Molecular Modification Of Acinetobacter Baumannii Endolysin PlyAB1

Acinetobacter baumannii is a Gram-negative conditional pathogen that is widely found in nature and in hospitals and can cause bacterial infections such as pneumonia and meningitis.With the increasing severity of bacterial drug resistance,there have been multiple drug resistant Acinetobacter,pan-drug resistant Acinetobacter and total drug resistant Acinetobacter.With the introduction of the policy of banning antibiotics and limiting the use of antibiotics in China,it is urgent to find new antibiotics.Phage lysins is the cell wall hydrolyzing enzyme produced by phages in the late stage of infection of the host.Compared with the traditional antibiotic therapy and phage therapy,the treatment of bacterial infection by lysins have many advantages,which are more acceptable to people,such as not easy to produce drug resistance,higher specificity,and no destruction of normal flora,etc.So at present,many researchers devote themselves to the development of lysins preparations.Compared with gram-positive bacteria,the gram-negative bacteria has an outer membrane which is composed of lipopolysaccharide.There are few endolysins that can kill Acinetobacter baumannii without the outer membrane penetrating agent,while PlyAB1 is one of them.PlyAB1 is the endolysin of phage Abp1 which is isolated from the MDRAB strain AB1.At first,we tried to express the enzyme in Escherichia coli,and made the determination of the basic enzymatic activity.We are sure that the optimum condition of the induce is 16?,0.5 mM IPTG,and we also detect the anti microbial activity use the killing curve and plate assay.Through the experiment of thermostability,we found that PlyABl is stable between 20? and 45?.And when we pretreat the target bacteria with EDTA,PlyAB1 shows strong lysis activity against most of the gram-negative bacteria such as E.coli DH5?,E.coli JM109,E.coli BL21(DE3)?Salmonella?KPneumoniae and A.baumannii ATCC19606.After understanding the basic enzymological properties of PlyAB1,we used protein engineering modification to optimize its properties.On one hand,through heterologous expression and antibacterial activity assay,we found that the antibacterial activity of PlyAB1 is two folder higher than LysAB2,but there are only 5 different amino acid sites between them.So we made five mutants:E33A,S41T,E96K,G100E,S103G.The result showed that the antibacterial activity of G100E decreased by 30%,so we considered that the 100th amino acid site was the key amino acid site that influence the antibacterial activity.Then we established the 100 site-saturation mutagenesis library and got some mutants that could increase the antibacterial activity.If amino acids are classified according to their polarity,they can be divided into non-polar amino acids,polar non-charged amino acids and polar charged amino acids,while polar charged amino acids can be divided into acidic amino acids and basic amino acids.By analyzing the experimental results,we found that when the amino acid mutant to acidic amino acids,the antibacterial activity decreases;when the amino acid mutant to basic amino acids,the antibacterial activity increases.We suspect that the reason for that is related to the way how endolysin act with the gram-negative bacteria.When the amino acid surface cation is contact with the negative iron of the lipopolysaccharide(LPS),the outer membrane will lost its balance and its stability so that let the endolysin arrved at the peptidoglycan layer and lyse the bacteria immediately.On the other hand,we made the rational design of the PlyAB1 in order to increase its thermostability.We made some mutants through software SpotWizard and before that we made the three-dimensional structure simulation of PlyAB1 and predict its conserved domain.At last,we got the mutant K69R and P84A that respectively increased by 50%and 150%.After we got the high antibacterial activity mutant and high thermostability mutant,we made the combination of the optimum mutants:G100R/K69R.The result showed that the antibacterial activity of the mutant G100R/K69R at 50? has increased by 172%,and the half-life of PlyAB1 increased by a factor of 25.We also tried to costruct the chimeric lysins and obtained a broader lysis spectrum chimeric lysin:Lysostaphin-GGGS-AB1.In conclusion,we got the higher antibacterial activity mutant and higher thermostability mutant by rational design and semi-rational design in this study.And then we made the combination design to acquire a mutant with both higher thermostability and higher lytic assays.It's sensable for the industrial production and drug strorage in the future.And We preliminarily investigated the possible mechanism of endolysins interaction with Gram-negative bacteria,which laid a foundation for future research in our laboratory.