Team:HIT-Harbin/project/part2

    Staphylococcus aureus is a major nosocomial pathogen that causes a range of diseases including endocarditis, osteomyelitis, pneumonia, toxic-shock syndrom, food poisoning, carbuncles, and boils. The emergence of antibiotic-resistant variants in the treatment of staphylococcal infections has been a serious problem. Above all, the increasing evidence of S.aureus resistance to various antibiotics has been well documented for MRSA. Apart from this, there has been emergence of clinical isolates of MRSA with reduced susceptibility to vancomycin. Moreover, S.aureus has been shown to be completely resistant to lysozyme. These problems have prompted a search for new and novel therapeutic agents active against S.aureus. Previous studies have shown lysostaphin to be an effective therapeutic agent for the treatment of various staphylococcal infections. Moreover, lysostaphin rapidly lyses actively growing and non-dividing cells including staphylococci in biofilms. So this time we chose lysostaphin as a new "tool" to kill S.aureus.

    Lysostaphin is a zinc metalloenzyme that has a specific lytic action against S.aureus. Lysostaphin has activities of three enzymes namely, endo-β-N-acetyl glucosamidase, N-acteyl-muramyl-L-alanine amidase, glycylglycine endopeptidase. Glycylglycine endopeptidase lyses staphylococcal cells by hydrolyzing glycylglycine bonds in the poly-glycine bridges； these bridsges which form cross links between glycopeptide chains in cell wall peptidoglycan of S.aureus cells[1]. The lytic principle of lysostaphin is a peptidase which liberates N-terminal glycine and alanine from S.aureus cell wall (Fig. 1).

    The peptidoglycan of S.aureus consists of a backbone made up of alternating β-1,4 linked N-acetylglucosamine and N-acetylmuramic acid residues. Tetrapeptide chains consisting of D-alanine, D-glutamine, L-lysine, and D-alanine are bound to the carboxyl groups of the muramic acid residues. These tetrapeptide chains are cross-linked by polyglycine bridges between the ε-amino group of the lysine residues of one chain and the D-alanyl carboxyl group of another chain (Fig. 1). The peptidoglycan is insoluble due to this cross linking of the polymers and hydrolysis of any single chemical linkage in sufficient number within the cross-linked network can bring about solubilization of the cell wall. The extreme mechanical strength of S.aureus cell walls is probably dependent on the high degree of cross linking of the pentaglycine bridge between the ε-amino group of lysine and the terminal D-alanine of an adjacent tetrapeptide.

    However, this is not the case with other staphylococcal species. The peptidoglycan of other staphylococcal species contains higher amount of serine than glycine and hence makes them less susceptible to lysostaphin. This unique property of S.aureus cell wall separates it from other staphylococcal species and makes lysostaphin as a novel therapeutic agent against antibiotic-resistant S.aureus infections.

    The wild-type lysostaphin gene encodes a preproenzyme which consists of three distinct domains The wild-type lysostaphin gene encodes a preproenzyme which consists of three distinct domains with a typical secretion signal peptide of 38-amino acid sequence, followed by a hydrophilic and highly ordered domain of 7 tandem repeats of a 13-amino acid sequence, followed by the hydrophobic mature lysostaphin itself, which contains 246 amino acids. The mature enzyme is a monomer of about 27kDa and contains no disulfide bonds. The conversion of prolysostaphin to mature lysostaphin occurs extracellularly and involves the removal of the hydrophilic tandem repeat portion of the proenzyme. In order to directly produce mature lysostaphin, we truncate the preprolysostaphin and prolysostaphin sequence (Fig. 2).

Fig.3 the biobrick of killing device. Lysostaphin is free of preprolysostaphin and prrolysostaphin

    We use a constitutive promoter T5 to conduct the gene luxR to express. In this way, protein luxR is produced. Then the signal molecular AHL that we just talked aboutintroduced above enters the engineered E.coli to become a complex with the luxR protein. This complex molecular is a inducer of the PluxI, an inducible promoter. Once the complex combines with PluxI, the expression of lysostaphin and merR gene is switched on. So we get both lysostaphin and merR accumulation at the same time. In consideration ofGiven that low concentration of lysostaphin may not kill Staphylococcus aureus, only a relatively high amount of merR can induce the PmerT to start the transcription of the lysis E7 gene. So upon the lysis of E.coli， it releases a large amount of lysostaphin to kill Staphylococcus aureus, only a relatively high amount of merR can induce the PmerT to start the transcription of the lysis E7 gene. So upon the lysis of E.coli， it releases a large amount of lysostaphin to kill Staphylococcus aureus. Primers B1(5’-GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-3’) and B2(5’-GTTTCTTCCTGCAGCGGCCGCTACTAGTA-3’) were used to PCR-amplify the whole sequence of the killing device(Fig. 4).

    Cultures were grown in LB to exponential phase(A600=0.2). Then cultures of the E.coli cells bearing the plasmid pLys were treated with 1mM IPTG. After 3h of treatment, cultures of the E.coli cells carrying the plasmid pLys and not carrying were harvested and subjected to SDS-PAGE. As shown in Fig. 5, it’s evident that lysostaphin in the E.coli cells bearing the plasmid pLys(lane2) was expressed in comparison with the control(lane3).