Abstract
BackgroundStaphylococcus aureus is a food-borne pathogen and the most common cause of infections in hospitalized patients. The increase in the resistance of this pathogen to antibacterials has made necessary the development of new anti-staphylococcal agents. In this context, bacteriophage lytic enzymes such as endolysins and structural peptidoglycan (PG) hydrolases have received considerable attention as possible antimicrobials against gram-positive bacteria.ResultsS. aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88) contains a virion-associated muralytic enzyme (HydH5) encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encoded by staphylococcal phages. The protein consists of 634 amino acid residues. Two putative lytic domains were identified: an N-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain (135 amino acid residues), and a C-terminal LYZ2 (lysozyme subfamily 2) domain (147 amino acid residues). These domains were also found when a predicted three-dimensional structure of HydH5 was made which provided the basis for deletion analysis. The complete HydH5 protein and truncated proteins containing only each catalytic domain were overproduced in E. coli and purified from inclusion bodies by subsequent refolding. Truncated and full-length HydH5 proteins were all able to bind and lyse S. aureus Sa9 cells as shown by binding assays, zymogram analyses and CFU reduction analysis. HydH5 demonstrated high antibiotic activity against early exponential cells, at 45°C and in the absence of divalent cations (Ca2+, Mg2+, Mn2+). Thermostability assays showed that HydH5 retained 72% of its activity after 5 min at 100°C.ConclusionsThe virion-associated PG hydrolase HydH5 has lytic activity against S. aureus, which makes it attractive as antimicrobial for food biopreservation and anti-staphylococcal therapy.
Highlights
Staphylococcus aureus is a food-borne pathogen and the most common cause of infections in hospitalized patients
S. aureus bacteriophage phiIPLA88 contains a structural component with a putative cell wall- degrading activity The virions of phage phiIPLA88 possess a structural component with lytic activity as was previously shown by zymogram analysis [23]
This lytic activity corresponded in size to that expected for the protein product of orf58 (72.5 kDa), which is located in the morphogenetic module with most of the phage head and tail structural genes
Summary
Staphylococcus aureus is a food-borne pathogen and the most common cause of infections in hospitalized patients. The increase in the resistance of this pathogen to antibacterials has made necessary the development of new anti-staphylococcal agents. In this context, bacteriophage lytic enzymes such as endolysins and structural peptidoglycan (PG) hydrolases have received considerable attention as possible antimicrobials against gram-positive bacteria. Bacteriophage lytic enzymes such as endolysins and structural peptidoglycan (PG) hydrolases have received considerable attention as possible antimicrobials against gram-positive bacteria Despite their relatively small size and apparent simplicity, double-stranded DNA bacteriophages propagate by a tightly programmed infection process which involves a number of steps. Virion-associated murein hydrolases appear to be widespread in bacteriophages infecting both Gramnegative and Gram-positive bacteria as shown by zymograms of fully assembled virions and homology analysis of sequenced phage/prophage genomes [3]. In the T7 bacteriophage, gp is an internal head protein with transglycosylase activity that is ejected into the cell at the initiation of infection but is required only when the cell wall is highly crosslinked [6]
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