The Effect of D-Ala Substitution

The reported effects of D-ala substitution on the immune responses to a number of strains are summarised in Table 10. When the dltD gene was knocked out in

L. rhamnosus GG, the D-ala substitution of the LTA of this mutant was completely abolished; nevertheless, the cytokine responses from PBMCs incubated with either the WT or the dltD- mutant were similar (Perea Velez et al. 2007). When the same dltD gene was inactivated in L. rhamnosus HN001, D-alanylation of LTA was eliminated, as in GG; however, the levels of TNF, IL-1ȕ, IL-8 and IL-10 secreted by PBMCs in response to the mutant bacteria were substantially increased compared to that of the WT, in contrast to GG (Table 10). One explanation for this dramatic difference in the impact of D-alanine depletion could be the presence of GlcNAc substituents in the LTAs of HN001 and its DltD- mutant (Table 9), and the absence of sugar substituents on L. rhamnosus GG LTA and its dltD- mutant (Table 10, Figure 35b). A dltA knockout of Streptococcus pyogenes resulted in mutant bacteria with LTA that were substituted at less than 7 % with D-ala, versus 65 % for the WT; however, both the purified LTA and whole cells of this mutant induced similar pro-inflammatory cytokine responses in human whole blood to the WT LTA and cells (Hasty et al. 2006). The LTAs from both

dltA mutant and WT strains of S. pyogenes have no saccharide substituents on the PGP chain, only D-ala esters (Table 10). The authors propose that the reason for the apparent

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lack of influence of D-alanylation of LTA on the cytokine response for S. pyogenes is because it is not glycosylated. Similarly, a dltA- mutant of Group B Streptococcus

(GBS) that contained LTA lacking D-ala esters induced similar cytokine concentrations as the WT (Table 10); the LTA of GBS is also not glycosylated (Henneke et al. 2005).

Staphylococcus aureus contains LTA that is substituted with GlcNAc (Figure 35a), and the cytokine inducing activity of this LTA is reduced after elimination of D-ala (Table 10) (Morath et al. 2001; Rockel et al. 2010). D-alanylation of LTA has a significant impact on the cytokine induction profile of Lactobacillus plantarum, which has glucose substituents on its LTA (Figure 35c) (Grangette et al. 2005; Jang et al.

2011), adding further weight to the hypothesis that the influence of D-alanylation of LTA is only seen when the PGP chain of LTA is also substituted with saccharide molecules.

While D-alanylation of LTA was shown to play an important role in the cytokine response induced by LTA from both the pathogen S. aureus and the probiotic HN001, reduction of D-ala substitution on LTA from each of these strains had quite different outcomes (Table 10). Elimination of D-ala on the LTA from HN001 brought about a large increase in TNF induction in PBMC by both the LTA and the bacteria, whereas removal of D-ala substituents from S. aureus LTA by alkaline hydrolysis resulted in a reduction of TNF response to purified LTA in whole blood (Morath et al. 2001). The alkaline hydrolysis may have also had other effects on the structure, such as removal of sugar substituents; however, the immune results were consistent with recent findings for LTA that lacked D-ala, isolated from a dltA- mutant of S. aureus (Rockel et al. 2010). The same was true for a non-pathogenic Bacillus subtilis, alkaline treated LTA from which induced less TNF in PBMCs than did intact LTA (Ryu et al. 2009); and an oral commensal Streptococcus gordonii LTA, which when de-D-alanylated (by creating a

dltA knockout), reduced TNF induction in DCs (Table 10) (Chan et al. 2007). HN001 is unusual in this sense, as it appears to be the only bacteria studied so far where removal of the D-alanyl esters from its LTA actually increases the induction of pro- inflammatory cytokines. Conversely, when a dltB- mutant of L. plantarum was created, reduction of the D-ala content of the LTA decreased the induction of the pro- inflammatory cytokines TNF, IL-1ȕ, IL-6 and IL-8 (Table 10), similar to what was observed for S. aureus and S. gordonii (Morath et al. 2001; Chan et al. 2007); however, the induction of anti-inflammatory IL-10 was increased for the dltB- mutant (Grangette

Šƒ’–‡”͛Ǧ‡•—Ž–•Ƭ‹•…—••‹‘

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et al. 2005). NMR analysis indicates that the LTA from HN001 contains unsaturated fatty acids; whereas LTAs from S. aureus and B. subtilis have only SFAs (section 3.5.4.5 and Figure 35a). Structural differences such as this may be partly responsible for the different effects after the elimination of D-ala. The fact that GlcNAc substituents are found on the LTA from HN001, which is unusual among LTA from LAB (section 3.5.4.3 and Figure 35b-d), may also explain the contrasting effects of D-alanine depletion of LTA in HN001 and L. plantarum.

Interestingly, both the L. plantarum mutant bacteria, and the DltD- mutant of HN001 in this study induced considerably more of the anti-inflammatory cytokine IL-10 than their respective wild type bacteria (Grangette et al. 2005). While the authors did not measure the IL-10 induced by the purified LTAs, it seems likely that the altered LTA is

Substituents Effect on Immune Response to:

Strain % D-Ala Sugar LTA Bacteria

HN001 * 79 GlcNAc

HN001 dltD- nil GlcNAc ½ ½

L. rhamnosus GG 74 nil

L. rhamnosus GG dltD- nil nil n.d. ** §

L. plantarum 42 nil L. plantarumdltB- 1 Glc ¾ pro-infl. IL-10 n.d. ¾pro-infl. ½IL-10 L. lactis 29 Gal n.d. L. lactisdltD- 6 Gal n.d. § S. aureus 70 GlcNAc S. aureusdltA- nil n.d. ¾ § S. pyogenes 65 nil S. pyogenes dltA- 7 nil § § GBS 20 nil GBS dltA- nil nil n.d. § S. gordonii †3.2μg/mg n.d. S. gordonii dltA- nil n.d. ¾ ¾

Table 10: Effects of dlt Mutations on Immune Responses

KEY: ½, increased; ¾, decreased; §, similar to WT; n.d., not determined.

Comparison of the effects after inactivation of genes of the dlt operon, which leads to decreased D-ala

substitution of LTA. The effect of these mutations on the immune response induced by isolated LTA and/or whole bacteria are indicated relative to the WT LTA and/or bacteria. * Weighted average of D-ala content, calculated using the data for the three pools and the proportions of total LTA for each pool. ** Recently shown to exhibit improved probiotic effects on colitis in mice compared to the WT (Claes

et al. 2010). † Dry wt of D-ala per dry wt of LTA. Strains: Lactobacillus rhamnosus GG (Perea Velez

et al. 2007), Lactobacillus plantarum NCIMB8826 (Grangette et al. 2005; Palumbo et al. 2006),

Lactococcus lactis MG1363 (Perea Velez et al. 2007; Kramer et al. 2008), Staphylococcus aureus S113

(Peschel et al. 1999; Rockel et al. 2010), Streptococcus pyogenes M type 1 strain 8004 (Hasty et al.

2006), Group B Streptococcus NEM316 (Henneke et al. 2005) and Streptococcus gordonii PM14 (Chan

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responsible for at least part of this response. The LTA from the L. plantarum mutant, while greatly reduced in D-alanine, was found to be substituted with glucose on 24 % of the GroP units, while the WT LTA had barely detectable amounts of glucose (Table 10). The LTA of both the WT HN001 and the DltD- mutant in this study were substituted with sugars on the PGP chain, but no increase in the amount of these sugar substituents were observed in the mutant LTA, in contrast to L. plantarum. Similarly, a

dltD- mutant of Lactococcus lactis was shown to have LTA with its D-ala content reduced to 6 % (from 29 % for the WT, Table 10), while substitution of the PGP chain with galactose was considerably increased from 12 % to 46 % (Kramer et al. 2008). This helps to demonstrate the complexity of the various factors involved in the immune response due to LTA, and it seems unlikely that any one structural component of the LTA is solely responsible for all of the differences seen between different strains of gram positive bacteria.

It has been shown that the D-alanine esters of LTA are able to migrate along the PGP chain, or between chains, after the initial esterification, independent of the D-alanylation enzymes (Childs et al. 1985); thus the positions of the D-alanines on the native LTA structure are not known. Nor is there knowledge of the distribution of the sugars along the PGP chain, although there is no evidence that they are redistributed between GroP units. The relative positions of the D-ala and saccharides on the PGP chain may be important for immune recognition, as receptors on immune cells may recognise a specific architecture made up from these molecules. If an LTA molecule was highly substituted with D-ala, the D-ala residues may be quite close together along the chain. In an LTA with low levels of D-ala substitution, however, the D-ala residues may be further apart. Therefore, altering the degree of D-ala substitution may alter this architecture, resulting in changes to the recognition of LTA.