Phage-encoded GP56 Trigger Lysis Of V.cholerae By Monitoring Host-produced Quorum-Sensing

In order to adapt to the environment and make the most efficient use of the limited resources for better survival,bacteria can communicate with each other to monitor cell density and coordinate group behavior,this process is called Quorum sensing(QS).The occurrence of Quorum sensing depends on the production,accumulation,release and recognition of signaling molecules called autoinducers(AI).Over the past half century,many studies have shown that Quorum sensing is a ubiquitous communication process,which is crucial for the survival of bacterial population.The scientists have found that quorum sensing is present in vibrio cholerae,the pathogenic microorganism that causes human diarrhea diseases.Multiple quorum sensing signaling pathways control the production of Vibrio cholerae toxic factors and the formation of Vibrio cholerae biofilms.The occurrence of vibrio cholerae quorum sensing is related to the production of two extracellular signaling molecules called CAI-1 and AI-2.These two autoinducers were synthesized by Cqs A and Lux S,respectively.After synthesis in the cytoplasm,these two autoinducers were secreted to the outside of the cell,then recognized and bound by the membrane-bound receptor protein Cqs S and Lux PQ respectively,we named them autoinducer-receptor pair,which further activated the downstream signaling pathway and affected the expression of related genes.Significantly,these two autoinducer-receptor pair share the same downstream signal transduction pathway and ultimately inhibit the formation of biofilms and the production of virulence factors.Beyond these two autoinducers,scientists found a new autoinducer in Vibrio cholerae,called DPO.DPO is made from threonine and alanine,and its synthesis depends on threonine dehydrogenase(Tdh,threonine dehydrogenase).After synthesis,DPO will be secreted to the outside of the cell immediately,and then,enter the intracellular of other cell.DPO can be recognized and combined by the transcription regulator protein Vqm A in the cytoplasm,then,this Vqm A-DPO complex can activates new quorum sensing signal pathway to regulate the expression of genes,and ultimately,inhibit the formation of biofilms and the production of virulence factors.Although DPO-mediated quorum sensing pathway is different from those mediated by CAI-1 and AI-2,these three autoinducers are not isolated from each other,instead,they connect with each other and eventually construct a complex quorum sensiting network.Recently,scientists found a special bacteriophage vp882,which can coordinate its behavior by monitoring the Quorum sensing of Vibrio cholerae.When QS occurs,the bacteriophage vp882 can change from lysogeny to lysis.So,how does vp882 sense the density of Vibrio cholerae? The findings show that bacteriophage vp882 can produce one transcription regulator protein GP56,which is homologous to the Vqm A,a transcription regulator protein encoded by Vibrio cholerae.GP56 can bind with DPO,the autoinducer produced by Vibrio cholerae,to form GP56-DPO complex.Then,this GP56-DPO complex can activate the downstream signal pathway of bacteriophage vp882 and change this phage from lysogeny to lysis,which finally causes the death of Vibrio cholerae.Moreover,the GP56-DPO complex can bind to the promoter region,a DNA sequence originally bound by the Vqm A-DPO,then,activate the downstream signal pathway to inhibit the formation of biofilms and the secretion of virulence factors.GP56 is the first quorum-sensing receptor which is encoded by phage but recognised by the DPO,the autoinducer produced by Vibrio cholerae.Based on the above characteristics,GP56-DPO complex can coordinate the lysogeny-lysis of phages vp882 by monitoring the QS of Vibrio choleraeNow,the molecular mechanism of the protein GP56 in this signaling pathway is not well understood,so,my work is aim to study the structure and function of GP56,a critical protein in this signaling pathway.Fortunately,we got the crystal structure of the GP56-DPO,to my knowledge,this is the first crystal structure formed by phage-encoded protein and bacteria-produced autoinducer.By analyzing the structure,we get the following conclusions: 1)GP56 is similar to Vqm A in overall structure,they all exist in the form of dimer.The crystal structure shows that GP56 contains an N-terminal PAS domain for binding autoinducer and a C-terminal DNA binding domain(DBD)for recognizing DNA sequence.2)By comparing the structure of GP56 and Vqm A,we found that their N-terminal PAS domain was quite consistent,especially in the core region for binding DPO.However,their C-terminal DBD domain that was used to bind promoter DNA was not conservative.This phenomenon indicates that they are specific in recognizing promoter sequences.3)These two monomers of Vqm A can form an intermolecular disulfide bond,which can stable and enhance the transcriptional activity of Vqm A,but phage protein GP56,a homologous protein of Vqm A,can not form the intermolecular disulfide bond,because it is a valine at this site.As far as I know,the cysteine used to form the disulfide bond in each monomer is conserved in almost all Vibrio species.On the whole,this is the first molecular-level view of the GP56-DPO,so,this complex provides the structural basis for the transcriptional regulation mechanism of GP56.Apart from analyzing the structure of GP56-DPO complex and comparing it with Vqm A-DPO,we also explored the function of GP56-DPO.Depending on the intracellular fluorescence experiment and dose-dependent experiment,we proved that DPO plays a decisive role for the transcriptional activity of GP56.Our results showed the following conclusions: 1)GP56,same as Vqm A,has ligand-dependent and ligand-independent two forms.2)The transcriptional activity of GP56 is deeply affected by binding DPO or not.Without binding with DPO,the transcriptional activity of GP56 is very low,only binding with DPO,the transcriptional activity of GP56 is up to highest.Generally,GP56 does not require DPO for folding or basal transcriptional activity,but does require DPO to drive maximal activity.When we found that DPO plays a decisive role for the transcriptional activity of GP56,we further explored how DPO affects the transcriptional activity of GP56.Firstly,the gel filtration chromatography and dynamic light scattering experiments show that GP56 exists in the form of dimer whether it binds DPO or not,it means that DPO can not affect the dimeric form of GP56,so,DPO does not affect the transcription activity of GP56 in this way.Secondly,we investigate whether GP56 has conformation change after binding with DPO,and explore whether this change affect the affinity of GP56 and the promoter DNA sequence,then,the affinity between GP56 and the promoter DNA finally affect the transcriptional activity of GP56.Our original intention is to get the crystal structure of GP56,we hope to analyze the conformational differences between GP56 and GP56-DPO by comparing their structures.Unfortunately,we were unable to obtain the crystal structure of GP56.As a last resort,we tried other methods,we did DNase Ⅰ foot printing experiment and EMSA experiment.Depending on the results of the above experiments,we identified the specific DNA sequence on the pqtip promoter which can be bound by GP56,and then,we test the affinity of GP56 and GP56-DPO with this specific DNA sequence through EMSA experiment.Contrary to our expectation,the experimental results show that the GP56 could bind to promoter DNA in the absence of DPO.To our surprise,the affinity between GP56 and promoter DNA can not be improved by binding DPO,so,how does DPO affect the transcriptional activity of GP56 is a question worth thinking about.We speculate that DPO enhance the stability of the protein.We did thermal shift assay to analysis the GP56’s stability.The experimental result shows that GP56 is thermally unstable,no matter it bind with DPO or not.Only in the case of binding DNA,GP56 can be thermally stable.In conclusion,GP56-DPO-DNA ternary complex is the most stable.After that,we did proteolytic digestion experiment.The experimental results suggested that the DPO did stabilize the GP56.Without binding with DPO,GP56 is not only easy to degrade,but also easy to precipitate.This result suggests that the stability of GP56 is likely related to its transcriptional activity.When GP56 binds with DPO,this complex is not easy to be digested by trypsin,which ensures the persistence of transcription.Depending on these experimental results,we propose such a hypothesis,both GP56 and GP56-DPO lack dense folding,so they are in a state of "OPEN",however,GP56 is in a state of "Inactive OPEN",GP56-DPO is in a state of "Active OPEN",this "Active OPEN" state is important for the transcriptional activity of GP56.Based on the crystal structure of GP56–DPO and biochemical results in this study,we confirmed that GP56,a transcription regulator protein encoded by vp882,can not only active the expression of Qtip by binding on the promoter region of phage qtip gene,then start the lysis process of phage to result in the death of Vibrio cholerae,but also can bind the promoter region of Vibrio cholerae vqm R gene,then active the signaling pathway to inhibit the formation of biofilms and the secretion of virulence factors of Vibrio cholerae,this indicates that GP56 has dual functions.The enhancement of bacterial resistance caused by the abuse of antibiotics has become a great challenge in clinical practice.Scientists are constantly looking for new treatments to deal with the antibiotic abuse.With the in-depth study of quorum sensing,scientists began to develop target antimicrobial therapy for the treatment of bacterial diseases.This antimicrobial therapy has many potential advantages: 1)Strong targeting and high specificity.2)The selection pressure is small,which can minimize the risk of bacterial resistance.3)It also has effects on strains which have antibiotic resistance.4)No harm to host cells.Taken together,the structural analysis and functional research of GP56 have important guiding significance for the treatment of cholera.The experimental results provide an important structural basis for the design of GP56 agonists.Phage protein GP56 has the dual functions of inhibiting the proliferation of Vibrio cholerae and promoting the lysis of Vibrio cholerae,which has become a major breakthrough in phage therapy.With the development of the study,phage therapy is likely to become a revolutionary antibacterial method.