Endopeptidase and Glycosidase Activities of the Bacteriophage B30 Lysin

Synthetic peptides corresponding to portions of group B streptococcalpeptidoglycan were used to show that the endopeptidase activityof bacteriophage B30 lysin cleaves between D-Ala in the stempeptide and L-Ala in the cross bridge and that the minimal peptidesequence cleaved is DL- ${gamma}$ -Glu-Lys-D-Ala-Ala-Ala. The only glycosidaseactivity present is that of N-acetyl-ß-D-muramidase.

INTRODUCTION

Top

Introduction

The ability of many phage endolysins to specifically lyse variousgram-positive bacteria exposed to the enzymes externally hasled to recent intense interest in using endolysins as antimicrobialagents (3-6, 9, 12-14, 18). Endolysins have been found thatare capable of cleaving most bonds in peptidoglycan (10). Theseinclude glycosidases, amidases, and endopeptidases. Glycosidasescleave the polysaccharide backbone of peptidoglycan and includeN-acetyl-ß-D-glucosaminidases, N-acetyl-ß-D-muramidases,and lytic transglycosylases. N-Acetylmuramyl-L-alanine amidasescleave the amide bond between the lactic acid side chain ofN-acetylmuramic acid (MurNAc) and the L-alanine of the stempeptide. Endopeptidases capable of cleaving either the stempeptide or cross bridges of peptidoglycan have also been described(8, 11, 17). Unfortunately, assignments of cleavage specificitiesof lysins are often based upon sequence homologies and, in theabsence of experimental evidence, may not be correct. The widespreadmisnaming of lysins with known cleavage specificities compoundsthis problem. For example, T7 phage amidase is often referredto as T7 lysozyme.

We previously described the cloning of the gene for a groupB streptococcal (GBS) bacteriophage lysin and partially characterizedthe expressed enzyme (14). The enzyme, termed GBS bacteriophageB30 lysin, lysed several beta-hemolytic streptococci, includinggroup A, B, C, E, and G streptococci. It possesses both glycosidaseand endopeptidase activities, since reducing end groups as wellas N-terminal alanine residues are generated during cell walllysis. Site-directed mutagenesis established that the endopeptidaseactivity was located in the N-terminal cysteine- and histidine-dependentamidohydrolase/peptidase (CHAP) domain (1, 15) and that theglycosidase activity was associated with the central acetylmuramidase(Acm) domain (Fig. 1A). The phage B30 lysin also contains aC-terminal SH3b putative cell wall-binding domain (19). Donovanand coworkers recently reported that enzymatic activity associatedwith the CHAP domain is primarily responsible for target celllysis from without (5).

View larger version (14K):
[in this window]
[in a new window]
FIG. 1. Domain structure of cloned bacteriophage B30 lysin (A) and structure of GBS peptidoglycan with sites of B30 lysin cleavage indicated (B). SH3b, putative bacterial cell wall-binding domain; His₆, hexahistidine affinity tag. The highlighted area depicts the minimum peptide structure required for endopeptidase cleavage.

Enzyme purification and substrates.

Top

Enzyme purification and...

Recombinant bacteriophage B30 lysin was expressed and purified,GBS strain 3331 cell walls were prepared, and an aliquot ofcell walls was acetylated as described previously (14). Peptidescorresponding to the portions of the stem peptide and crossbridges of GBS peptidoglycan shown in Table 1 (16) were customsynthesized by Genscript Corp. (Piscataway, NJ). Ala-Ala andD-Ala-Ala-Ala were obtained from Sigma Chemical Co. (St. Louis,MO). For some experiments, peptide substrates were N-acetylatedby dissolving each peptide (<20 µg) in 40 µlwater, followed by the addition of 140 µl ethanol, 20µl triethylamine, and 20 µl acetic anhydride. After30 min at room temperature, the mixture was rapidly dried byvacuum centrifugation and redissolved in 95 µl water plus5 µl triethylamine. After being dried again, the acetylatedpeptide was dissolved in water at approximately 2 µg ofpeptide/µl.

View this table:
[in this window]
[in a new window]
TABLE 1. Susceptibilities of synthetic peptides to cleavage by phage B30 endopeptidase^a

Digestion of peptides by phage B30 lysin.

Top

Digestion of peptides by...

Peptides (10 µg) were incubated with lysin (5 µg)in 0.05 M sodium acetate, 10 mM CaCl₂, pH 6.0 (total volumeof 25 µl), at 30°C for 18 h. Digests were dilutedwith water (200 µl), and peptide digestion products wereseparated using an HP capillary electrophoresis (CE) system(Hewlett-Packard Co., Palo Alto, CA). Samples were injectedhydrostatically (20 s at 5 kPa) onto an uncoated silica capillary(50 µm by 72 cm). Electrophoresis was done at 20 kV ina phosphate-borate-sodium dodecyl sulfate buffer, pH 9.3, at40°C for 15 min (2).

CE analysis of a digest of Ala-D- ${gamma}$ -Glu-Lys-D-Ala-Ala-Ala, correspondingto the stem peptide and cross bridge of GBS peptidoglycan (Fig.1), gave peaks at 8.7 and 11.4 min, which corresponded to Ala-D- ${gamma}$ -Glu-Lys-D-Alaand Ala-Ala, respectively (Fig. 2A). Coelectrophoresis of thedigest with the substrate showed that digestion was complete(Fig. 2B). Electrospray mass spectrometry of the digest, carriedout on a Micromass Q-TOF 2 mass spectrometer, confirmed thepresence of peptides with the expected masses of 417 Da forAla-D- ${gamma}$ -Glu-Lys-D-Ala and 160 Da for Ala-Ala.

View larger version (10K):
[in this window]
[in a new window]
FIG. 2. Digestion of Ala-D- ${gamma}$ -Glu-Lys-D-Ala-Ala-Ala by phage B30 endopeptidase and analysis of products by capillary electrophoresis at pH 9.28. (A) The digest contains two peptides, revealed by their absorbance levels at 200 nm. They are Ala-D- ${gamma}$ -Glu-Lys-D-Ala (II) and Ala-Ala (III). (B) Digest supplemented with authentic Ala-D- ${gamma}$ -Glu-Lys-D-Ala-Ala-Ala (I). AU, absorbance units.

The peptide D- ${gamma}$ -Glu-Lys-D-Ala-Ala-Ala, which lacks the N-terminalAla, also was cleaved by the enzyme. However, a truncated peptidelacking the C-terminal Ala, Ala-D- ${gamma}$ -Glu-Lys-D-Ala-Ala, was notcleaved. Substituting an L-Glu for D-Glu gave ${gamma}$ -Glu-Lys-D-Ala-Ala-Ala,which was also cleaved by the enzyme. However, replacing the ${gamma}$ -linked Glu with ${alpha}$ -linked Glu gave Ala-D-Glu-Lys-D-Ala-Ala-Ala,which was not cleaved. Replacing the D-Ala with L-Ala gave D- ${gamma}$ -Glu-Lys-Ala-Ala-Ala,which was not cleaved. Lys-D-Ala-Ala-Ala and D-Ala-Ala-Ala werealso not cleaved (Table 1).

The above experiments showed that the minimum peptide sequencenecessary in a substrate for the phage B30 lysin is DL- ${gamma}$ -Glu-Lys-D-Ala-Ala-Ala.There is also an absolute requirement for D-Ala, but eitherD-Glu or L-Glu may be present, provided that they are ${gamma}$ -linked,not ${alpha}$ -linked, to the Lys of the stem peptide. In addition, bothalanines of the cross bridge must be present for cleavage tooccur.

Identification of reducing sugar termini exposed by digestion of GBS cell walls with phage B30 lysin.

Top

Identification of reducing sugar...

Cell wall suspensions (1.0 ml) in 20 mM ammonium acetate, 5mM CaCl₂, pH 6.8, with an initial optical density at 550 nm(OD₅₅₀) of 1.0 were incubated with 0.76 mg of lysin (40 µlof 19-mg/ml lysin) for 2 h or 18 h at 37°C, and the reductionin turbidity (i.e., OD₅₅₀) was measured (Table 2). Reducingend groups in cell wall peptidoglycan were reduced by incubationat 4°C for 16 h with an equal volume of ice-cold 20-mg/mlNaBH₄ in 0.2 M NaHCO₃. Borohydride and bicarbonate were decomposedby carefully adding an excess of methanol-acetic acid (9:1 [vol/vol])to the samples, which were then dried by vacuum centrifugationin a SpeedVac (Savant Instruments, Hicksville, NY). Borate wasremoved as methyl borate by adding acidified methanol and dryingthe samples four more times. The samples were resuspended in1.0 ml of water, and 200-µl aliquots were dried undervacuum in 2-ml glass vials, resuspended in 0.4 ml propanolicHCl, and placed in a heating block at 80°C for 18 h. PropanolicHCl was freshly prepared by carefully adding 0.5 ml acetyl chlorideto 4.5 ml 1-propanol and allowing the mixture to react for 30min prior to use. After propanolysis, insoluble NaCl in thesample was removed by centrifugation, and an aliquot of thesupernatant (200 µl) was dried by vacuum centrifugation.Re-N-acetylation of amino sugars in the samples was accomplishedby dissolving them in 200 µl methanol and adding 20 µlacetic anhydride followed by 20 µl pyridine. After 30min at room temperature, the samples were dried under vacuum,redissolved in 100 µl of methanol, and transferred toglass conical inserts in sample vials. After evaporation todryness, samples were trimethylsilylated by dissolving themin 50 µl of Tri-Sil (Pierce Chemical Co., Rockford, IL)under argon. Derivatized sugars and alditols were separatedand quantitated on an HP 5890 gas chromatograph (Hewlett Packard)equipped with a 30-m HP-1 wide-bore fused-silica column coatedwith a 0.88-µm layer of cross-linked methylsilicone gum.Aliquots (1 µl) were applied to the column with an automaticsample injector, and peaks were monitored using a flame ionizationdetector. After injection, the oven temperature was maintainedat 100°C for 5 min and then increased to 275°C at arate of 20°C per min. Retention times for derivatized N-acetylglucosaminitol,N-acetylglucosamine, N-acetylmuramitol, and N-acetylmuramicacid were 12.888, 13.187, 14.009, and 14.383 min, respectively.

View this table:
[in this window]
[in a new window]
TABLE 2. Gas chromatographic analysis of amino sugars and alditols in cell walls after B30 lysin digestion

Approximately one-fifth of N-acetylmuramic acid residues inundigested cell walls, but no N-acetylglucosamine residues,were reduced (Table 2). Presumably, this is because peptidoglycanchains are biosynthesized via a lipid-linked disaccharide-peptideprecursor (lipid II) that has an N-acetylmuramic acid residueat its reducing terminus (7) and because the carbohydrate chainsare relatively short. Digestion of GBS cell walls with phageB30 lysin for 2 h increased the percentage of MurNAc that wasreduced from 19.7% to 74.7%, and overnight digestion resultedin 94.3% reduction (Table 2). Figure 3 shows the gas chromatogramobtained for the 2-h digest. These data clearly show that phageB30 lysin cleaves N-acetylmuramic acid-N-acetylglucosamine bondsin GBS peptidoglycan and is therefore an N-acetyl-ß-D-muramidase.

View larger version (19K):
[in this window]
[in a new window]
FIG. 3. Gas chromatographic separation of the reduction products of a 2-h phage B30 lysin digest of GBS cell walls. It can be seen that after 2 h of digestion, most of the N-acetylmuramic acid present could be reduced with borohydride. Details of the analytical procedure are described in the text.

Donovan and coworkers recently described the construction andproperties of chimeric lysins containing both lysostaphin andeither the entire phage B30 lysin or only its endopeptidasedomain (4). These chimeric lysins displayed lytic activity againstall three major mastitis-causing pathogens of dairy cattle,i.e., Streptococcus agalactiae (GBS), Streptococcus uberis,and Staphylococcus aureus. Knowledge of the precise cleavagespecificities of phage lysins will be important for the rationaldesign of such engineered and chimeric enzymes.

ACKNOWLEDGMENTS

This work was supported by funds from Public Health Servicegrant AI054897 from the National Institute of Allergy and InfectiousDiseases.

Mass spectrometry was performed by Marion Kirk of the Universityof Alabama at Birmingham Comprehensive Cancer Center Mass SpectrometryShared Facility.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry & Molecular Genetics, MCLM 552, University of Alabama at Birmingham, 1530 3rd Ave. S, Birmingham, AL 35294-0005. Phone: (205) 934-6023. Fax: (205) 934-6022. E-mail: davidp1{at}uab.edu.

REFERENCES

Top