Study On Monitoring And Killing Pathogenic Bacteria By Engineering Probiotics

Natural probiotics maintain colony balance by regulating intestinal flora,thus beneficial to the health of the host.With the development of synthetic biology,the artificial design of gene circuits with different functions in the host of probiotics to construct engineering probiotics for the diagnosis and treatment of diseases has become a research focus in recent years.As the next generation of active therapeutics,engineered probiotics modified in a specific way can play a specific physiological role for specific diseases.For example,the homeostasis of the intestinal microenvironment,metabolic disease detection,and tumor-targeted therapy.In this paper,Lactobacillus plantarum WCFS I and Escherichia coli Nissle1917 were used as the engineering probiotic chassis.Construction of whole-cell biosensors responding to Staphylococcus aureus,Pseudomonas aeruginosa,and Yersinia enterocolitica;To realize the monitoring of intestinal pathogenic bacteria by engineering probiotics;The whole cell biosensor was used to control the killing system to realize the killing of intestinal pathogenic bacteria by engineering probiotics.1.Construction of engineered L.plantarum WCFS I for monitoring and treatment of S.aureus infectionS.aureus is a common gram-positive pathogenic bacterium that utilizes its unique Agr quorum sensing system(AgrQS system)to sense the population density of the community.The AgrQS system secretes autoinducing peptides(AIPs)as signal molecules for population sensing.In this chapter of the study,we first analyzed the mechanism of S.aureus secretion of AIP,overexpressed the ligand-binding protein AgrC and transcriptional activator AgrA that recognizes AIP,and used the naturally regulated promoter P3 of AgrA to initiate the expression of reporter genes.We constructed a whole-cell biosensor based on AIP production and recognition in L.plantarum WCFS I,and ultimately designed the biosensor for sensing and killing S.aureus.To validate the effectiveness of the whole-cell biosensor that we constructed,we co-expressed the two-component system AgrC/AgrA and the regulated promoter P3(containing an Agr operator)induced by AIP from S.aureus in the sensing module on the vector.We controlled the expression of the green fluorescent protein gene(gfp)using the P3 promoter,which was activated by the AIP signal.When the AgrC receptor protein in the whole-cell biosensor bound to the external AIP,it promoted the phosphorylation of the AgrA transcription factor,which could activate the expression of the gfp gene controlled by the P3 promoter.In order to improve the output fluorescence intensity and response range,we modified the P3 promoter to optimize the whole-cell biosensor we constructed.We replaced the-10 and-35 regions of the P3 promoter with those of another strong promoter,P11.The optimized whole-cell biosensor had a maximum fluorescence intensity output approximately 3.6 times higher than the unmodified version.When the concentration of AIP added was 100nM,the maximum fluorescence output of the biosensor was 400a.u,which confirmed that the sensing module could detect external AIP at the nanomolar level and achieve engineering monitoring of S.aureus in L.plantarumWCFS I.In the killing module,we used the whole-cell biosensor to control the expression of the lysostaphin gene(lss)which encodes a bacteriolytic enzyme,which could effectively inhibit S.aureus growth when induced by AIP.Through S.aureus growth dynamics detection and observation of inhibition zones,we found that the induced lysostaphin produced by engineered L.plantarumWCFS I had the most significant antibacterial effect against S.aureus when the concentration ofA IP added was 10μM,achieving the killing of S.aureus.The engineered L.plantarumWCFS I constructed in this study can detect AIP at the nanomolar level and kill S.aureus,laying the foundation for the development of biosensors for monitoring intestinal pathogenic bacteria and high-throughput drug screening in the future.At the same time,it provides a new treatment strategy for the prevention and treatment of S.aureus diseases.2.Construct engineering probiotic Escherichia coli Nissle1917 for monitoring and treating intestinal multibacterial infectionP.aeruginosa and Y.enterocolitica are gram-negative pathogenic bacteria that can cause intestinal diseases.Both rely on quorum sensing systems to regulate their population density.P.aeruginosa uses the Las(LasI/R type)QS system,which is highly sensitive to the 30C12HSL signal molecule,while Y.enterocolitica uses the Esa(EsaI/R type)QS system,which is highly sensitive to the 3OC6HSL signal molecule.Based on these two endogenous QS systems,we overexpressed the receptor proteins LasR and EsaR in vitro and constructed corresponding whole-cell biosensors in E.coli Nissle 1917 to validate their functions.We used the Plas promoter,regulated by 3OC12HSL,to control the expression of the blue fluorescent protein gene(bfp)and the Pesa promoter,regulated by 3OC6HSL,to control the expression of the red fluorescent protein gene(mCherry).We constructed a dual signal molecule "Or" logic gate to separately monitor P.aeruginosa and Y.enterocolitica.Based on this,we designed a concise gene circuit "AND" logic gate based on dual signal inputs,which can simultaneously detect these two bacteria.We used a constitutive promoter to initiate the LasR and EsaR genes,and designed a synthetic promoter with double QS signal inputs,placing the LasR DNA binding site(DBS)upstream of the synthetic promoter35 region,and placing the EsaR DBS between the-35 and-10 regions of the promoter.This resulted in a synthetic promoter(Pl&e)that can be simultaneously regulated by the 3OC12HSL and 3OC6HSL signals,controlling the expression of the gfp gene and allowing for characterization of the dual signal "AND" gate,to achieve simultaneous detection of P.aeruginosa and Y.enterocolitica.Then,we optimized the whole-cell gene cascade pathway responding to two signal molecules we constructed by using global transcriptional regulatory factor cAmp receptor protein(Crp),and increased the expression level of fluorescent protein.When exogenous 100 nM 3OC12HSL and 3OC6HSL were simultaneously added,gfp produced the maximum fluorescent output.Compared with before optimization,the response range of the dual-signal system was increased by about 10 times.Afterwards,we verified the directed chemotaxis ability of engineered E.coli Nissle1917 by knocking out the flagellar protein gene CheZ and the motor protein gene motA,and then regulating the expression of motA and CheZ genes through the signal molecule-controlled whole-cell gene cascade pathway to make E.coli Nissle1917 move in a directed chemotaxis manner in the concentration gradient of signal molecules 3OC12HSL and 3OC6HSL,and finally enriched around the target pathogenic bacteria.Finally,by fusing the antimicrobial peptide gene McsS into the artificially constructed whole-cell gene cascade pathway,engineered E.coli Nissle 1917 can be co-cultured with two pathogenic bacteria,and it was found that the growth of pathogenic bacteria was inhibited,thereby achieving the killing of target pathogenic bacteria.In this study,we aimed to construct an engineered probiotic E.coli Nissle1917 that can detect two pathogenic bacteria P.aeruginosa and Y.enterocolitica,providing a new approach for the future clinical treatment of infections caused by these or other intestinal pathogenic bacteria.