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.
Highlights
Peptidoglycan hydrolases, including bacterial autolysins and bacteriophage endolysins, contain generally a cell wall-binding domain (CWBD), responsible for their high affinity and specificity toward target cell walls
Sequence Analysis of L. casei BL23 Prophage Endolysins—By sequence similarity search with well characterized bacterial Peptidoglycan hydrolases (PGHs), we identified previously in the genome sequence of L. casei BL23 two prophage-encoded endolysins, Lc-Lys and Lc-Lys-2, each with a neighbor holin [14]
Contradictory results were previously published regarding the hydrolytic specificity of these two endolysins with MurNAc-L-Ala-amidase specificity assigned to PL-1 [21] and D-Ala-D-Asp-endopeptidase specificity assigned to LysA2 [13], both endolysins contain the same PF01510 (Amidase_2) catalytic domain
Summary
Peptidoglycan hydrolases, including bacterial autolysins and bacteriophage endolysins, contain generally a cell wall-binding domain (CWBD), responsible for their high affinity and specificity toward target cell walls. 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 ␥-D-glutamyl-L-lysyl endopeptidase. The display of proteins, including antigens by noncovalent anchoring at the surface of LAB, considered as generally recognized as safe organisms, constitutes an attractive alternative to construction of genetically modified organisms [12] Because of their high affinity for the cell wall, endolysin CWBDs constitute potential anchoring domains [13]. We found that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, and Lc-Lys-2 is a ␥-D-glutamylL-lysyl endopeptidase Both lysins were able to lyse bacterial cells containing PG with an amidated D-Asn crossbridge, including food LAB such as Lactococcus lactis and the pathogen Enterococcus faecium. We showed that their C-terminal CWBD exhibits an original and not previously described binding specificity toward amidated D-Asp (i.e. D-Asn) of PG cross-bridge
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