The Effect of the Glycolipid Anchor

The average length of the FA chain of LTA from the strains in this thesis was estimated to be approximately 15 carbons (section 3.5.4.5), which is in agreement with the 14 - 16 carbon length typically found in the membrane anchor of LTA (Morath et al. 2001; Perea Velez et al. 2007). It seems that the average FA chain length is not a major determinant of the immune response to LTA, although other differences in the composition of the FA chain may be important. Structural changes in the lipid anchor may alter the recognition and binding of LTA by PRRs, including co-receptors such as CD36, which is known to interact with ligands containing diacyl chains (section 1.2.5). Further analyses of the lipid moieties of the LTAs in this study are required before the role of the lipid part in cytokine induction can be described in detail.

FACING PAGE:

Figure 35: Structures of LTA from S. aureus, L. rhamnosus GG, L. plantarum and L. lactis

Schematic diagrams of four Type I LTA structures as determined by NMR and MS analysis, reproduced

with permission. Structural elements and their relative average proportions are shown where appropriate.

(a) LTA from Staphylococcus aureus DSM20233 (Morath et al. 2001). (b) LTA from

Lactobacillus rhamnosus GG, which contains no sugar substitution of GroP (Perea Velez et al. 2007).

(c) LTA from Lactobacillus plantarum KCTC 10887BP (Jang et al. 2011). NB: L. plantarum

NCIMB8826 (not pictured) contained LTA with 22 GroP units, 42 % D-ala substituted, whereas a dltB-

mutant of this strain had LTA with 63 GroP units substituted with 1 % D-ala and 24 % glucose

(Grangette et al. 2005). (d) LTA from Lactococcus lactis G121, which contains lactobacillic acid (a

(d) Lactococcus lactis LTA (c) Lactobacillus plantarum LTA (b) Lactobacillus rhamnosus GG LTA

GroPn = 48 %D-ala = 70 %GlcNAc = 15 FA = C15 Double bonds = 0 GroPn = 50 %D-ala = 74 FA = C14 Double bonds = 1-2 GroPn = 7 - 29 %D-ala = 0 FA = C16 Double bonds = 1-2 FA = C15-22 Double bonds = 0-2 FA = C14-16 Double bonds • 1 Lactobacillic acid ~14 %

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NMR analysis indicated the presence of unsaturated fatty acids (UFAs) in LTAs of all three strains in this thesis (section 3.5.4.5). The presence of olefinic bonds in fatty acids can alter the structure of membrane lipids. In particular, cis-unsaturated carbon-carbon bonds are known to introduce kinks in the acyl chain, changing the structure dramatically. They also increase membrane fluidity and decrease the tightness of packing in the cell membrane and UFAs have also been shown to be less likely to be incorporated into lipid rafts than SFAs (Zhang and Rock 2008); all of these may change the accessibility of LTA on cells to PRRs. The presence of UFAs in the glycolipid anchor of LTA may alter the recognition by PRRs of both isolated LTA and LTA on cells, and/or the binding affinity between ligand and receptor(s). The activation of TLR2 by lipopeptides has been shown to be potentiated by SFAs, but suppressed by UFAs (Lee et al. 2003). Similar modulatory effects were also demonstrated for activation of TLR4 by LPS. It may be expected then, that TLRs may interact differently with LTA containing UFAs, compared with those containing only SFAs. It has been suggested that the degree of saturation of the FA in LTA is a major structural difference between highly pro-inflammatory LTAs, such as those from S. aureus and B. subtilis and LTAs that are less pro-inflammatory or that have anti-inflammatory properties, for example, those from commensal or probiotic bacteria such as L. plantarum and L. rhamnosus

(Jang et al. 2011). LTA from both S. aureus and B. subtilis have LTA that contain only SFAs, while both L. rhamnosus and L. plantarum have LTA containing UFAs and SFAs (Figure 35). The identification of UFAs in all fractions of the LTA from all three strains in this thesis may help to explain some of the immunomodulatory properties of HN001 and IM126.

The glycolipid anchor of LTA most commonly has two acyl chains, as found in

S. aureus and B. subtilis (Morath et al. 2001; Neuhaus and Baddiley 2003); however, this is not always the case (Figure 35). LTA from L. rhamnosus has been previously shown to contain both diacyl and triacyl glycolipids, at a ratio of 60 % to 40 % of the LTA, respectively (Fischer et al. 1980). However, this has not yet been established for the L. rhamnosus strains studied here. Recently, L. plantarum was shown to synthesise both diacyl- and triacyl-anchored LTAs (Jang et al. 2011). Listeria monocytogenes has been reported to produce two different LTA structures, one with a DAG anchor, and another simultaneously possessing two DAG chains, a total of four acyl chains attached to the glycolipid (Dehus et al. 2010). These and other variations in acylation of the

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glycolipid may continue to be found in the LTA of other strains, and may have a significant impact on the immune recognition of LTA. The PRR, TLR2 is known to be important for the immune recognition of lipopeptides. It has been shown that diacylated lipopeptides require the formation of a TLR2/TLR6 heterodimer to be recognised by TLR2, whereas recognition of triacylated lipopeptides occurred only after formation of a TLR2/TLR1 heterodimer (Farhat et al. 2008; Kang et al. 2009). As TLR2 has been implicated in the recognition of LTA, it is quite possible that the formation of TLR heterodimers could also be impacted by the number of acyl chains in the glycolipid moiety of LTA, with potentially different immune outcomes. CD36 is known to recognise diacylated lipid compounds, and is thought to be a co-receptor in the recognition of LTA by TLR2 (Hoebe et al. 2005; Jimenez-Dalmaroni et al. 2009). It is possible that the roles of co-receptors such as CD36 and CD14 are not the same in the case of the triacylated ligand, which may lead to differences in the recognition and response to diacylated and triacylated LTAs. The presence of triacyl membrane anchors in the LTA of probiotic strains of L. plantarum and L. rhamnosus may therefore be partly responsible for the unique immune responses to these bacteria, when compared to highly pro-inflammatory strains such as S. aureus, which contain only diacylated LTA. This requires further investigation.

Changes to the structure of LTA such as the saturation of the fatty acids or the number of acyl chains anchoring the molecule in the membrane are also likely to impact on the fluidity of LTA in the membrane. As a consequence, the formation of lipid rafts and other complexes involving LTA may be affected. Presentation of ligands such as LTA on a scaffold, where multiple ligands are clustered on the surface, is known to increase the specificity and stability of interactions with receptors, compared with single ligand- receptor binding. Thus, the efficiency of recognition by immune cells of LTA on the bacterial surface may be influenced by the ability of LTA to migrate across the membrane. The interaction of multiple receptors and ligands is common in immune signalling. Thus, a structural change in one component of an immune recognition complex may alter the overall specificity or stability of the complex, and result in different signalling.

LTAs with different lipid anchor structures may be released into the environment at different rates, which may have an impact on immune responses to bacterial cells. This

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study found that a considerably higher concentration of purified LTA was required than that naturally occurring on HN001 cells to induce a similar level of cytokine response from PBMCs (section 3.4.5). Therefore, released LTA is unlikely to have much influence. It is not clear however, what impact any increased release of free LTA along with other bacterial components, such as peptidoglycan or nucleic acids, might have on this response.

For IM126, the approximate lengths of the PGP and FA chains, and the degree of substitution with D-ala and GlcNAc were found to be similar to HN001 (Table 9). Although the LTA from IM126 generally demonstrated a weaker cytokine inducing activity than LTA from HN001, the concentrations of cytokines induced in response to LTAs from HN001 and IM126 were more similar than those measured for LTAs from the DltD- mutant (Figure 25). The pattern of cytokines induced in PBMCs was also quite similar for HN001 and IM126 LTAs (Figure 26 A and C; Figure 27 A and C). The biggest structural difference between these two LTAs revealed by NMR spectroscopy was an extra 5 – 7 repeating units in the PGP chain of IM126 LTA. It is possible that there may be differences between the glycolipid moieties in the two LTAs that were not identified in this study, or there may be sugars other than GlcNAc attached to some of the GroP units. The small differences between the structures of the LTA from HN001 and IM126 seem, however, to correlate with the small differences between the cytokine responses to these two LTAs. This generally supports the idea that differences in the structure of LTA are responsible for the differences in immune response, as the more obvious differences between the structures of LTAs from the DltD- mutant and WT HN001, compared to the minor differences between IM126 and HN001 LTAs, were reflected in the relative immune responses in PBMCs.