Dynamic Changes Of Biological Molecules Of Escherichia Coli Responded To Oligochitosan Treatment

Chitosan is a deacetylated derivative of chitin mainly composed of glucosamine units,2-amino-2-deoxy-β-D-glucose. This natural compound is biodegradable and nontoxic, and hasa positive charge that confers to this polymer numerous physiological and biologicalproperties with great potential in diverse industries. Many studies have shown that chitosanand its degradation products oligochitosan can effectively inhibit the growth of Escherichiacoli. Several antimicrobial laws have been proposed for chitosan and oligochitosan. Severalstudies suggested that chitosan neutralizes the electronegative charges on cell surfaces and thecell permeability is changed, therefore, this interaction causes the leakage of intracellularmaterial of the cell. Chitosan can cause an increase in the content of active oxygen species ofmicrobial cell. However, up till now, it is still not clear in the antibacterial mechanism ofchitosan and oligochitosan. The present paper utilizing biochemical analysis methods andproteomics technology carries out research about dynamic changes of biological molecules ofE. coli responded to oligochitosan treatment. The results are as follows.According to the method of the viable cell count, the inhibitory effect of oligochitosan onE. coli was determined at24h cultivation. The inoculated plates were cultured for24h at37oC and the number of formed colonies were counted and the viable cell counts were calculated.By measuring changes in the number of viable cell counts, it showed that0.5%oligochitosantreatment caused the number of viable cell counts increased at first and then decreased during24h, meanwhile,1%oligochitosan treatment reduced the number of viable cell countsgradually during24h, the inhibitory effect of oligochitosan on E. colii was obvious. Todetermine the cell integrity of the E. coli under the treatment of0.5%and1%oligochitosan,the leakage of intracellular enzymes, alkaline phosphatase, lactic dehydrogenase andβ-galactosidase, was detected. Results showed that cell integrity of the E. coli was destroyedin some degree. The apoptosis of E. coli was measured by flow cytometry at the culture of2h,4h and6h, it showed that apoptosis increased gradually and cell necrosis was basically stable.The results of apoptosis and of the number of viable cells, also the results of cell integrity of the E. coli under oligochitosan treatment, together they showed that apoptosis was increasingand cell necrosis was basically stable during the growth of E. coli inhibited by oligochitosan,it can be explained that oligochitosan mainly induced apoptosis of E. coli to achieve theinhibitory effect.The content of reactive oxygen species of E. coli that was treated with0.5%and1%oligochitosan increased rapidly and then decreased gradually,1%oligochitosan treatmentshowed significantly different trend compared with0.5%oligochitosan treatment, and at thesame time showed higher content of active oxygen species than0.5%oligochitosan treatment.In the determination of oxidative damage, the0.5%and1%oligochitosan treatment showedserious lipid peroxidation and protein damage. The content of MDA, protein carbonyl andprotein nitration was signigicantly higher than control. The0.5%oligochitosan treatmentinduced that the content of MDA, protein carbonyl and protein nitration increased graduallyin0-4h, and decreased in4-16h. The1%oligochitosan treatment induced that the content ofMDA, protein carbonyl and protein nitration increased gradually during24h. The DNAoxidative damage, at0.5%and1%oligochitosan treatment was significant compared with thecontrol in2h and4h, but there was no difference in6-24h. These results indicated thatoligochitosan treatment can inhibit the growth of E. coli by inducing the accumulation ofintracellular active oxygen and leading to oxidative damage of biological molecules.Proteome analysis has been widely used to establish the cellular functions in response tovarious external stimuli and the variation of protein expression level reflects biologicalresponses. A proteomic approach involving two-dimensional polyacrylamide gelelectrophoresis and mass spectrometry was applied to detect changes in quantitative proteinprofiles of E. coli in response to the stimulus of0.5%and1%oligochitosan. The E. colicultured for4h, out of a total number of approximately1150protein spots were got from totalprotein separated by two-dimensional electrophoresis. According to the two times proteinexpression differences and the actual situation of protein spots on the gel, there was42proteins spots was selected to identify by mass spectrometry, it showed40proteins spots weresuccessfully identified and these were35kinds of proteins. The expression levels of28protein spots increased significantly and10decreased significantly under oligochitosantreatment group compared with the control. Also, there are two particular points showed different under0.5%oligochitosan treatment. Among the identified proteins were those thatwere mainly involved in metabolism, protein synthesis and oxidation-reduction and so on.Among them,0.5%and1%oligochitosan treatment compared with the control, all ofredox-related protein were up-regulated in expressed proteins, most of metabolism relatedproteins and synthesis related proteins were up-regulated in expressed proteins.This study investigated dynamic changes of biological molecules of E. coli responded tooligochitosan treatment, the results showed that oligochitosan induced the accumulation ofreactive oxygen species, elicited the apoptosis and oxidative damage, identified proteinsmainly involved in metabolism and protein synthesis were significantly altered expressionlevels, these may the way to inhibit the growth of E. coli by oligochitosan.