A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated D-Asp Cross-bridge, Identified in Lactobacillus casei Bacteriophage Endolysins

Krzysztof Regulski (1) , Pascal Courtin (1) , Saulius Kulakauskas (1) , Marie-Pierre Chapot-Chartier (1)

1 MICALIS - MICrobiologie de l'ALImentation au Service de la Santé

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 gamma-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.

Mots clés: LACTIC-ACBACTERIA; LACTOCOCCUS-LACTIS; STAPHYLOCOCCUS-AUREUS; MUREIN HYDROLASES; SURFACE DISPLAY; HIGH-AFFINITY; CELL-SURFACE; WALL; PROTEINS; LYSINS

Domaines: Sciences agricoles

Krzysztof Regulski, Pascal Courtin, Saulius Kulakauskas, Marie-Pierre Chapot-Chartier. A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated D-Asp Cross-bridge, Identified in Lactobacillus casei Bacteriophage Endolysins. Journal of Biological Chemistry, 2013, 288 (28), pp.20416 - 20426. ⟨10.1074/jbc.M112.446344⟩. ⟨hal-01003290⟩