Structural analysis of Bacillus megaterium KM spore peptidoglycan and its dynamics during germination

604

602

Nod1 and Nod2 are mammalian proteins implicated in the intracellular detection of pathogen-associated molecular patterns. Recently, naturally occurring peptidoglycan (PG) fragments were identified as the microbial motifs sensed by Nod1 and Nod2. Whereas Nod2 detects GlcNAc-MurNAc dipeptide (GM-Di), Nod1 senses a unique diaminopimelate-containing GlcNAc-MurNAc tripeptide muropeptide (GM-Tri DAP ) found mostly in Gram-negative bacterial PGs. Because Nod1 and Nod2 detect similar yet distinct muropeptides, we further analyzed the molecular sensing specificity of Nod1 and Nod2 toward PG fragments. Using a wide array of natural or modified muramyl peptides, we show here that Nod1 and Nod2 have evolved divergent strategies to achieve PG sensing. By defining the PG structural requirements for Nod1 and Nod2 sensing, this study reveals how PG processing and modifications, either by host or bacterial enzymes, may affect innate immune responses. Peptidoglycan (PG)1 is a major constituent of the cell wall of Gram-positive bacteria and consists of glycan chains of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) that are cross-linked to each other by short peptides, allowing the formation of a rigid polymer that surrounds the bacterial cell. During bacterial life cycle, peptidoglycan is constantly degraded by specific hydrolases, and newly synthesized subunits are integrated into the polymeric structure, allowing biological processes such as cell division. In Gram-negative bacteria, a thin layer of peptidoglycan is also found in the periplasmic space. Apart from the thickness and the degree of stem peptides cross-linking, an important difference between Gram-positive and Gram-negative peptidoglycans resides in the nature of the third amino acid of the peptides. In Gram-positive bacteria, this amino acid is commonly a lysine, whereas a diaminopimelic acid is found in most Gramnegative bacteria. Extensive analysis from many bacteria has revealed that, in steady state conditions, peptidoglycans from each bacterial strain have somehow fixed proportions of di-, tri-, tetra-, or pentapeptide substituted to the MurNAc sugar moiety.

Section: Resultssupporting

confidence: 73%

Peptidoglycan Molecular Requirements Allowing Detection by Nod1 and Nod2

Girardin

Travassos

Hervé

et al. 2003

604

602

“…The presence of non-acetylated aminosugars in the peptidoglycan is not limited to pneumococci (25,26,28,29,33,34), and the increased activity of lysozyme after N-acetylation of partly deacetylated peptidoglycan was also reported in B. cereus (35,36). The data search suggests that, similar to pneumococci in bacilli and other bacterial species, the presence of non-acetylated amino sugars in the peptidoglycan is related to the activity of PgdA-like enzymes.…”

Section: Identification Of N-deacetylated Amino Sugars In the Pneu-mentioning

confidence: 71%

“…Putative Peptidoglycan Deacetylases in Other BacteriaNon-acetylated hexosamine residues have been reported to be present in the peptidoglycans of many bacteria, especially in Bacillus species, although no genes encoding for such an enzyme were identified (25)(26)(27)(28)(29). In searching the data bases for hypothetical proteins similar in sequence to the pneumococcal PgdA protein we found other putative peptidoglycan deacetylases in bacterial species having partially deacetylated peptidoglycan: three hypothetical proteins in Bacillus subtilis (GenBank TM accession numbers AAC46306, BAA23389, and CAA74511) and one NodB homolog protein in both Bacillus stearothermophilus (GenBank TM accession number B47692) and Bacillus cereus (GenBank TM accession number CAB40600).…”

Section: Identification Of N-deacetylated Amino Sugars In the Pneu-mentioning

confidence: 99%

The pgdA Gene Encodes for a PeptidoglycanN-Acetylglucosamine Deacetylase in Streptococcus pneumoniae

Vollmer

Tomasz

2000

228

125

Analytical work on the fractionation of the glycan strands of Streptococcus pneumoniae cell wall has led to the observation that an unusually high proportion of hexosamine units (over 80% of the glucosamine and 10% of the muramic acid residues) was not N-acetylated, explaining the resistance of the peptidoglycan to the hydrolytic action of lysozyme, a muramidase that cleaves in the glycan backbone. A gene, pgdA, was identified as encoding for the peptidoglycan N-acetylglucosamine deacetylase A with amino acid sequence similarity to fungal chitin deacetylases and rhizobial NodB chitooligosaccharide deacetylases. Pneumococci in which pgdA was inactivated by insertion duplication mutagenesis produced fully N-acetylated glycan and became hypersensitive to exogenous lysozyme in the stationary phase of growth. The pgdA gene may contribute to pneumococcal virulence by providing protection against host lysozyme, which is known to accumulate in high concentrations at infection sites.The unusual complexity and diversity of the cell surface of Streptococcus pneumoniae is apparent both in the capsular structure and in the underlying cell wall structure of this bacterium, and this complexity and diversity may be related to the multiplicity of interactions of this microbe and its human host. S. pneumoniae is capable of producing at least 90 chemically distinct capsular polysaccharides (1). The cell wall of this microorganism contains a teichoic acid of unusually complex chemistry (2, 3) the components of which include ribitol phosphate, galactosamine, trideoxydiaminohexose, and covalently linked phosphocholine residues (4). The peptidoglycan of S. pneumoniae is also unusual because its stem peptides are cross-linked in both a direct and an indirect manner (5). Furthermore, the proportion of distinct linear and branched muropeptides in the peptidoglycan is clonally related (6). The pneumococcal cell wall is a potential target for components of the first line host defense such as lysozyme. In addition, the peptidoglycan and teichoic acid may represent bacterial ligands recognized by the innate immune system of the host.The recent introduction of high resolution analytical techniques and genetic approaches began to shed light on the determinants and biological functions of the pneumococcal cell wall. In this study we describe the identification of a genetic determinant, pgdA, of the first bacterial peptidoglycan GlcNAc deacetylase. We show that the innate activity of this enzyme is responsible for the high proportion of non-acetylated hexosamine residues in the peptidoglycan that appears to play a role in the resistance of S. pneumoniae to the activity of exogenous lysozyme, an enzyme that is known to accumulate in high concentrations at infection sites. EXPERIMENTAL PROCEDURESBacterial Strains, Plasmids, and Growth Media-Cultures of S. pneumoniae R36A, a non-encapsulated laboratory strain from the Rockefeller University collection, were grown in a casein-based semi-synthetic medium (C ϩ Y) containing 1 mg/ml yeast extract (7) o...

“…Previously Atrih et al (4) described that the vegetative peptidoglycan in B. subtilis includes (136)-anhydro-N-acetylmuramic acid. Thus, it is possible that lytic transglycosylase, which digests MurNAc-GlcNAc linkage with synthesis of a 1,6-anhydro bond in the N-acetylmuramic acid (5), hydrolyzes the vegetative peptidoglycan.…”

mentioning

confidence: 99%

Identification and Characterization of Novel Cell Wall Hydrolase CwlT

Fukushima

Kitajima

Yamaguchi

et al. 2008

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to DL-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of D-␥-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a DL-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."Many microorganisms have peptidoglycan as a major component of the cell wall, which consists of glycan strands crosslinked by peptides. Bacteria have various cell wall lytic enzymes. For Bacillus subtilis, more than 30 candidate peptidoglycan hydrolases are proposed on the basis of amino acid sequence similarity (1), and these enzymes are important in various cellular processes during vegetative growth, sporulation, and germination (1, 2). Several groups of peptidoglycan hydrolases are enzymatically identified as follows: N-acetylglucosaminidases (digesting GlcNAc-MurNAc 3 linkage), N-acetylmuramoyl-Lalanine amidases (digesting MurNAc-L-alanine linkage), DL-endopeptidases (digesting D-glutamic acid-meso-A 2 pm linkage) (1, 2), and LD-endopeptidase (digesting L-alanine-D-glutamic acid linkage) (3). However, the characterization of the group of DD-endopeptidase (digesting the cross-linked D-alanine-meso-A 2 pm linkage) has not been reported in B. subtilis. Interestingly, the groups of muramidase and lytic transglycosylase (digesting MurNAc-GlcNAc linkage) in B. subtilis are still not characterized, even many hydrolases in those groups, including hen egg white lysozyme, have already been identified.Previously Atrih et al. (4) described that the vegetative peptidoglycan in B. subtilis includes (136)-anhydro-N-acetylmuramic acid. Thus, it is possible that lytic transglycosylase, which digests MurNAc-GlcNAc linkage with synthesis of a 1,6-anhydro bond in the N-acetylmuramic acid (5), hydrolyzes the vegetative peptid...