| With the evolution and spread of drug-resistant genes,most traditional antibiotics are helpless against multidrug-resistant bacteria.It is well known that Gram-negative bacteria are more resistant to some antibiotics than Gram-positive bacteria due to their unique outer membrane structure.Among them,the β-barrel outer membrane proteins(OMPs)located on the outer membrane of bacteria undertakes the main physiological functions of cells,such as nutrient uptake,metabolic waste efflux and drug resistance mechanism formation.Since the β-barrel outer membrane protein precursor is generated in the cytoplasm,it needs to cross the bacterial inner membrane and the periplasmic space(without ATP)to reach the bacterial outer membrane,undergo a difficult process from a hydrophobic environment to a hydrophilic environment and end up to a hydrophobic environment.Due to their important physiological functions and difficult complex biosynthetic processes,OMPs has became popular targets for new drugs.Based on the transportation of the outer membrane protein OmpF in the periplasmic space and the folding and membrane insertion assisted by the BAM complex,our research group has completed the design and development of a novel antibacterial short peptide in the early stage,with/without a targeting sequence coupled to its N-terminus.Based on this,Firstly,we verified that the polypeptides exhibit antibacterial activity against the wild-type E.coli BW25113 strain and the special imp4213 strain.Then we treat the bacteria with the antimicrobial peptide in different media at different processing time to discover the optimum treating condition.It was found that the bactericidal effect of the novel polypeptide does not depend on the processing time,but increases with the concentration within limits.The study also explored the bactericidal mechanism of Bam A-H7,Bam D-H7 and OmpF7 polypeptides by biochemical and molecular biology techniques.It was found that :(1)OmpF7 exhibits significant interaction with Sur A,and with a large amount of Sur A expressed,the bactericidal effect of OmpF7 is weakened.(2)In vivo photocrosslinking as well as chemical crosslinking demonstrated that OmpF7 was able to interact with both the periplasmic quality control factor Sur A and Skp,suggested that the polypeptide OmpF7 was able to occupy both Sur A and Skp and interfere the transport process of outer membrane proteins.(3)It is also suggested that exogenous addition of polypeptides OmpF7,Bam A-H7 and Bam D-H7 may interfere the folding of OmpF.(4)Further investigation observed the destruction of the bacterial cell membrane by the polypeptide using GFP tracing and SEM.After the peptide OmpF7 treatment,the bacteria maintain the basic shape and only slightly invaginate on the surface,while the OmpF with guiding sequence significantly damages the cell membrane,causing the bacteria to shrink.Therefore,it is believed that the novel polypeptide kills bacteria by attacking its target rather than damaging the outer cell membrane.However,when the guiding sequence is coupled to form a fusion peptide,the bactericidal effect should be the result of the combination power with both the destruction of the cell membrane itself and the mechanism of attacking the target.In addition,the study also tested the antibacterial effect of OmpF7 with guiding sequence on E.coli separated from clinics.It was found that most of the log-phase drug-resistant bacteria and even several of the stationary drug-resistant bacteria were sensitive to OmpF7(with guiding sequence).In conclusion,the novel antibacterial peptide OmpF7(with guiding sequence)exhibits bactericidal effect by interrupting the transportation,folding and insertion process of outer membrane protein as well as damaging the cell membranes.The combined antibacterial effect kills the standard strains E.coli in different phase and even exhibit significant antibacterial effect on some clinical drug-resistant E.coli. |
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