Characterization of O-acetylation of N-acetylglucosamine: a novel structural variation of bacterial peptidoglycan

Abstract

Peptidoglycan (PG) N-acetyl muramic acid (MurNAc) O-acetylation is widely spread in gram-positive bacteria and is generally associated with resistance against lysozyme and endogenous autolysins. We report here the presence of O-acetylation on N-acetylglucosamine (GlcNAc) in Lactobacillus plantarum PG. This modification of glycan strands was never described in bacteria. Fine structural characterization of acetylated muropeptides released from L. plantarum PG demonstrated that both MurNAc and GlcNAc are O-acetylated in this species. These two PG post-modifications rely on two dedicated O-acetyltransferase encoding genes, named oatA and oatB, respectively. By analyzing the resistance to cell wall hydrolysis of mutant strains, we showed that GlcNAc O-acetylation inhibits N-acetylglucosaminidase Acm2, the major L. plantarum autolysin. In this bacterial species, inactivation of oatA, encoding MurNAc O-acetyltransferase, resulted in marked sensitivity to lysozyme. Moreover, MurNAc over-O-acetylation was shown to activate autolysis through the putative N-acetylmuramoyl-L-alanine amidase LytH enzyme. Our data indicate that in L. plantarum, two different O-acetyltransferases play original and antagonistic roles in the modulation of the activity of endogenous autolysins.

FIGURE 1.

Structure of PG and modulation of PGH activity in L. plantarum. Two disaccharides are represented. The different identified modifications (O-acetylation, amidation, and cross-linking) are highlighted in gray, and their occurrences (%) are indicated in parentheses. The O-acetylation of GlcNAc on C6-OH is predicted by analogy to MurNAc O-acetylation. Modulation of PGH activities by O-acetylation are indicated by an arrow (cleavage) or a broken arrow (no cleavage). Lyso, lysozyme (N-acetylmuramidase); Acm2, N-acetylglucosaminidase; LytH, N-acetylmuramoyl-l-alanine amidase; meso-A₂ pm, meso-diaminopimelic acid; CL, cross-linking index.

FIGURE 2.

RP-HPLC separation of muropeptides from L. plantarum PG. WT (A), oatA mutant (B), oatB mutant (C), and oatA oatB mutant (D). Peak 12 (supplemental Table S3), the dissacharide tripeptide with O-acetylated MurNAc, is indicated by an asterisk, and peak 14 (supplemental Table S3), the dissacharide tripeptide with O-acetylated GlcNAc, is indicated by an arrow.

FIGURE 3.

Fragmentation of O-acetylated dissacharide tripeptides by MS-MS. A, localization of the O-acetyl group on MurNAc. Peak 12 (supplemental Table S3) yielded fragments resulting from a mass loss of 203.08 Da (GlcNAc residue) and 319.13 Da (MurNAc-OAc residue). B, localization of the O-acetyl group on GlcNAc. Peak 14 (supplemental Table S3) yielded fragments resulting from a mass loss of 245.10 Da (GlcNAc-OAc residue) and 277.11 Da (MurNAc residue). Fragmentation was performed on the [M + H]⁺ ion at m/z 911.4. The indicated m/z values correspond to ions obtained by cleavage of peptide bonds as represented on the chemical structures. The indicated masses correspond to [M + H]⁺.

FIGURE 4.

Comparison between OatA and OatB. A, schematic representation of the topology of OatA and OatB. The position and the orientation of transmembrane segments are drawn based on HMMTOP prediction. The numbers denote sizes (in amino acids) of the different loops. The putative C-terminal acetyltransferase domain (AT) is predicted as surface-exposed. Regions displaying identity and similarity (% in parentheses) with N. gonorrhoeae PatA and PatB are presented in blue and red, respectively. B, partial sequence alignment of the AT domain of OatA and OatB. Conservation based on the PRALINE algorithm is represented by a color code where dark blue and red represent the less and most conserved residues, respectively. Similarity scores are indicated below the alignment. Values in parentheses denote the residue number of OatA from L. plantarum used as reference, and black asterisks indicate the putative catalytic residues. Abbreviations (locus tag) are as follows: OatA_Lpl, L. plantarum (lp_0856); OatA_Wpa, W. paramesenteroides (HMPREF0877_0552); OatA_Lla, L. lactis (llmg_2391); OatA_Sau, Staphylococcus aureus (SAV2567); OatA_Lsa, L. sakei (LSA_1044); OatA_Efa, E. faecalis (EF_0783); OatA_Lmo, Listeria monocytogenes (lmo1291); OatB_Lpl, L. plantarum (lp_0925); OatB_Lsa, L. sakei (LSA_0646); OatB_Wpa, W. paramesenteroides (HMPREF0877_1514); PatB_Ngo, N. gonorrhoeae (NGO0533).

FIGURE 5.

Involvement of MurNAc O-acetylation in lysozyme resistance. A, comparison of lysozyme resistance of O-acetyltransferase mutants by serial dilutions (10⁰ to 10⁻⁵) on MRS medium supplemented or not with lysozyme (2 mg/ml). For complementation experiments (bottom panels), chloramphenicol (10 μg/ml) and nisin (20 ng/ml) were added to MRS medium. OatA⁻, oatA mutant; OatB⁻, oatB mutant; OatAB⁻, oatA oatB mutant; OatA⁻/ctl, oatA mutant carrying the empty plasmid pNZ8048 (control); OatA⁻/OatA⁺, oatA mutant complemented with plasmid pGIEB003 (OatA^WT), WT/ctl, WT carrying the empty plasmid pNZ8048 (control). B, RP-HPLC separation of muropeptides resulting from PG digestion by lysozyme. I, WT; II, oatA mutant; III, oatB mutant. Asterisks indicate GlcNAc-OAc containing muropeptides (peaks 14 and 33, supplemental Table S3).

FIGURE 6.

Effect of GlcNAc O-acetylation on autolysis and Acm2 activity. A, autolysis of L. plantarum and its mutant derivatives in presence of Triton X-100 (0.05%). Wild-type is represented by a line, oatA mutant by circles, oatB mutant by triangles, and oatA oatB mutant by squares. Mean values ± S.D. of one representative experiment of three independent experiments (n = 5 for each). B, zymogram with cell extracts of B. subtilis (Bsu), L. plantarum wild-type (Lpl or Lpl WT), and acm2 mutant (Lpl Acm2⁻) against dead cells of WT or derivative strains either lacking OatA (OatA⁻) or OatB (OatB⁻). C, Acm2 activity against autoclaved cells of L. plantarum. Mean values ± S.D. of one representative experiment of two independent experiments (n = 3 for each). Symbols are as described in A.

FIGURE 7.

Effect of MurNAc over-O-acetylation on autolysis. A, autolysis curves (Triton X-100-induced) of oatA^WT overexpressing strains L. plantarum WT (dashed line), oatA mutant (open circles), and oatB mutant (open triangles) compared with control strains (empty vector) of WT (line), oatA mutant (filled circles), and oatB mutant (filled triangles). Mean values ± S.D. (n = 3). B, autolysis curves of oatA^WT overexpressing strains: WT (dashed line), acm2 mutant (open diamonds), and lytH mutant (asterisks) compared to control strains of WT (line), acm2 mutant (filled diamonds), and lytH mutant (crosses). WT (line) and lytH mutant (asterisks and crosses) overlap. Mean values ± S.D. of one representative experiment of two independent experiments (n = 6 for each).