that LTA purified using the optimised chromatography protocol (as shown in Figure 19) was used to determine the CMC, not the LTA of uncertain purity shown in Figure 15. Experiments for all strains with the LTA in RPMI cell-culture medium demonstrated that the CMCs were lower at room temperature than at 37 ºC (the immune cell assay is incubated at 37 ºC, section 2.2.8.2). For this reason, the CMCs were determined at room temperature, the lowest temperature expected to be encountered during the set-up of the immune assay. As each of the samples contained a heterogeneous mixture of LTAs which may have different individual CMCs, the CMC for each sample was determined to be the lowest concentration at which micelles appeared to form. The critical micelle concentrations determined in RPMI media, in terms of phosphate concentration, are listed in Table 3. The values obtained are in a similar range to those for S. aureus LTA found in the literature of 28 – 60 μg/mL in PBS at ambient temperature (Courtney et al.
1986). It seems therefore unlikely that the specific immune responses induced by these bacteria are due to the CMCs of their respective LTAs.
CMC in RPMI Media
CMC
(μM LTA-PO
4)
CMC
(μg/mL LTA)
(at room temp.)
HN001
266 64
DltD- Mutant
221 49
IM126
158 31
Table 3: Critical Micelle Concentrations of LTA Purified from L. rhamnosus Strains
The CMC of LTA purified from each of the strains HN001, the DltD- mutant and IM126 was determined by measuring the absorbance of Coomassie Brilliant Blue R-250 dye in solutions containing LTA at a range of concentrations. The CMCs shown were determined in RPMI media, the closest approximation to
the conditions of the PBMC assay. Each concentration is shown both in units of μM LTA-PO4, as
referred to predominantly in this thesis, and also in μg/mL, for comparison with literature values. The total combined LTA was used for each strain and the CMC represents the lowest concentration where micelle formation was observed.
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Using the information in Table 3, the concentrations of the fractions 29 – 45 in Figure 16 were determined to be above the CMC for LTA from HN001. The formation of micelles in these samples is the probable explanation for the lower than expected TNF responses to these fractions.
3.3.3.1Optimised HIC of LTA
Based on the results of the preliminary HIC purification of LTA, the HIC separation protocol was optimised to provide high purity LTA. To eliminate the problem of column overloading, trials were undertaken to establish the optimum load for HN001 crude LTA, which was determined to be 15 μmoles of total phosphate (PNH). The FPLC elution program was modified to include a longer wash step of seven column volumes, (compared to five), to ensure separation of the LTA-phosphate peak from the unbound material. Fractions of 5 mL were collected and the phosphate concentration of each was measured as described. The results are shown in Figure 19. Monitoring of the UV signal again revealed a large peak in the early unbound fractions that correlated with a large unbound phosphate peak. It also showed that the LTA-phosphate peak was free of detectable contaminating proteins and nucleic acids (data not shown). The LTA- phosphate peak is now well resolved from the unbound phosphate-containing material with no evidence of column overloading, and consists of the main peak (fractions 43 - 52, Figure 19), and a small shoulder (fractions 53 – 57, Figure 19). Immune cell assays of the fractions shown in Figure 19 revealed that TNF inducing activity was now greatest for fractions in the LTA-phosphate peak, and that there was little TNF induced by the flow-through fractions (section 3.4.1). This HIC profile is typical for LTA purified from gram positive bacteria reported in a number of published studies. The apex of the main peak was sometimes observed to be split, as in Figure 19, which may indicate the presence of two closely overlapping peaks. Although this split peak was not always apparent, its shape was taken into consideration later when pooling fractions for parallel NMR and immune response experiments (section 3.3.4.1).
Trials were carried out to determine the optimum loading amount for HIC of LTA from the DltD- mutant and IM126 strains. For IM126, as with HN001, loading was 15 μmoles of phosphate (PNH), but for the DltD- mutant, 90 μmoles (PNH) of DltD-
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crude LTA in terms of phosphate was loaded, as the starting material from this mutant strain appeared to contain much less LTA-phosphate per total phosphate loaded. The elution profiles of LTA from IM126 and the DltD- mutant were similar to that of HN001 seen in Figure 19, when the same FPLC protocol was applied.