Screening Of Marine Fungi With Quorum Sensing Inhibitory Activity And Studies On Quorum Sensing Inhibitor From Strain QJ012

Pseudomonas aeruginosa is an increasingly prevalent opportunistic pathogen that can cause a wide range of acute and chronic infections in human beings.It is the third most common pathogen found in nosocomial and lifethreatening infections of immunocompromised patients.P. aeruginosa infections are difficult to cure since P. aeruginosa bears innate resistance to many antibiotics and its antibiotic susceptibility is decreased when it is present in biofilms.Recent studies have elucidated that the pathogenicity of P. aeruginosa is regulated by a microbial cell-cell signaling system that was known as "quorum sensing" (QS). Quorum sensing is an important global regulatory system that controls the expression of genes in a population density-dependent manner. It uses small diffusible molecules as signals to coordinate behaviors of P. aeruginosa including the production of virulence factors and development of biofilms.Therefore,the inhibition of QS may provide a novel strategy for treating Pseudomonas aeruginosa infections.In order to design and construct novel systems to screen high-performance P. aeruginosa quorum sensing inhibitors, two SacB-based screening systems, QlasI (pZ-1, PAO-MW1) and QrhlI (pZ-2,PAO-MW1) were developed.The plasmid pZ-1 harbors the fragment PlasI-sacB along with Plac-lasR,while pZ-2 harbors the fragment PrhlI-sacB along with PrhlR-rhlR.Both plasmids are maintained in P. aeruginosa MW1 (a lasl, rhll mutant unable to produce AHL signals).QlasI and Qrhll can be used for the rapid and effective screening of microbial,plant extracts and pure compounds.The marine fungal strains were isolated from the seawater and mud of Jiaozhou Bay. All of the cultural extracts were screened by the two screening systems mentioned above. Three fungal extracts were found to exhibit quorum sensing inhibitory activity with QlasI screening system.The most active strain QJ012 was selected for further study. Strain QJ012 was affiliated to Penicillium atramentosum according to the Biolog system and its ITS sequence was 100% identical with Penicillium atramentosum.The fermentation broth was separated by the means of modern chromatographic methods in a bioassay-guided separation manner to obtain pure bioactive compound. From the bioactive extract of QJ012 fermentation broth,one polyketide compound with quorum sensing inhibitory activity was isolated. By means of spectroscopic method (MS, 1H NMR,13C NMR,DEPT), structure of the compound was elucidated as 4,6-dimethyl-7-[(1R,2E,4aS,6S,7R,8R,8aR)-1,2,4a,5,6,7,8,8a-octahydro-7-hydroxy-2-(3-hydroxy-2-oxopropylidene)-3,6,8-trimethyl-1-naphthalenyl]-(2E,4E,6E)-Heptatrienoic acid. The compound had a negative effect on elastase and pyocyanin productions and biofilm formation, as well as on the expression of the key QS regulatory genes lasl, lasR and of the QS-regulated genes lasB and toxA.In conclusion, we constructed two rapid and effective P. aeruginosa quorum sensing inhibitors screening systems.Three fungal extracts were found to inhibit P. aeruginosa las quorum sensing system.The most active strain QJ012 was affiliated to Penicillium atramentosum.To our knowledge, no Penicillium atramentosum strains were reported to exhibit quorum sensing inhibitory activity previously. From the bioactive extract of QJ012 fermentation broth, one polyketide compound with quorum sensing inhibitory activity was isolated.And this is believed to be the first report of this compound acing as P.aeruginosa quorum sensing inhibitor. Our work has practical significance in finding new approaches to the treatment of bacterial infections.