title: A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.
abstract: Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.
authors.affiliation: INRA, UMR1319 Micalis, 78352 Jouy-en-Josas, France.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Regulski
authors.forename: Pascal
authors.initials: P
authors.lastname: Courtin
authors.forename: Saulius
authors.initials: S
authors.lastname: Kulakauskas
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Chapot-Chartier
mesh_terms: D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics / Q000821:virology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism*; D039002:Prophages / Q000201:enzymology / Q000235:genetics; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D000577:Amides; D010457:Peptidoglycan; D001224:Aspartic Acid; D001216:Asparagine; D010450:Endopeptidases; C574540:gamma-D-glutamyl-L-lysyl-endopeptidase, Lactobacillus casei; C001143:endolysin; D009238:N-Acetylmuramoyl-L-alanine Amidase
keywords: Bacteria; Bacteriophage; Cell Wall; Cell Wall-binding Domain; Endolysin; Hydrolases; Lactobacillus; Lysis; Peptidoglycan; Peptidoglycan Cross-bridge