Bacteriophage derived CHAP domain protein, P128, kills Staphylococcus cells

21 P128 is an anti-staphylococcal protein consisting of Staphylococcus aureus phage K derived tail 22 associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with cell wall binding 23 SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of 24 resistance to P128, we isolated mutants of Staphylococcus spp, including MRSA, resistant to 25 P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of 26 P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the 27 mutants tested was found to be reduced in virulence in animal model of S. aureus septicemia 28 suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed 29 that the mutants derived from MRSA strains had become sensitive to methicillin and other β30 lactams. Interestingly, the mutant cells were resistant to lytic action of phage K, though the 31 phage was able to adsorb to these cells. Sequencing of femA gene of three P128 resistant mutants 32 showed either a truncation or deletion in femA, suggesting that improper cross bridge formation 33 in S. aureus could be causing resistance to P128. Using GST fusion peptides as substrates it was 34 found that both P128 and Lys16 were capable of cleaving a pentaglycine sequence, suggesting 35 that P128 might be killing S. aureus by cleaving pentaglycine cross bridge of peptidoglycan. 36 Moreover, peptides corresponding to reported cross-bridge of S. haemolyticus (GGSGG, 37 AGSGG) which were not cleaved by lysostaphin, were cleaved efficiently by P128. This was 38 also reflected in high sensitivity of S. haemolyticus to P128. This showed that in spite of sharing 39 a common mechanism of action with lysostaphin, P128 has unique properties which allow it to 40 act on certain lysostaphin resistant Staphylococcus strains. 41