The Antibacterial Activity And Mechanism Of RNAâ…¢-inhibiting Peptide Derivative RIP1183

AIM:As the widespread overuse of antibiotics, drug-resistant bacterial infections are becoming increasingly serious. It has become the leading cause of death in severe infections. In the course of the contest with antibiotics, some bacteria have evolved to resist multiple antibiotics. The "super bacteria" like methicillin-resistant Staphylococcus aureus(MRSA), are multiply resistant to various antimicrobial agents. Since it is extremely difficult to treat, the mortality rate of the infection is quite high. There are also not effective antimicrobial agents for clinical treatment of MRSA infections. It has become extremely difficult to treat infections of MRSA. Therefore, it is necessary to explore new antibacterial strategies and develop new antibacterial drugs with low resistance.Quorum sensing system(QSS) is a bacterial communication process that depends on the bacterial population density. With the help of this process, bacteria regulate myriad activities such as virulence, biofilm formation, pathogenicity. There is a need for development of novel strategies to combat the infections caused by multidrug resistant organisms. Quorum sensing system inhibitors do not affect the growth of bacteria, no selective pressure on bacteria, less induce bacterial resistance. Hence, targeting QS systems is a more promising strategy than traditional approaches in antibacterial research.agr system is widely found in Staphylococcus aureus. The system is activated, and expressions of RNAⅢ start to increase, which regulate the expression of multiple virulence factors. RNAⅢ-inhibiting peptide(RIP) could effectively inhibit the QS system in staphylococci. The RIP derivative reduced lethality in combination with some antimicrobial agents in mouse models of staphylococcal sepsis. However, all the agr inhibitor has only antibacterial activity in vivo. The internal rules and antibacterial mechanisms of RIP are almost not completely clear, which hindered the RIP-depth study as an antimicrobial agent.In our study, amino acid substitutions and terminal modification or oligomerization methods were used to design different RIP derivatives. The antibacterial activity of and the antibacterial mechanism of RIP was proposed.METHODS:1. Design and synthesis of RIP derivativesBased on the sequence of RIP-I(YKPITNF), amino acid substitutions and terminal modification or oligomerization method were used to design RIP derivatives. Five RIP derivatives(RIP-V、RIP-L, RIP1181, RIP1182, RIP1183) were designed. RP-HPLC method was used to analyze the purity of peptides. And MALDI-TOF mass spectrometry or ESI mass spectrometry was used to identify the molecular weight of synthetic polypeptide.2. Screening for antibacterial activity of RIP derivatives in vivoMouse model of staphylococcal sepsis was established to screen the antibacterial activity of RIP derivatives. The number of bacterial in mice tissues, pathological damage in tissue and survival rate of infectious mice were analyzed.3. Evaluation of antibacterial effect RIP1183 in vitroMinimum inhibitory concentration(MIC) and growth curves at different time points against 6 staphylococci were tested. Two standard strains and four drug resistant strains were used in our study.4. Evaluation of antibacterial effect RIP1183 in different mouse model caused by MRSA in vivoThree mouse models with HA-MRSA or CA-MRSA(sepsis, necrotizing pneumonia and skin infection model) were used to evaluate the activity of RIP1183. The survival rate of mice, the number of bacteria in tissues, pathological damage, skin abscesses area, and other indicators were detected to assess the protective effect of RIP1183 against MRSA.5. Antibacterial mechanism of RIP in vivo(1) Neutrophils deficient mice were used to establish of MRSA sepsis model, protective effect of RIP1183 were observed. The role of neutrophil in this process was analyzed.(2) Neutrophils isolated from human peripheral blood were incubated with RIP1183; MPO test kit was used to assay the activity of MPO in neutrophils. The effect of RIP1183 on MPO of neutrophils was analyzed.(3) Isolating MRSA from mice, the expression levels of RNAⅢ and virulence factor PVL, HLA, PSMα and PSMβ in MRSA were detected by using real-time quantitative PCR method.(4) LAC, bacterial supernatants or HKSA(heat-killed S. aureus), incubated with human neutrophils. To observe the effect of LAC on neutrophil necroptosis, western blot was used to detect the expression levels of p MLKL.(5) PSMα or PSMβ defective bacterial supernatant was incubated with human neutrophils. To observe the effect of PSM on neutrophil necroptosis, western blot was used to detect the expression levels of p MLKL.(6) Seven kinds of PSM polypeptide respectively, was incubated with human neutrophils, or pretreated MLKL inhibitor NSA. Western blot to detect the expression levels of p MLKL; after neutrophil treated by PSM, morphology of neutrophil were obversed by transmission electron microscopy.(7) Wild type strain LAC or the genetic defect strains of LAC was adopted to establish necrotizing pneumonia mouse model. Mice were administered intraperitoneally RIP1183 or Nec after infection. Immunofluorescence staining in lung tissue, methods of flow cytometry and western blot were used to detect the necroptosis of neutrophil.(8) Wild type strain LAC or the genetic defect strains of LAC was adopted to establish necrotizing pneumonia mouse model. Body weight, survival rate, lung injury(HE staining) in lung tissue and the number of bacteria were observed.6. Safety and pharmacokinetics of RIP1183(1) The general signs, body weight and food intake of animal were analyzed, after SD rats or beagles receiving single intravenous injection of RIP1183 at the maximum dose.(2) After RIP1183 intravenously administered to mice or beagles, the effect of RIP1183 on the central nervous system of mice, or on the cardiovascular and respiratory systems of beagles was observed.(3) Beagle dogs received single intravenous injection administered 2 mg/kg, 4 mg/kg and 8 mg/kg of RIP1183, the concentration of RIP1183 in beagle plasma at different time points were detected and pharmacokinetic parameters were calculated.RESULTS:1. Design and synthesis of RIP derivatives.RIP-I and five RIP derivatives were synthesized. RIP-L(YKPLTNF-CONH2) and RIP-V( YKPVTNF-CONH2) were obtained by amino acid substitutions based on the sequence of RIP-I. RIP1181( CH3CO-YKPVTNF-ST-YKPVTNF-CONH2), RIP1182( YKPVTNF-ST-YKPVTNF-CONH2) and RIP1183( CH3CO-YKPVTNF-CONH2) were obtained by terminal modification or oligomerization method.The purity of these synthetic peptides analysed by reverse-phase high-performance liquid chromatography were more than 95%. MALDI-TOF or ESI mass spectrometry analysis showed that measured molecular weight values of all peptides are in line with the theoretical value.2. Screening for antibacterial activity of RIP derivatives in vivoEstablishment of sepsis model induced by MRSA XJ75302 to screen RIP derivatives, RIP-V showed better antibacterial activity than that of RIP-I and RIP-L in vivo. Furthermore, after the end of the modified RIP-V, RIP1183 significantly improved the survival rate and survival time of sepsis mice, than that of RIP-V, RIP1181 and RIP1182.3. Evaluation of antibacterial effect RIP1183 in vitroMIC values of RIP1183 against 6 staphylococci were all greater than 256 μg/ml. And growth curve of RIP1183 against 6 staphylococci showed that RIP1183 could not inhibit the growth of 6 bacteria at concentration of 250-1000 μg/ml.4. Evaluation of antibacterial effect RIP1183 in different mouse model caused by MRSA in vivoIn sepsis model caused by HA-MRSA or CA-MRSA, RIP1183 increased the survival rate of infected mice by 40% and 60%, respectively. And RIP1183 significantly reduced the number of bacterial, and reduced the pathological damage in infected animal tissue. In necrotizing pneumonia model, RIP1183 significantly reduced the number of bacteria in the lungs of infected animals and decreased pulmonary edema and pathological damage. Skin infection model, RIP1183 significantly reduced the abscess area and the count of S. aureus in the abscess, and promoted wound healing.5. Antimicrobial mechanisms of RIP in vivo.(1) Protective effects of RIP1183 disappeared on sepsis mice after neutrophils depletion. That indicating antibacterial activity of RIP1183 may be associated with the function of neutrophil.(2) RIP1183 had no effect on MPO activity of neutrophils, which indicate that RIP1183 affects neutrophil function in an indirect way.(3) RIP1183 decreased the expression levels of RNAⅢ and virulence factor( PVL, HLA, PSMα and PSMβ).(4) LAC bacterial supernatant could induce necroptosis of neutrophil; the affect could be inhibited by MLKL inhibitor NSA.(5) Compare to wild strain LAC, PSMα or PSMβ defective bacterial supernatants decreased the expression levels of p MLKL.(6) Cell necroptosis typical characteristics could be observed by TEM, after the neutrophils treated with PSMα1. And NSA could block the effect of PSMα1 on neutrophils.(7) RIP1183 or Nec significantly reduces p MLKL levels in neutrophil isolated from bronchoalveolar lavage fluid, and decrease necroptosis of neutrophil in lung tissue of infected mice. Necroptosis of neutrophil in mice lung tissue also less been induced by genetic defect strains of LAC(8) Mice were administered intraperitoneally RIP1183 or Nec after infection, or mice infected by LAC after gene deletion of psmα, could significantly decrease the mortality rate of mice, significantly decrease lung injury and promote bacterial clearance in mice lung tissue.6. Safety and pharmacokinetics of RIP1183(1) Single-dose intravenous toxicity study of RIP1183 in SD rats or beagle dogs, the results show that, RIP1183 have no effect on the food take, body weight and general signs of rat or beagle dogs, at dose of 100 mg/kg or 50 mg/kg.(2) The safety pharmacology experiment showed that RIP1183 had no effect on the central nervous system of mice, respiratory and cardiovascular systems of beagle dogs.(3) The plasma elimination half-life of RIP1183 is about 20 minutes in dogs.CONCLUSION:1. QS inhibitor RIP1183 is an efficient, safe, less toxic antibacterial agent. The plasma elimination half-life of RIP1183 is about 20 minutes in dogs.2. RIP1183 can significantly reduce the expression of virulence factors in bacterial isolated from mouse tissues, like RNAⅢ, PSMα and PSMβ.3. Neutrophils are closely related RIP1183 antibacterial activity and RIP1183 likely to exert indirect influence of neutrophils in vivo.4. Hypothesis of antibacterial mechanism of RIP1183: RIP1183 inhibited S. aureus agr QSS, decrease expression of virulence factors and reduce the damage caused by the virulence factors. RIP1183 could inhibit expression of PSM, decrease the neutrophil necroptosis caused by PSM and indirectly promote neutrophil scavenging bacteria in vivo.