Study On The Regulation Mechanism Of The BDSF Quorum Sensing System In Burkholderia Cenocepacia And Screening For QS Inhibitors

Quorum sensing(QS)is widely utilized by bacterial pathogens to regulate biological functions and pathogenicity.Recent evidence has shown that QS is subject to regulatory cascades,especially two-component systems that often respond to environmental stimulation.At least two different types of QS systems regulate pathogenesis in Burkholderia cenocepacia.However,it remains unclear how this bacterial pathogen controls these QS systems.Here,we demonstrate a novel two-component system Rqp SR(Regulating Quorum sensing and Pathogenicity),which plays an important role in modulating QS and pathogenesis in B.cenocepacia.We demonstrate strong protein-protein binding affinity between Rqp S and Rqp R.Mutations in rqp S and rqp R exerted overlapping effects on B.cenocepacia transcriptomes and phenotypes,including motility,biofilm formation and virulence.In trans expression of rqp R rescued the defective phenotypes in the rqp S mutant.Rqp R controls target gene expression by direct binding to DNA promoters,including the cis-2-dodecenoic acid(BDSF)and N-acylhomoserine lactone(AHLs)signal synthase gene promoters.These findings suggest that the Rqp SR system strongly modulates physiology by forming a complicated hierarchy with QS systems.This type of two-component system appears to be widely distributed and coexists with the BDSF QS system in various bacterial species.B.cenocepacia employs a molecular mechanism in which the cis-2-dodecenoic acid(named Burkholderia diffusible signal factor,abbreviated as BDSF)QS system regulates N-acyl homoserine lactone(AHLs)signal production and virulence by modulating intracellular levels of cyclic diguanosine monophosphate(c-di-GMP).Thus,inhibition of BDSF signaling may offer a non-antibiotic-based therapeutic strategy against BDSF-regulated bacterial infections.In this study,we report the synthesis of small molecule mimics of the BDSF signal and evaluate their ability to inhibit BDSF QS signaling in B.cenocepacia.A novel structural analogue of BDSF,14-Me-C_16:?2(cis-14-methylpentadec-2-enoic acid),was observed to inhibit BDSF production and impair BDSF-regulated phenotypes in B.cenocepacia,including motility,biofilm formation and virulence,while it did not inhibit the growth rate of this pathogen.14-Me-C_16:?2also reduced AHL signal production.Genetic and biochemical analyses showed that 14-Me-C_16:?2inhibited the production of the BDSF and AHL signals by decreasing the expression of their synthase-encoding genes.Notably,14-Me-C_16:?2attenuated BDSF-regulated phenotypes in various Burkholderia species.These findings suggest that 14-Me-C_16:?2could potentially be developed as a new therapeutic agent against pathogenic Burkholderia species by interfering with their QS signaling.In the late growth stage of B.cenocepacia liquid culture,a large amount of BDSF signal degradation can be observed.However,there has been no systematic overall report on BDSF signal degradation mechanism.Some reports indicate that Rpf B was closely related with DSF signal transduction in Xanthomonas campestris pv.campestris.In this study,we carried out a preliminary study on the function of Rpf B homologous protein Nqs R in B.cenocepacia H111.The experimental results showed that deletion of nqs R enhanced swarming motility,biofilm formation and protease activity.Complementation of nqs R mutants by in trans expression of nqs R restored swarming motility,biofilm formation and protease activity to wild-type strain levels.In addition,we tested B.cenocepacia wild-type and nqs R mutants in a human cell line infection model.As expected,deletion of nqs R led to a significantly enhanced virulence in the A549 cell line infection model.As we known,biological function of swarming motility,biofilm formation,protease activity,and virulence are regulated by the BDSF QS systems in B.cenocepacia,which indicates that Nqs R is closely related to the BDSF system in B.cenocepacia H111.The regulation network between Nqs R and BDSF QS system needs further study.We then analyzed and compared the transcriptomes of the wild-type strain and the nqs R mutants using RNA-Seq.Differential gene expression analysis showed that51 genes were increased and 157 genes were decreased(greater than or equal to two folds)in the nqs R mutant compared with their expression in the wild-type H111 strain.These differentially expressed genes are associated with a range of biological functions,including fatty acid metabolism,membrane proteins,amino acid metabolism and transport,energy metabolism and transport,ferritin synthesis and transport,signal transduction and transcriptional regulation.Transcriptome data results was consistent to the above phenotype results,indicating that Nqs R is closely involved in the regulation of biological function in B.cenocepacia H111.These results of this chapter laid a foundation for the subsequent study of BDSF signal conversion mechanism,and it has further deepened our understanding of the virulence regulation network of B.cenocepacia.