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Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and Lys K SH3b cell wall-binding domain is a potent agent against Staphylococcus aureus in four functional assays. [ABSTRACT FROM AUTHOR]

Abstract: Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset harbors C-terminal SH3b_5 cell wall binding domains. These C-terminal domains have been shown to be necessary for accurate cell wall recognition and subsequent staphylolytic activity for some endolysins. Over fifty proteins of staphylococcal or phage origin containing SH3b domains were aligned, yielding five highly repetitive groups of proteins. Representative C-termini from these five groups, and six staphylococcal proteins for which no homologues have been identified, were aligned, revealing two distinct SH3b_5 subgroups with overlapping but differentially conserved residues. A premise behind this research is that there may be unique cell wall binding properties conferred by these staphylococcal domains that could be exploited to specifically enhance anti-staphylococcal efficacy in heterologous protein fusion constructs. To identify functional differences between the two subgroups, the native Cpl-7 cell wall binding domains of the streptococcal LambdaSa2 endolysin were replaced by staphylococcal SH3b domains from both subgroups. SH3b domains from either lysostaphin (bacteriocin) or LysK (phage endolysin) resulted in a ∼5× increase in staphylolytic activity conferred on the streptococcal endopeptidase domain, and surprisingly these same fusions maintained significant streptolytic activity suggesting that the staphylococcal SH3b domains are not always staphylococcal-specific. A comparison of the differences in lytic activity conferred on the LambdaSa2 endopeptidase domain by either LysK or lysostaphin SH3b domain differed by no more than a factor of two. Through the collection of peptidoglycan hydrolase sequences, three new putative intron-containing phage endolysin genes were identified in public data sets for the phages G1, X2 and 85. [Copyright &y& Elsevier]

LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage betweend-alanine of the stem peptide and glycine of the cross-bridge peptide, and N-acetylmuramoyl-l-alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK. [ABSTRACT FROM AUTHOR]

LysK is the endolysin from the staphylococcal bacteriophage K, and can digest the cell wall of many staphylococci. Lysostaphin is a bacteriocin secreted by Staphylococcus simulans to kill Staphylococcus aureus. Both LysK and lysostaphin have been shown to lyse methicillin-resistant S. aureus (MRSA). This study describes optimal reaction conditions for the recombinant His-tagged LysK protein (pH range pH 6–10, and 0.3–0.5 M NaCl), and C-His-LysK MIC (32.85±4.87 μg mL−1). LysK and lysostaphin demonstrate antimicrobial synergy by the checkerboard assay. [ABSTRACT FROM AUTHOR]

Streptococcal pathogens contribute to a wide variety of human and livestock diseases. The routine use of antibiotics to battle these pathogens has produced a new class of multidrug-resistant streptococci. Thus, there is a need for new antimicrobials. Bacteriophage endolysins (peptidoglycan hydrolases) comprise one group of new antimicrobials that are reportedly refractory to resistance development. The LambdaSa2 prophage endolysin gene was recently isolated from a Group B streptococcal genome, expressed on an Escherichia coli plasmid, and shown by homology screening and biochemical analysis to harbor an amidase-5 (endopeptidase) domain, an amidase-4 (glycosidase) domain, and two Cpl-7 cell wall-binding domains. In this study, turbidity reduction and plate lysis assays indicate that this hydrolase shows strong lytic activity toward Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus equi, GES, and GGS. Deletion analysis indicates that the N-terminal endopeptidase domain with both Cpl-7 domains can lyse with a higher specific activity than the full-length protein (against some strains). This dual Cpl-7 domain truncated version also shows weak lytic activity against methicillin-resistant Staphylococcus aureus (MRSA) and the coagulase negative staphylococci, Staphylococcus xylosus. The truncated constructs harboring the glycosidase domain are virtually inactive, showing only minimal activity on plate lysis assays. [ABSTRACT FROM AUTHOR]

The article discusses the two conflicting primary nucleotide sequences of the Staphylococcus aureus. Details related to the nucleotide differences and alternative translational start site assignments that result in three non-identical protein sequence predictions for the phi11 amidase. It is suggested that it is wise to verify the correct phi11 amidase protein sequence since various versions of the amidase gene have been subcloned, performed deletion analysis including the discussion of the experimental use.

The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative staphylococci ( Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus), making it a strong candidate protein antimicrobial. This lytic activity is maintained at the pH (6.7), and the ‘free’ calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins containing only the endopeptidase domain also lyse staphylococci in the absence of the SH3b-binding domain. [ABSTRACT FROM AUTHOR]

The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity. [ABSTRACT FROM AUTHOR]

Rainbow trout (Oncorhynchus mykiss) have two types of lysozyme. Type II lysozyme differs from type I by only one amino acid, but only type II lysozyme has significant bactericidal activity. Due to this novel antibacterial property, lysozyme type II appears to be a candidate gene for enhancing disease resistance in fish as well as livestock species. Using polymerase chain reaction the lysozyme type II gene was amplified from genomic DNA isolated from rainbow trout. Two amplified fragments of 2041 and 2589 bp were observed. Sequencing revealed both amplicons were lysozyme genes having nearly identical nucleotide sequences, except the longer fragment has 548 base pairs inserted in intron 2 at nucleotide position 513 and a few point mutations within intron 2. Both versions of trout lysozyme type II gene were comprised of four exons and three introns. We also demonstrated that trout lysozyme these two different genes. [ABSTRACT FROM AUTHOR]

Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics.