Screening And Study On The Function Of Peptide Conjugated With Accessory Gene Regulator Of Staphylococcus Aureus

Staphylococcus aureus is the most frequent positive coccobacteria in lower respiratory tract infection, which pathogenic mechanism is correlated with its secretion of extracellular enzymes and exotoxins, which can causes multi-organs infection and therefore result in high morbidity and mortality. It becomes focal point of infection with Staphylococcus aureus because of the characteristics of high infection rate, drug fast and easy to shift from acute infection to chronicity, persistency and recurrent infection. Accessory gene regulator( agr) is an important quorum sensing system and regulatory system of virulence factors, which participate the biofilm formation and dispersion, as well as control the expression of virulence factors. Many researches indicate that agr system of Staphylococcus aureus is a quorum sensing system which plays a great part in regulating to the virulence factors.AgrC is a signal transduction factor in the tow-component systems of Staphylococcus aureus, which is a histidine protein kinase, including a transmembrane domain and a histidine protein kinase region intracellular, which can be activated or inhibited by homologization or heterogenesis AIPs. AgrC can regulate the transcription of operons RNAII and RNAIII because of its specificity recognization of AIP, thus to regulating the toxicity in the agr system.Because of widespread using and abusing the antibiotics, many bacteria emerge drug resistance, which results in difficulties to exploitation of neotype antibiotics with new antibiosis mechanisms. Studies revealed that many analogues of quorum sensing signals could inhibit Staphylococcus aureus to produce causative agent or to growth. For this reason, to interfere with the quorum sensing system of Staphylococcus aureus to degrade its toxicity, prevent causative agent producing and biofilms formation becomes a new breakthrough to prevent and treat the Staphylococcus aureus infection, which provides a target to solve the drug resistance to traditional antibiotics. So, it is important to taking agrC as a target to finding short peptides specific binding to agrC to blocking agr system activation, so as to suppress the Staphylococcus aureus toxicity, and to treat Staphylococcus aureus infection ultimately.It provides an original route to screening polypeptide drugs to prevention and cure Staphylococcus aureus infection with the technology of phage display. The objective of the present study was to screening peptides conjugated with agrC from phage random 12-mer peptide library and determine the function of agrC.Methods:1,The DNA of Staphylococcus aureus was amplified by PCR and cloned into expression vector pET28α(+). The verified recombinant was transformed into E.coli DH5α. After inducing with isopropyl-β-D-thio- galaetopyranoside(IPTG), the recombinant protein was purified via His Tag magnetic stock.. The recombinant proteins were analyzed with SDS-PAGE and Western blot.2,Phage display technique was employed in our study. After 4 sequential rounds of biopanning to phage random 12-mer peptide library, some phage clones which specific conjugated with agrC were obtained.3,They were selected randomly and amplified. The binding activity of these phage clones were identified by ELISA. Single stranded genomic DNA from the positive clones were extracted for sequencing as described in manual, and then were sequenced. The peptide sequences were deduced from DNA sequences of these phage clones. Trough Blast, the peptide sequences were compared for homology analysis.4,The screening bioactive peptide was synthesized by the solid-phage method, and its further explored functional properties were identified.Results:1,The agrC was successfully constructed and the recombinant agrC protein was expressed in E.coli at a high level . And the purified protein had reached 90%.2,Using agrC as target, 4 rounds of biopanning were performed as described in methods. The bound phages were eluted by the glycine solution at the first tow rounds, and were concentrated at the third and fourth round.3,60 phage clones were selected randomly and amplified. 22 phage clones had high affinity to agrC. Single stranded genomic DNA from the positive clones were extracted and then were sequenced. The peptides sequences of 22 clones were deduced for 5 different sequences. The core peptide sequence of the 5 was (XX)L(X)Q(XX)L(XX)L(X). By homology analysis, one high homologous sequence was found as CRLEQERLSPLI.4,The synthetic peptide with the sequence of CRLEQERLSPLI could inhibit the ability of biofilm formation and the toxcity of Staphylococcus aureus.Conclusions:1,agrC protein was expressed in E.coli at a high level and purified.2,The core peptide sequence of the positive phage clones was (XX)L(X)Q(XX)L(XX)L(X).and the peptide sequence with high ability to bind to agrC was CRLEQERLSPLI.3,The synthetic peptide with the sequence of CRLEQERLSPLI could inhibit the ability of biofilm formation and the toxcity of Staphylococcus aureus. These results may lead to a better understanding of how agrC regulate the toxicity of Staphylococcus aureus and provide a new pathway to explore peptide antibiotics to solve drug resistance in Staphylococcus aureus infection.