Screening And Activity Evaluation Of Bacterial Quorum Sensing Inhibitory Factor

With indiscriminate use of antibiotics, there has been an alarming increase in thenumber of antibiotic resistant pathogens. Antibiotics are no longer the magic bulletsthey were once thought to be and therefore there is a need for development of newantibiotics and/or other novel strategies to combat the infections caused by multidrugresistant organisms. Quorum sensing (QS) is a bacterial communication process thatdepends on the bacterial population density. It involves small diffusible signalingmolecules which activate the expression of myriad genes that control diverse array offunctions like bioluminescence, virulence, biofilm formation, sporulation, to name afew. As QS is not directly involved in processes essential for growth of the bacteria,inhibition of QS does not impose harsh selective pressure for development ofresistance as with antibiotics. Therefore, QS is an obvious target for a novel class ofantimicrobial drugs which would function to efficiently block reception of the cognateQS signals in vivo, and thereby be capable of inducing attenuation of pathogens.The purpose of this study is to search for novel quorum sensing inhibitors (QSI)from secondary metabolites in marine microorganisms and Chinese herbal extractsusing QS biosensors. Several QS-inhibitory compounds have been identified andanalysis the biological activity.More than170marine fungi, actinomycetes and yeast isolated from marineseaweed, mud and water collected from Sanya District in Hainan were screenedagainst the QS indicator strain Pseudomonas aeruginosa QSIS-lasI, Pseudomonasaeruginosa PQSI-pqsA and Chromobacterium violaceum CV026. The isolates fungiSY004, SY012and SY029exhibited very good anti-QS activity, with SY029beingthe most promising one. The fragment of18S rDNA of SY029was compared withthose of reference strains deposited in GenBank (NCBI) databases by a BLASTsearch. Results show that the sequences of SY029exhibites high identity withAspergillus sp, named Aspergillus sp. SY029. The bioactivator with anti-QS activity was separated by bioactivity-guided fractionation from the crude extract, usingsilica-gel column chromatography, Sephadex LH-20gel column chromatography andHPLC, and was identified as butyrolactone I based on spectroscopic analysis. Thiscompound could attenuate pathogen virulence factors of P. aeruginosa such asactivity of elastase and pyocyanin production, without affecting bacterial growth inthe effective range of concentration. In order to exclude the P. aeruginosaenvironment and other QS system interference, two heterologous Escherichia colibiosensors, E. coli MG4/pKDT17and E. coli pEAL08-2, were used to identify theeffect of butyrolactone-I on las and PQS quorum sensing system. The results showthat butyrolactone-I can specifically inhibit the las and PQS quorum sensing systems.RT-PCR shows that it can significantly reduce the mRNA level of QS-related genes inP. aeruginosa. Butyrolactone-I also inhibits violacein production in C.violaceumwhich is regulated by QS.Traditional Chinese herb is commonly used by people as an alternative as well assupplement to mainstream health care. QSI compounds are shown to be present intraditional Chinese herb and these medicines may be a rich source of compounds tocombat pathogenic bacteria. Preliminary screened of70commonly available herbsextracts, moutan bark and ash bark were selected for further anti-QS activities due tothe effective quorum sensing inhibition. The major active constituent of the two herbswere identified as paeonol and fraxetin, using bioautographic TLC, preparative TLC,and HPLC analysis. It is the first time to report that these compounds can interferewith bacteria quorum sensing system. Paeonol can inhibit the violacein production,biofilm formation and the mRNA level of QS-related genes in C. violaceum atsub-inhibitory concentrations. Fraxetin showing inhibition of quorum sensing isaccessed for downregulation of QS-mediated virulence factors like elastase activity,pyocyanin production and swarming motility in P. aeruginosa PAOI. It also reducesviolacein production in C.violaceum.In this study, the screening source of novel QSI is mainly secondary metabolites inmarine microorganisms and Chinese herb. Marine microorganisms is an importantsource of screening novel QSI because of the species and genetic diversity. Butyrolactone-I, a secondary metabolite produced by Aspergillus sp. SY029candownregulate the quorum sensing system of P. aeruginosa. As a putative signalingmolecule, butyrolactone-I regulates fungal biological function. It is the first time toreport that these compounds can interfere with bacteria quorum sensing system. It canbe a precursor compound for further study of novel antibacterial based on structuralmodification. As a valuable resource of our country, Chinese herb are low toxic andbiological diversity. The clinical drugs paeonol and fraxetin show great quorumsensing inhibitory activity, so they may have a good application prospect with highsafety and stability.