Isolation And Activity Avaluation Of A Marine Bacterial Quorum Sensing Inhibitory Factor

Quorum sensing(QS)refers to the density of bacteria to coordinate the behavior itself.When the bacterial density increases,the signal molecules(autoinducers)also increased to bind with receptor and intracellular protein and regulate the expression of specific genes.These genes regulate many physiological functions of bacteria,such as bioluminescence,biofilm formation,bacterial aggregation,extracellular polysaccharide production and virulence factor secretion.Compared to the antibiotics,quorum sensing inhibitors(QSIs),functioned as an anti-virulence agent,mainly attenuate the virulence without killing bacteria or inhibiting their growth.Therefore,anti-QS therapies could avoid evolutionary pressure of bacteria and the resulting drug resistance problem.In this article,the cultivating condition of marine microorganisms named Raoultella sp.Pan22 x was optimized to improve the quality of thecrude extract and quorum sensing inhibitory activity.At the same time,the mutation assay of the strain named Pan22 x was carried out to generate the new type of quorum sensing inhibitor with high activity,and the resulting mutants were screened by separation and purification.The main results of this paper are as follows:(1)The cultivating condition of marine microorganisms Raoultella sp.Pan22 x was optimized Single factor experiment and orthogonal experiment were carried out to generate the result that the optimum medium is LB medium with starch 3%,yeast extract 0.3%,peptone 4%,NaCl 1%.The optimal fermentation conditions were temperature 30?,initial pH 7.5,rotate speed 150 rpm,fermentation time 96 h,inoculation rate 10%,medium capacity 120 mL/250 mL.The quality of crude extract was increased by about 400 times and the activity was increased by about30.2% compared with that before optimization.(2)After 60 L fermentation of Raoultella sp.Pan22 x,38.87 g crude extract was obtained and was poured onto MCI column.Six fractions were obtained,and the fraction C,fraction D,fraction E,and fraction F had quorum sensing inhitory activity.A total of ten compounds were isolated in those fractions and nine of them were identified,including one alkaloid named tryptophol(1)and one amino acid named Tryptophan(5),and seven cyclic dipeptides named Cycle(Pro-Tyr)(2),Cycle(Phe-Pro)(3),Cycle(4-OH-Phe-Pro)(4),Cycle(Pro-Trp)(6),Cycle(Pro-Ile)(7),Cycle(Pro-Leu)(8)and Cycle(Trp-Gly)(9).The resulting nine compounds can significantly inhibit the production of violacein,compound 1,and 4-9can significantly inhibit the production of pyocyanin,compound 1,5 and 9can reduce biofilm formation.The variety of the structures had significant effect on the quorum sensing inhibitory activity.(3)Raoultella sp.Pan22x was mutagenized by ultraviolet,gamma ray and antibiotics.Virulence factors such as pyocyanin,elastase and biofilm production were functioned as targets.Raoultella sp.Pan22x-3,Raoultella sp.Pan22x-5 and Raoultella sp.Pan22x-6 were positive mutants,and the quorum sensing inhibitory activity was stronger than that of Raoultella sp.Pan22 x,while Raoultella sp.Pan22x-1,Raoultella sp.Pan22x-2,Raoultella sp.Pan22x-4 and Raoultella sp.Pan22x-7 were negative mutants,Raoultella sp.Pan22x-1,Raoultella sp.Pan22x-2 and Raoultella sp.Pan22x-4 had no quorum sensing inhibitory activity,and Raoultella sp.Pan22x-7 activity was lower than Raoultella sp.Pan22x.