LambdaSa1 and LambdaSa2 Prophage Lysins of Streptococcus agalactiae

Abstract

Putative N-acetylmuramyl-L-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 lysins exhibited ␥-D-glutaminyl-L-lysine endopeptidase activity. The endopeptidase cleavage specificity of the lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 lysin also displayed ␤-D-N-acetylglucosaminidase activity.Bacteriophages often lyse their bacterial hosts at the end of an infective cycle and release their progeny into the surrounding medium. Phages of gram-positive bacteria usually accomplish this by employing a two-component lytic system consisting of a holin and a lysin (11). The holin makes the bacterial cell membrane porous, thereby exposing the peptidoglycan layer of the cell wall to the degradative action of the lysin. Phage lysins and the related endogenous bacterial cell wall hydrolases that cleave many different bonds in peptidoglycan have been found (11,12). They include ␤-D-N-acetylmuramidases, ␤-D-N-acetylglucosaminidases, lytic transglycosylases, N-acetylmuramyl-L-alanine amidases, and various endopeptidases that cleave either the stem peptide or cross bridge peptide of peptidoglycan (11,12).The observation that many phage lysins specifically lyse susceptible gram-positive bacteria which have been exposed to the enzyme externally has led to recent intense interest in using them as antimicrobial agents. For example, Nelson and coworkers showed that nasopharyngeal colonization of mice with Streptococcus pyogenes could be cleared using the C 1 phage lysin from a group C streptococcus (14). Schuch et al. reported that the phage enzyme PlyG killed vegetative cells of Bacillus anthracis and protected mice from an otherwise fatal intraperitoneal infection with Bacillus cereus (16). Similarly, Jado and coworkers showed that a single intraperitoneal injection of either of the pneumococcal phage lysins Pal, an amidase, and Dpl-1, a lysozyme, was sufficient to protect mice in a murine model of pneumococcal sepsis (7). These workers also demonstrated a synergistic effect on pneumococcal killing when the two enzymes were administered together. A similar synergism among different peptidoglycan cleavage activities may be why many phage lysins evolved with more than one type of lytic activity. The different peptidoglycan cleavage activities are usually located on distinct enzymatic domains, often along with one or more cell wall binding domains (11, 12). We recently described the cloning and elucidation of cleavage specificities of the B30 lysin from a bacteriophage of Streptococcus aga...

X-ray crystal structure of the streptococcal specific phage lysin PlyC

“…Although the endopeptidase domain was active by itself (20,21), the amidase domain was shown to be silent by deletion analysis (21). Likewise, the LysK lysin has an active endopeptidase domain and an inactive amidase domain (22,23), and the streptococcal λSA2 lysin contains an active D-glutaminyl-L-lysine endopeptidase and an inactive N-acetylglucosaminidase domain (24,25). Thus, PlyC is one of the first characterized lysins with two active and distinct catalytic domains.…”

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…Likewise, the prophage LambdaSa2 endolysin has two EADs, an endopeptidase that is a highly catalytic and a poorly catalytic glycosidase. 19 Removal of the entire glycosidase EAD resulted in improving the truncated enzyme's streptolytic activity over the larger parental endolysin. 32 However, the hypothesis that smaller enzymes are more efficient at navigating the peptidoglycan matrix making them catalytically more potent has not been demonstrated.…”

“…Nevertheless, an intraperitoneal injection of PlyPy can still protect the majority of S. pyogenes bacteremic mice from death, similar to PlyC and PlySs2, but at a much lower dose of 0.5 mg (»20 mg/kg). 60 There are several other endolysins that have been reported to have bacteriolytic activity against S. pyogenes, such as the Streptococcus agalactiae phage endolysins B30 27,63 and LambdaSa2 endolysin, 19 the Streptococcus uberis phage endolysin Ply700 64 and the S. pneumoniae endolysin Cpl-7. 65 However, these have yet to be tested in an animal model of S. pyogenes infection.…”

“…23 The number of EADs in the modular endolysins from Gram-positive-infecting phage also varies, with several having two, each encoding a different catalytic activity. 19,24,25 For instance, the staphylococcal phage phi11 endolysin has both an endopeptidase and an amidase. 26 Likewise, the streptococcal phage endolysin B30 has both an endopeptidase and a muramidase EAD.…”

“…10 In some cases, sequence analysis homologies have turned out to be inconsistent with biochemically verified catalytic sites. 19 (For an overview of endolysin types and activities, see refs.) 10,11 Endolysin derived from phages that infect Gram-positive hosts typically have a modular domain structure composed of Nterminal enzymatically active domain (EAD), and a C-terminal cell wall binding domain (CBD) connected by a short linker region (Fig.…”