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.

Keywords: Bacteria; Bacteriophage; Cell Wall; Cell Wall-binding Domain; Endolysin; Hydrolases; Lactobacillus; Lysis; Peptidoglycan; Peptidoglycan Cross-bridge.

FIGURE 1.

A, schematic representation of endolysins. Lc-Lys, Lc-Lys-2, and two truncated derivatives of Lc-Lys corresponding to its catalytic domain (Lc-LysCD) and its cell wall-binding domain (Lc-LysBD) are shown. Numbers refer to amino acid positions in the complete sequences. Amidase, catalytic domain according to Pfam designations; CD, catalytic domain; BD, binding domain; CWBD, cell wall-binding domain; His₆ tag and Strep-tagII, tags added to recombinant proteins. B, sequence alignments of the C-terminal CWBD of Lc-Lys and Lc-Lys-2 endolysins with the C-terminal region of other proteins. Enterolysin A was from E. faecalis (M23 peptidase_Efa) (AF249740); amidase was from C. aerofaciens (Amidase_Cae, COLAER_00250); amidase was from L. coryniformis (Amidase_Lco, LcortK3_010100004007); endolysin was from L. lactis bacteriophage 949 (LaPh949_gp055); and glycosylhydrolase family-like lysozyme was from E. eligens (EUBELI_01316). Accession numbers correspond to those of the img database (//img.jgi.doe.gov/). Multiple alignments were done with ClustalW. Black boxes, identity in at least 6 of the 7 sequences. Consensus sequence found in each of the three detected modules is indicated.

FIGURE 2.

SDS-PAGE and zymogram analysis of purified recombinant endolysins and derivatives. A, Lc-Lys; B, Lc-Lys-2; C, Lc-Lys catalytic domain (Lc-LysCD); D, Lc-Lys binding domain (Lc-LysBD). After SDS-PAGE, gels were stained with Coomassie Blue (lanes 1). Zymogram assays of purified proteins were performed on autoclaved cells of L. casei BL23 (lanes 2), L. lactis MG1363 (lanes 3), L. lactis asnH (lanes 4), and L. lactis aslA(pmurMN+) (lanes 5).

FIGURE 3.

Analysis of the hydrolytic specificity of Lc-Lys and Lc-Lys-2 endolysins on purified L. casei PG. A, PG digested with mutanolysin as a control for the muropeptide profile obtained with a muramidase (14); B, PG digested with recombinant endolysin Lc-Lys; C, PG digested with recombinant endolysin Lc-Lys-2. Soluble muropeptides were separated by RP-HPLC. Peaks marked with numbers and letters are identified in Table 3. D, schematic representation of L. casei PG structure showing positions of amide bonds cleaved by Lc-Lys (arrows) and Lc-Lys-2 (dashed arrows).

FIGURE 4.

Lytic activity of purified recombinant Lc-Lys and Lc-Lys-2 toward bacteria with different PG cross-bridges and schematic representation of the cross-bridges. A, Lc-Lys; B, Lc-Lys-2 tested on L. casei BL23 (black diamonds), L. lactis MG1363 (black squares), L. lactis asnH (black triangles), L. lactis aslA(pmurMN+) (crosses), L. fermentum (black circles), E. faecium D344S (white diamonds), and E. faecium D344M512 mutant (white circles). C, schematic structure of the PG cross-bridges of the different bacterial strains tested. (a) L. lactis MG1363 wild-type (75% d-Asn and 25% d-Asp), L. lactis asnH mutant (100% d-Asp), and L. lactis VES4240 aslA(pmurMN+) (l-Ala-(l-Ser/l-Ala)), L. casei BL23 (≈100% d-Asn), and E. faecium D344S (96% d-Asn); (b) L. fermentum; (c) ampicillin-resistant E. faecium mutant M512 with 3→3 cross-links and mainly d-Asn (96%) in its cross-bridge.

FIGURE 5.

Binding of Lc-LysBD to L. casei and L. lactis cells. L. casei BL23 (A–C), L. lactis MG1363 (D and E), and L. lactis asnH mutant (F and G) cells harvested in stationary phase were treated with TCA and incubated successively with purified Strep-tagII-tagged Lc-LysBD, anti-Strep-tagII antibody, and secondary antibody coupled to Alexa Fluor 555. Bright field pictures (A, D, and F) were overlaid with the corresponding fluorescent images (B, E, and G, respectively). Additional staining of L. casei was performed with DAPI (blue) and overlaid with the corresponding fluorescent image (C).

FIGURE 6.

Binding of purified Lc-LysBD to purified PG containing different cross-bridge amino acids. PG samples extracted from L. casei BL23 (Lc), L. lactis MG1363 (Ll), and L. lactis asnH and L. lactis aslA(pmurMN+) were incubated with different amounts of Lc-LysBD: 15 μg (I), 30 μg (II), and 45 μg (III). Protein bound to PG was analyzed by SDS-PAGE. PG cross-bridge structure of the different strains is shown on Fig. 4C.