Lytic Activity of Recombinant Bacteriophage 11 and 12 Endolysins on Whole Cells and Biofilms of Staphylococcus aureus

Infections caused by Staphylococcus aureus and Staphylococcus epidermidis still play a major role in human and animal disease. In particular staphylococcal biofilms on indwelling devices are difficult to treat due to their inherent antibiotic resistance (5, 7, 26). In this context, it is important to develop alternative treatment strategies to combat staphylococcal infections, particularly in view of the ability of staphylococci to acquire resistance to commonly used antibiotics (10, 23, 29). Phage lysins, or endolysins, have received considerable attention as possible antimicrobial agents against gram-positive bacteria and have been applied to a variety of pathogens, such as Bacillus anthracis (21), Streptococcus pneumoniae (9), and S. aureus (25). Endolysins are active against dead cells and even living, planktonic cells (9, 25), but their ability to lyse the complex structure of staphylococcal biofilms has not yet been investigated. In this approach, we cloned and heterologously overexpressed the lysis genes of the bacteriophages 11 and 12 of S. aureus NCTC8325 in Escherichia coli for subsequent analysis of the lytic activity of the enzymes and their single subdomains on cell walls, whole cells, and biofilms. Knowledge of the lytic activity of both endolysins is limited. Their nucleotide sequences have been published (16), and the 11 endolysin has been shown to possess a D-alanyl-glycyl endopeptidase and an N-acetylmuramyl-L-alanine amidase activity on crude cell walls of S. aureus OS2 (24). Sequence comparison, cloning, and overexpression of 11 and 12 endolysins. The 11 and 12 endolysin sequences were BLAST searched against the NCBI protein database. Both endolysins are modular enzymes which consist of three distinct domains coding for an N-terminal CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain with hydrolytic function, a central amidase domain (N-acetylmuramyl-L-alanine amidase), and a C-terminal SH3b domain, which is involved in cell wall recognition (1). In spite of their similar domain architecture, these endolysins show low sequence identity (26.9%). The endolysin genes of the bacteriophages 11 (open reading frame [ORF] 53; 1,473 bp; accession number NC_004615) and 12 (ORF 49; 1,455 bp; accession number NC_004616) were amplified by PCR from genomic DNA of S. aureus NCTC8325, using the primers listed in Table 1. In order to test the activity of the 11 endolysin subunits, the endopeptidase unit ( 11endo, amino acids [aa] 1 to 180) and the amidase unit ( 11ami, aa 180 to 371) as well as each unit plus the cell wall binding domain ( 11endo/CBD, aa 1 to 180/371 to 490, and 11ami/CBD, aa 180 to 490) were constructed separately (Fig. 1). In addition, the cell wall binding module was deleted from the 11 endolysin ( 11endo/ami, aa 1 to 371). The amplification products were cloned into the multiple cloning site of the expression vector pET22b (Novagen) without the pelB leader tag to inhibit protein transport to the periplasm of the expression host. The resulting plasmids, pETer 11, pETer 12, pETendo11, pETendoCBD11, pETami11, pETamiCBD11, and pETendo/ami11, were used to overexpress each endolysin as a C-terminal six-His-tagged fusion protein. After subcloning of the plasmids in E. coli JM109, E. coli BL21(DE3) was used as a host for expression of each six-His-tagged endolysin. Expression cultures were grown in Luria-Bertani (LB) broth containing ampicillin (40 g/ml) to an optical density at 600 nm (OD600) of 0.6. Then protein expression was induced by addition of isopropyl-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Expression cultures were harvested after 4 h followed by protein purification steps under native conditions via nickel-nitrilotriacetic acid affinity chromatography (Fig. 2). Protein purification was also performed with cells harboring the empty vector, and the eluate served as a control in the activity tests. Lytic activity of 11 endolysin modules. The lytic activities of the full-length 11 endolysin and its deletion variants (each applied in a concentration of 20 g/ml) were examined photometrically at 600 nm employing Staphylococcus simulans 22 cell walls (purified with sodium dodecyl sulfate [SDS]) (4) resuspended in incubation buffer (50 mM Tris-HCl, 100 mM NaCl; pH 7.5) to an OD600 of 0.3. All lysis experiments were performed in triplicate. The full-length enzyme showed efficient lysis of the peptidoglycan compared to the control (emptyvector eluate), and both the endopeptidase and the amidase module plus cell wall binding domain ( 11endo/CBD and 11ami/CBD) were active and able to lyse cell walls. However, the full-length enzyme was more active than the isolated sub* Corresponding author. Mailing address: Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Phone: (49)228-287-1-9103. Fax: (49)228-2871-4808. E-mail: [email protected]. Published ahead of print on 3 November 2006.