Optimum Designing Of New Potent Antimicrobial Peptides And Their Effects On Multidrug Resistant Bacteria

AIM:Continous emerging of multi-drug resistance in pathogens has put huge threat to human health. The current worldwide development of bacterial resistance to antibiotics showed trend in multidrug resistance, including methicillin-resistant Staphylococcus aureus (MRSA), bacteria producing extended-spectrumβ-lactamases (ESBLs), as well as the spreading multi-drug resistant Mycobacterium tuberculosis. What is more serious is that bacteria resistant to all antibiotics (pan resistant bacteria) have emerged and human being may have no cure for the situation once they infect this type of bacteria. Faced with the rapid spread of drug-resistant bacteria, the grim situation that mortality rates of infectious disease continue to climb, governments in different countries have made controlling the harm caused by infection of drug-resistant bacteria focus of attention, and the development of new strategies and new drugs to effectively control drug-resistant bacterial infections have become imperative aim of research for scientists all over the world. Classic key strategies in antibiotic research and development (R&D) are to develop new antibiotics derivatives from various categories of existing antibiotics. With the extensive use of antibiotics, an increasing number of bacteria exists cross-resistance to antibiotics, which makes structural modification and improvement in antibiotic R&D quite limited, the opportunity to find new antibiotics scarce, and the rate of antibiotic R&D slower. Clinical application of these new antibiotics failed to prevent the emergence of drug resistance, and bacteria quickly develop resistance to these new antibiotics. The current emerging rate of antibiotic resistant strains has far exceeded the speed of new antibiotics R&D. In order to effectively fight against the growing bacterial resistance, we must find a new breakthrough.Antimicrobial peptide not only has the broad-spectrum bactericidal activity, but also plays a central role in the recruitment of macrophages and enhancement of the innate immune system. The most noteworthy is that the bactericidal activity of antimicrobial peptide is mainly through the physical permeability act on the bacterial membrane, and it is not easy for bacteria to develop resistance to antimicrobial peptide given that it is very difficult for microorganisms to change their own phospholipid bilayer membrane structure. Antimicrobial peptide has become most promising new generation of the drug candidates in the anti-drug-resistant bacteria study.There are a few problems in the study of antimicrobial peptide, mainly in the following areas: First, most antimicrobial peptides are highly toxic, with low selectivity to target cells, which make red cell hemolysis easier. Second, a number of antimicrobial peptides with significant antibacterial activity in vitro lost their antibacterial activity under the physiological salt and plasma concentrations. Third, the majority of antimicrobial peptides are composed of more than 20 amino acids, which makes production costs high.In response to these problems, we have adopted two design methods, which are de novo design of antimicrobial peptide with new structures and modification of antimicrobial peptides with known structure. Computer-aided design was used to design these two types of antimicrobial peptide which were synthesized by solid-phase synthesis methods. Experiments in vitro and in vivo for efficacy study have been carried out to screen new type of peptides with high activity, low toxicity, stability, and uneasiness to develop drug resistance against the seven most common multidrug resistant organisms with high drug resistance incidence and intensity and their antimicrobial mechanism was further explored.METHODS:1. Computer-aided design of new antimicrobial peptides: based on physical and chemical properties of known antimicrobial peptides and theory of their structure-activity relationship, de novo and modified design have been adopted to obtain new antimicrobial peptide with fatty acid chains of different lengths at N-terminal, named FA1, FA2, FA3, FA4, FA5, FA6, respectively; Accelrys / InsightII molecular modeling software has been applied to do computer-aided analysis, and antimicrobial peptides, named US1, US2 , US3, US4, US5, US6, US7, US8, US9, US10, and DS1, DS2, DS3, DS4, DS6, DS7, DS8, DS9, DS10, have been design by structural modification according to theγ-core motif structure of insect originated antimicrobial peptides thanatin. Both types of antimicrobial peptides above have been amidated at carboxyl-terminal to obtain a series of peptide sequence with C-terminal amidated.2.Synthesis, purification and identification of new antimicrobial peptides: solid-phase synthesis of polypeptide chain with C-terminal acid amination are proceeded from C-(carboxy-terminal) to the N-(N-terminal) on the Rink resin by Fmoc protection ofαamino acids and HOBt / DIC as condcensing agent. The molecular weight of crude peptides is determined by MALDI-TOF mass spectrometry and its purification by RP-HPLC chromatography; molecular weight of final pure peptide is determined by ESI-MS. The method of air oxidation is used to form intramolecular disulfide bonds for cyclization of polypeptide chain. RP-HPLC is used to analyze the continuous change of peptide chromatography oxidation and reduction peaks in the process of peptide oxidation; the process of peptide oxidation is monitored and oxidized peptides is purified by RP-HPLC; the molecular weight of final pure peptide before and after oxidation is determined by ESI-MS.3.In vitro and in vivo pharmacodynamic studies of antimicrobial peptide against multidrug-resistant bacteria: the relationship between plate colony forming units (CFU) and optical density (OD600) for ESBLs-EC and MRSE was tested; the minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 25 antimicrobial peptide against seven drug-resistant bacteria were tested; the effect of antimicrobial peptide DS1, US1 and DS7 on growth of ESBLs-EC and MRSE was observed by measuring growth curve at different time points; the effect of antimicrobial peptide DS1, US1 and DS7 on growth inhibition of ESBLs-EC and MRSE was observed by measuring the plate counts of CFU; the hemolytic toxicity of FA4, DS1, US1 and DS7 was assessed by hemolytic experiment; the stability of DS1 and DS7 was assessed in 50% plasma; the drug-resistance of DS1 and DS7 was assessed by drug-resistance test. ESBLs-EC induced sepsis model of BALB/c mice and MRSE induced sepsis model of ICR mice were established, and the effect of DS7 and DS1 on survival time and rate of mice was observed; the effect of DS1 on the growth inhibition of ESBLs-EC in BALB/c mouse organs was observed by measuring the CFU; the protective effect of the DS1 on the lungs and spleens of BALB/c mice was observed by lung and spleen tissue HE staining.4. Study on the functional mechanism of antimicrobial peptide: the change in surface morphology of bacteria was observed by scanning electron microscopy at different times (30, 60, 90 min) after incubation of DS1 and DS7 with ESBLs-EC and MRSE respectively. The ultrastructure changes in bacteria in vivo was observed by transmission electron microscopy at 90 min after incubation of 6MIC concentrations of DS1 and DS7 with ESBLs-EC and MRSE respectively. The intensity and distribution of fluorescence were observed by fluorescence microscopy at 90 min after incubation of different concentrations (4, 12, 24μg/ml) of DS1 and DS7 with ESBLs-EC and MRSE respectively. RESULTS:1. Computer-aided design of new antimicrobial peptide: 6 new peptides with different lengths of fatty acids at N-terminal have been designed by computer-aided de novo design; 10 peptide sequences withγ-core motif but no disulfide bonds formation and 9 peptide sequences withγ-core motif as well as disulfide bonds formation have been designed by computer-aided modification of the antimicrobial peptide thanatin containing genetic marker (γ-core motif).2. Synthesis, purification and identification of new antimicrobial peptides: MALDI-TOF mass spectrometry analysis showed that measured molecular weight values of all crude peptides are in line with the theoretical value; the purity of all crude peptide purified by RP-HPLC reached 95% above; ESI-MS-MS identification results showed that measured molecular weight value of pure peptide are in line with theoretical values. RP-HPLC analysis showed that the oxidation peak area of peptide gradually increased and reduction peak area decreased with time in the process of intramolecular disulfide bond formation; purity of all cyclization peptide reached 95% by RP-HPLC purification; ESI-MS-MS identification results showed that molecular weight difference ofγ-core motif containing polypeptide chain before and after oxidation was 2.3. In vitro and in vivo pharmacodynamic studies of antimicrobial peptide against multidrug-resistant bacterial: 3 out of 19 polypeptides withγ-core motif were screened with antibacterial activities, and peptide US1 and DS1 had obvious and equal antibacterial activity (MIC) against gram negative bacteria; DS7 showed better antibacterial activity against gram positive bacteria, especially its best activity against MRSE. 4 out of 6 peptides with fatty acids were screened with antibacterial activity, and FA4 with C16 soft acid showed the best antibacterial effect. MBC of FA4, US1 and DS1 against the 9 bacteria were 2-fold MIC, and MBC of DS7 against the 9 bacteria was≥2-fold MIC. FA4, DS1, US1 and DS7 at 128μg/ml showed no hemolysis in 2% and 10% plasma, whose HC50 is much higher than that of the positive control melittin. The plasma stability experiment results show that DS1 and US1 were transformed into forms with stronger activity after metabolism in plasma, and DS7 was very stable in plasma, while the positive control S4 (1-16) was seriously degraded in plasma. ESBLs-EC and MRSE are easy to develop resistance to antibiotics but difficult to DS1 and DS7. DS1 could significantly prolong survival time of the BALB/c mice (p<0.01) and increase their survival rate (p<0.01); it could remarkably inhibit the growth of bacteria in organs (p<0.01) and inhibit lung and spleen damage caused by ESBLs-EC in mice. DS7 could significantly prolong survival time of the ICR mice (p<0.01) and increase their survival rate (p<0.01).4. Study on the functional mechanism of antimicrobial peptides: increasingly severe edema, and ultimate cell rupture into irregular pieces was observed by scanning electron microscope after respective acting of DS1 and DS7 to ESBLs-EC and MRSE. Serious damage of ESBLs-EC cell wall, leakage of chromatic thread and other contents with bacteria being only a hollow shell were observed under transmission electron microscope after DS1 acting. After DS7 acting, MRSE showed abnormal unequal splitting, increasing of cell surface buddings, increasing of number and volume of vacuoles in bacteria, broken bacterial wall and ring screw dense bodies in bacteria. With propidium iodide as red fluorescent indicator and SYTO 9 as green fluorescent indicator, fluorescence intensity was observed under fluorescence microscope. With increasing of DS1 concentration, intensity and numbers of red fluorescence points which reflect the extent of bacterial membrane damage gradually increased, while the intensity and numbers of green fluorescence points which reflect the extent of bacterial membrane integrity gradually decreased; both red and green fluorescence points showed small or large aggregation. With increasing of DS7 concentration, the intensity of red fluorescence (propidium iodide) which reflects the extent of bacterial membrane damage showed no obvious increasing.CONCLUSION:1. De novo and modified design have been established to design and synthesize a series of new antimicrobial peptide in our study and they provided new ideas for antimicrobial peptide design and study.2. In vitro pharmacodynamics experiments showed that antimicrobial peptide FA4, US1 and DS7 have merits in high efficiency, low toxicity, stability, and uneasiness to develop drug-resistance. It is proved for the first time that disulfide bond inγ-core motif is not necessary for antimicrobial peptides functioning.3. In vivo pharmacodynamics experiments showed that DS1 and DS7 can effectively extend the survival time of BALB/c mice and ICR mice in mouse septicemia model, respectively. DS1 can effectively suppress ESBLs-EC growth in organs of infected BALB/c mice and alleviate lung and spleen injury.4. The mechanism of DS1 is to kill bacteria by penetrating through the membrane. And the mechanism of DS7 is to inhibit bacterial growth by disrupting normal bacterial division seriously.