Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan

Abstract

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other b-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the phage was able to adsorb to these cells. Sequencing of the femA gene of three P128-resistant mutants showed either a truncation or deletion in femA, suggesting that improper cross-bridge formation in S. aureus could be causing resistance to P128. Using glutathione S-transferase (GST) fusion peptides as substrates it was found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting that P128 might be killing S. aureus by cleaving the pentaglycine cross-bridge of peptidoglycan. Moreover, peptides corresponding to the reported cross-bridge of Staphylococcus haemolyticus (GGSGG, AGSGG), which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing a common mechanism of action with lysostaphin, P128 has unique properties, which allow it to act on certain lysostaphin-resistant Staphylococcus strains.

Efficient Killing of Planktonic and Biofilm-Embedded Coagulase-Negative Staphylococci by Bactericidal Protein P128

“…The poor potency of lysostaphin on CoNS strains has been attributed to its inability to cleave serine-or alanine-containing peptide cross-bridges in the peptidoglycan (40). The potent activity of P128 on CoNS can be explained by the fact that this protein can cleave serine-or alanine-containing pentapeptides (41). The rate of killing of CoNS strains by P128 in broth or serum is much higher than that of the SoC antibiotics vancomycin and daptomycin.…”

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

“…Protein P128, a chimera of the N-terminal endopeptidase of the structural lysin from staphylococcal K phage and the cell wall-binding SH3 of lysostaphin, was demonstrated to possess strong antistaphylococcal activity and was successfully used in decolonization of rat nares from staphylococcal infection, reducing the bacterial load by two orders of magnitude after treatment. Moreover, P128-resistant mutants were shown to lose the β-lactam drug resistance phenotype and become hypersensitive to β-lactams (Paul et al 2011; Saravanan et al 2013; Sundarrajan et al 2014). Two other chimeric proteins, i.e., HydH5Lyso and HydH5SH3b, were prepared based on the HydH5 from S. aureus -specific phage ΦIPLA88.…”

Antibiofilm Activity and Synergistic Inhibition of Staphylococcus aureus Biofilms by Bactericidal Protein P128 in Combination with Antibiotics

“…P128 possesses potent antistaphylococcal activity against sensitive and drug-resistant strains of S. aureus grow-ing as planktonic cells or in biofilms (14,19,20). The mechanism of killing of staphylococci by P128 involves cleavage of the pentaglycine cross bridge of peptidoglycan (21). The absence of a pentaglycine in species other than staphylococci makes P128 inactive on other bacteria.…”