title: 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 β-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.
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
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authors.initials: S
authors.lastname: Sundarrajan
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
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authors.lastname: Raghupatil
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
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authors.initials: A
authors.lastname: Vipra
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
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authors.initials: N
authors.lastname: Narasimhaswamy
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
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authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
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authors.initials: C
authors.lastname: Appaiah
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
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authors.initials: N
authors.lastname: Poonacha
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
authors.forename: Srividya
authors.initials: S
authors.lastname: Desai
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Nair
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
authors.forename: Rajagopala Narayana
authors.initials: RN
authors.lastname: Bhatt
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
authors.forename: Panchali
authors.initials: P
authors.lastname: Roy
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
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authors.initials: R
authors.lastname: Chikkamadaiah
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
authors.forename: Murali
authors.initials: M
authors.lastname: Durgaiah
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
authors.forename: Bharathi
authors.initials: B
authors.lastname: Sriram
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
authors.forename: Sriram
authors.initials: S
authors.lastname: Padmanabhan
authors.affiliation: GangaGen Biotechnologies Pvt. Ltd, No. 12 5th cross, Raghavendra layout, Tumkur road, Yeshwantpur, Bangalore 560022, India.
authors.forename: Umender
authors.initials: U
authors.lastname: Sharma
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D004195:Disease Models, Animal; D024881:Drug Resistance, Bacterial; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018805:Sepsis / Q000382:microbiology; D013210:Staphylococcus / Q000187:drug effects* / Q000254:growth & development / Q000302:isolation & purification; D013204:Staphylococcus Phages / Q000201:enzymology*; D014774:Virulence
chemical_list: D000900:Anti-Bacterial Agents; D010457:Peptidoglycan; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases