Screening Of Quorum-Sensing Inhibitors From Marine Microorganisms And Study On Their Action Mechanisms

With the widespread appearance of antibiotic-resistant bacteria, there is an increasing demand for novel strategies to control infectious diseases. Conventional antibiotics possess broad-spectrum toxic or growth-inhibitory effects on organisms. Therefore, an increased frequency of bacterial mutations has resulted in a significantly increased incidence of antibiotic resistance. Obviously, the problem of the increasing bacterial antibiotic-resistance could not be resolved absolutely by antibiotics against traditional targets. Hence, the development of new approaches to the treatment of bacterial infections constitutes a focal point of research. The discovery of bacterial communication systems (Quorum-Sensing, QS), which orchestrate important temporal events in the infection process, have afforded a novel opportunity to ameliorate bacterial infection by means of methods other than growth inhibition. Recent research showed that Quorum-Sensing system is widespread and involves complex networks that serve as fine-tuner of the performance of diverse behaviors, such as pathogenic factor production, biofilm formation, and so on. According to the estimation of National Institutes of Health (NIH), biofilms may be involved in up to 80 percent of human infections. Studies showed that biofilms play important roles in drug resistance and many chronic infectious diseases relapse repeatedly due to the biofilm formation. Therefore, the interference with QS is of great significance for the research and development of new antimicrobial drugs.In the present study, an effective method was developed for screening of marine bacteria isolates capable of inhibiting the N-acyl- homoserine lactones (AHL) mediated quorum-sensing process of many gram-negative bacteria. The action mechanism of such inhibition was also investigated.Our protocol is based on sole source of energy method and AHL reporter strains method, which was used for the rapid and sensitive screening of quorum-sensing inhibitor from marine bacteria. Many bacterial strains with QS inhibiting activities were isolated, and one of them, which showed the highest activity, was identified as Stenotrophomonas sp.A8.The partial sequence of an AHL quenching enzyme AQ1 was cloned by screening A8 genomic library with AHL sole source of energy method. The complete AQ1 gene was cloned by inverse PCR. The gene is 747bp in length, and is predicated to encode a protein of 248 amino acids with a molecular mass of 31.2 kDa, which is 12% and 13% amino acid sequence similar with those of Pseudomonas aeruginosa AHL-quenching enzyme PvdQ and QuiP, respectively. The AQ1 showed quenching activities to many AHL molecules with different side chain lengths, especially to the 3OC6HSL.Interestingly, from the bioactive partition of A8 fermentation broth, three compounds, CP1, CP2 and CP3, was isolated by reverse-phase high performance liquid chromatography and their activities were assayed by AHL reporter strains. By means of spectroscopic methods (MS, 1H NMR, 13C NMR), the structures of compounds CP1, CP2 and CP3 were elucidated. All these three compounds showed activities of inhibiting the binding of AHLs to their receptors. CP2 can also inhibit biofilm formation of gram-positive bacteria, Staphylococcus aureus and Staphylococcus epidermidis. The mRNAs of agrA, SarA and Hld gene were analyzed with real-time quantitative polymerase chain reaction. All of them were regulated by QS system in S.aureus RN4220. The results showed that mRNAs of these three genes decreased by CP1, so the effect of attenuating biofilm formation can be attributed to the depression of QS system.In conclusion, an effective method was developed for screening of environmental bacterial isolates capable of inhibiting the AHL-mediated quorum-sensing system. The AHL-quenching enzyme AQ1 gene was cloned from strain Stenotrophomonas sp.A8. Recombinant AQ1 showed activities to many AHL molecules with different side chain lengths. Meanwhile, three Antagonistic cyclic peptides of Quorum-Sensing were isolated from the bioactive partition of fermentation broth of A8. And it was reported for the first time that the cyclic peptide CP2 can inhibit biofilm formation of gram-positive bacteria, S.aureus and S.epidermidis. Analysis of three QS regulated genes demonstrated that the reduction of biofilm formation is related to the depression of QS system.Research into quorum sensing and its inhibition, may provide a mean of treating many common and damaging chronic infections without the use of growth-inhibitory agents, such as antibiotics, preservatives and disinfectants which unavoidably select for resistant organisms. To the large number of bacteria that employ quorum-sensing communication systems, attenuation of unwanted bacterial activities rather than bactericidal or bacteriostatic strategies may find application in many different fields, e.g., medicine, agriculture and food technology.