It is possible that the amount of LTA present in each cell is another factor in the immunomodulating ability of bacteria. Purified LTA from IM126 was shown to be a poor inducer of cytokine secretion from PBMCs when compared to LTA from HN001; however, IM126 cells were estimated to contain more than three times the amount of LTA per cell as HN001 (section 3.4.4). A number of bacteria have been shown to vary the total cellular LTA content in response to environmental changes (section 1.2.2). It may be that the greater amount of LTA in IM126 cells compensates for the weaker cytokine inducing activity of the LTA to give the significant cytokine response induced by IM126 bacteria in PBMC. The DltD- mutant strain was estimated to have ~ 4 times the molar amount of LTA per cell as the wild type HN001, and slightly more than IM126 cells. If the amount of LTA per cell alone were responsible for the LTA-induced immune response of bacteria then it would be logical to expect IM126 to induce the same levels of cytokine secretion from PBMCs seen for the DltD- mutant. This is not
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the case, so it appears that the differences in cytokine inducing activity between these three strains is not due solely to the amount of LTA in the cell.
Using the data for the amount of LTA per bacterial cell, the estimated final concentrations of cell-associated LTA for each of the whole cell samples: HN001, the DltD- mutant and IM126, were calculated (Table 11). Each of these samples contained intact cells at 1×106 cfu/mL, which is equivalent to 1×109 cfu/L. The amount of LTA- phosphate per cfu calculated for each strain in section 3.4.4 was divided by 1×109 cfu/L to give approximate concentrations of cell-associated LTA in the whole cell samples of 0.016, 0.045 and 0.060 μM phosphate for HN001, the DltD- mutant and IM126, respectively. Using HN001 as an example, the amount of LTA present in the whole cell sample in Figures 26A and 27A containing 0.016 μM PO4 is approximately 3000 times
less than that present in the 50 μM LTA-PO4 purified LTA sample. Interestingly, the
whole cells induced roughly similar levels of secreted TNF, IL-1ȕ, IL-8 and IL-10
Estimated Amount of LTA per Cell
0.00E+00 2.00E-12 4.00E-12 6.00E-12 8.00E-12 1.00E-11 1.20E-11 1.40E-11 HN001 DltD- IM126 Dry w t (mg/ cfu) 0 5E-13 1E-12 1.5E-12 2E-12 L T A (μ mo l/c fu )
Dry wt pri_0 sec_0 LTA
Figure 28: Estimated Amount of LTA per Cell
The amount of LTA per cell for HN001, the DltD- mutant and IM126, as estimated from the yield of LTA from purification, and the number of colony forming units in the bacterial culture used in the preparation. Blue bars: the dry weight of LTA in mg, left-hand axis. Dark green bars: the amount of LTA in μmoles (calculated by dividing the amt of phosphate by the number of GroP per chain, see Table 11), right-hand axis. The concentrations of phosphate per cfu for HN001, the DltD- mutant and
IM126 were 3.6×10-13, 9.2×10-13 and 9.5×10-13 μmol/cfu, respectively. The concentrations of LTA in
the graph are calculated by dividing the concentrations of LTA-PO4 by the estimated PGP chain
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cytokines as the 50 μM LTA-PO4 sample (Figures 26A and 27A), suggesting that the
LTA alone is a much less potent inducer of these cytokines than when it is on cells. It is possible that localised LTA concentrations on the bacterial cell surface may be significantly higher, increasing the effective concentration per cell and leading to a greater immune response. LTA on the bacterial cell surface is anchored to a scaffold, and thus may be presented to immune receptors in a very different way than is purified LTA, potentially increasing the efficiency of the immune response. Immobilisation of purified S. aureus LTA on a polystyrene surface has been shown to increase the induction of the pro-inflammatory cytokines IL-1ȕ, TNF and IL-6 and the chemokines IL-8 and G-CSF by LTA in whole blood, when compared to soluble LTA (Deininger
et al. 2008). Interestingly, the induction of the anti-inflammatory cytokine IL-10 was unaffected by immobilisation. It may be that the interaction of host cell receptors with LTA on the surface of bacterial cells leads to localised increases in signalling and/or cross-linking between receptors, resulting in a more efficient immune response. There are MAMPs other than LTA on L. rhamnosus, such as peptidoglycan, that are also able to interact with PRRs on PBMCs, and the presence of these other molecules may have a synergistic effect on the cytokine response that would not be possible for purified LTA.
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3.5
Structural Analysis of LTA
Purified lipoteichoic acids from HN001, the DltD- mutant and IM126 were prepared in three pools per strain, as described in section 3.3.4.1, that is, for each strain the main LTA-PO4 peak was divided in half to create Pools 1 and 2, and the smaller, later eluting
LTA-PO4 peak was collected as Pool 3. Each of these nine fractions was assayed
separately in the immune assay, and also individually analysed by NMR spectroscopy. A representative 1D 1H NMR spectra with the structural components labelled is shown in Figure 29. The 1D 1H spectra for all three fractions of LTA from each of HN001, the DltD- mutant and IM126 are shown in Figures 30, 31 and 32, respectively. The NMR profile in each case revealed that the samples contained Type I lipoteichoic acid, consisting of a polymer of glycerol-phosphate linked to a glycolipid anchor, based on comparison with published NMR studies of LTA (Morath et al. 2001; Neuhaus and Baddiley 2003). Many of the peaks in the 1H NMR spectra are broader than those seen in published spectra for S. aureus (Morath et al. 2001), which makes the assignment of structural elements from the spectra more difficult. This is indicative of a more complex sample, and suggests that the samples of LTA examined in this thesis may be more heterogeneous than those from S. aureus. As a result, identification of the specific LTA structure(s) that are responsible for immune activity may prove to be difficult. It must be remembered that the NMR data for each sample represent an average structure for a heterogeneous mixture of different LTA molecules. The structural heterogeneity of LTA may play a role in the immune response to bacteria. If, for instance, a particular structural motif was responsible for the immunomodulation, then it could be possible for a strain with more homogeneous LTA to contain a greater proportion of LTA with this motif, compared to a strain with greater structural variation in its LTA.