Characterization of AtlL, a bifunctional autolysin ofStaphylococcus lugdunensiswithN-acetylglucosaminidase andN-acetylmuramoyl-l-alanine amidase activities

Abstract

The nucleotide sequence of atlL, a gene encoding a putative Staphylococcus lugdunensis peptidoglycan hydrolase, was determined using degenerate consensus PCR and genome walking. This 3837-bp gene encodes a protein, AtlL, that appears as a putative bifunctional autolysin with a 29-amino acid putative signal peptide and two enzymatic putative centres (N-acetylmuramoyl-l-alanine amidase and N-acetylglucosaminidase) interconnected with three imperfect repeated sequences displaying glycine-tryptophan motifs. In order to determine whether both lytic domains were functional, and verify their exact enzymatic activities, gene fragments harbouring both putative domains, AM (N-acetylmuramoyl-l-alanine amidase enzymatic centre plus two repeated sequences) and GL (N-acetylglucosaminidase enzymatic centre plus one repeated sequence), were isolated, subcloned, and expressed in Escherichia coli. Purified recombinant AM and GL protein truncations exhibited cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis, and S. lugdunensis. AtlL is expressed during the whole growth, with an overexpression in the early-exponential stage. Liquid chromatography-mass spectrometry analysis of muropeptides generated by digestion of B. subtilis cell walls demonstrated the hydrolytic bond specificities and confirmed both of the acetyl domains' activities as predicted by sequence homology data. AtlL is the first autolysin described in S. lugdunensis, with a bifunctional enzymatic activity involved in peptidoglycan hydrolysis.

Multi-Virulence-Locus Sequence Typing of Staphylococcus lugdunensis Generates Results Consistent with a Clonal Population Structure and Is Reliable for Epidemiological Typing

“…Indeed, AtlL might play a role in virulence, as it has been shown in vitro through biofilm formation and in vivo in the C. elegans model (41). As AtlL presents high identity percentage with all staphylococcal bifunctional autolysins (40,42,43), it might be involved in the internalization of the bacteria in the eukaryote cell (59). Furthermore, the agr quorum-sensing and signal transduction system, which has been described as a global regulator of virulence gene expression in S. aureus, was also studied (60).…”

“…We selected (27,30) and IsdJ, a protein of an iron acquisition system, which of the CoNS, is only used by S. lugdunensis (30,32). Besides theses virulence-associated loci, we analyzed the polymorphisms of the repeated sequences (atlL R2 and atlL R3 ) of the putative CWA domains of the AM and GL autolysins, respectively, generated by the proteolytic cleavage of the bifunctional murein hydrolase, AtlL (40). Indeed, AtlL might play a role in virulence, as it has been shown in vitro through biofilm formation and in vivo in the C. elegans model (41).…”

“…Like AtlA and AtlE, AtlL of S. lugdunensis displays two enzymatic domains separated by three repeat domains (AtlL R1 to AtlL R3 ) and undergoes proteolytic processing to generate two extracellular peptidoglycan hydrolases, an N-acetylmuramoyl-L-alanine amidase (AM) and an N-acetylglucosaminidase (GL) with two (AtlL R1R2 ) and one (AtlL R3 ) repeated sequences harboring glycine tryptophan motifs, respectively (40). We recently reported that a ⌬atlL mutant showed a significant reduction in biofilm formation and virulence attenuation using the Caenorhabditis elegans model (41).…”

Endo-N-Acetyl-β-D-Glucosaminidases and Peptide-N4-(N-acetyl-β-D-Glucosaminyl) Asparagine Amidases: More Than Just Tools

“…Among the numerous sequences (>2500) included in the CAZy GH73 family, which is described as peptidoglycan hydrolases with ENGase specificity (EC 3.2.1.-), 20 were characterised from the following bacteria: Bacillus subtilis [26], Clostridium difficile [27], Clostridium perfringens [28], Enterococcus feacalis [29], Enterococcus hirae [30], Lactobacillus plantarum [31], Lactobacillus lactis [32,33], Listeria monocytogenes [34], Sphingomonas sp [35], Staphylococcus aureus [36], Staphylococcus lugdunensis [37], Staphylococcus warneri [38], Streptococcus pneumoniae [39].…”

Characterization of Acp, a Peptidoglycan Hydrolase of Clostridium perfringens with N -Acetylglucosaminidase Activity That Is Implicated in Cell Separation and Stress-Induced Autolysis

“…These enzymes may also be implicated in antibiotic-induced lysis (39) and may contribute to bacterial pathogenesis by generating inflammatory cell wall degradation products (32, 40), by releasing virulence factors (4), or by mediating bacterial adherence (1,20,21). The roles of PGHs in bacterial physiology, and probably in bacterial pathogenicity, further reinforce the importance of understanding bacterial autolysis.Autolytic systems of several Gram-positive low-GϩC bacteria have been studied (5,13,34,43,54,55). Belonging to this phylum is Clostridium perfringens, a common agent of food poisoning and implicated in infectious diseases initiating from the digestive tract (peritonitis, bacteremia, etc.…”