The Researches Of Staphylococcus Aureus Phage GH15and Three-dimensional Structure, Mechanism Of Its Lysin

Staphylococcus aureus (S. aureus) is an important zoonotic pathogen that can cause a varietyof local and systemic infections. Treatment of these infections has become ever more difficult dueto the emergence of multidrug-resistant strains, especially methicillin-resistant S. aureus (MRSA).Lytic bacteriophages are viruses that infect bacteria, hijack their machinery, replicateintracellularly and are finally released by host cell lysis. The phage lysin is synthesized at the endof the phage lytic life cycle to lyse the host cell. Phage and lysin are highly efficient in lysingbacteria. Thus, they might be an alternative treatment strategy for infections caused byantibiotic-resistant strains.In this study, the staphylococci phage and its lysin were studied. First, a lot of MRSA strainshave been isolated from clinical and were used as host bacteria to isolate and screen lytic phages.Finally, one phage, named GH15, was obtained and it showed broad-spectrum and high-efficientlytic activity to S. aureus. The genome of phage GH15has been sequenced. It indicated that thephage GH15genome is a139.8Kb contiguous sequence of linear, double-stranded DNA, with anoverall G+C content of30.23%, and encodes a predicted214ORFs. Analysis revealed that GH15is closely related to G1, ISP, A5W, Sb-1, and K. Interestingly, all the introns present in thehomologous phages listed above are absent in GH15. A comparison of the intron-related genesdemonstrated a clear distinction in the location of the insertion site between intron-containing andintron-free alleles. The introns which existed in the lysin gene and polymerase gene of most classIII phages do not share homology with any gene in other phages or bacterial genomes. The uniquenature of these introns may support the speculation that these introns originated in the phages andthere were intron-loss events in GH15. By SDS-PAGE and mass spectrometry, ten structuralproteins were identified and most of them are capside-or tail-related proteins.LysGH15, the lysin of GH15, was identified in the genome by sequence analysis and wasexpressed by using E. coli. LysGH15displayed broad spectrum and high-efficient lytic activity toS. aureus. The optional temperation and pH of LysGH15are37°C and7.0respectively. The bacteremia mouse model was used to detect the bactericidal activity of LysGH15. It indicated thata single intraperitoneal injection of LysGH15(50μg) administered1h after MRSA injections atdouble minimum lethal dose, was sufficient to protect mice. Administration of LysGH15significantly reduced the number of bacteria in spleen and blood compared with those in untreatedmice. At the same time, LysGH15redueced the levels of IL-6, IL-4, and IFN-γ mRNA in spleencells.In order to reveal the mechanism of LysGH15, the three-dimensional structure of LysGH15would be resolved. However, SAXS and secondary structure prediction of LysGH15indicated thatthree spherical domains are linked by two long linkers and the LysGH15shows high flexible. Sothe protein crystal of LysGH15could not be obtained. Since, the structures of three domains ofLysGH15(CHAP, amidase-2and SH3b) were resolved respectively. High-quality crystals ofsingle CHAP domain and amidase-2domain were obtained by screening and optimization ofcrystallization conditions. The structures of single CHAP domain and amidase-2domain wereresolved by using Se-SAD and I-SAD respectively. Structure-guided mutagenesis, ICP-AES, ITC,CD, thermal shift, and lytic activity of the CHAP domain indicated that: residues D45, D47, Y49,H51, and D56coordinate a central Ca2+ion and the side chains of D45, D47, and D56playessential role in binding the calcium; the CD spectroscopy showed that the presence/absence ofCa2+does not affect the secondary structures of the LysGH15CHAP domain, thefluorescence-based thermal shift assays indicated that Ca2+has a slight contribution to the proteinthermostability, the ITC demonstrated that the equilibrium dissociation constant of the LysGH15CHAP domain for Ca2+is approximately27μM; the calcium ion plays an important role as aswitch that modulates the CHAP domain between the active and inactive states;C54-H117-E134-N136is the activity site, the–SH of C54plays important role in attackingfunction. The structure of amidase-2indicated that there is a zinc ion coordinated by H214, H324and C332. Structure-guided mutagenesis of the amidase-2domain revealed that both the zinc ionand E282are required in catalysis and enable us to propose a catalytic mechanism. Due to that thecrystal of SH3b could not be obtained, the structure of SH3b domain was resolved by using NMR.The NMR titration identified the interface and key residues of SH3b domain interacted withpeptide “AGGGGG”.Additionally, the C54S mutation or D45A/D47A/D56A in LysGH15induced the lytic activityloss of LysGH15which indicated that the CHAP domain plays an important role in the activity of LysGH15. However, the E282A mutation or mutation of the zinc-binding residues (H214A,H324A, or C332A) does not affect the lytic activity of full-length LysGH15. The bactericidalactivity of LysGH15was reduced when the key residues (identified by NMR titration) in SH3bdomain were mutated to alanine. Thus, both the binding activity of the SH3b domain and thecatalytic activity of the CHAP contribute to the high-efficient lytic activity of LysGH15.