Screening Of Binary Toxin Aptamers Of Clostridium Difficile And Bioinformatics Analysis Of Multidrug-resistant Stains

Objective A new laboratory diagnosis method for the accurate diagnosis of highly virulent C.difficile infections was provided by constructing an 80bp ssDNA random library and using SELEX technology to screen aptamers with high affinity for binary toxins A chain and B chain of C.difficile.Two strains of multi-drug resistant C.difficile genomes were performed high-throughput sequencing using the Illumina and Pac Bio technology platform to analyze the virulence factors and composition forms,drug resistance genes,and mobile genetic elements,which can understand the resistance mechanisms and pathogenic mechanisms in the multi-drug resistant strains CDT4 and CD161.MethodFirst:Construction of pET-28b-CDTa-_C and pET-28b-CDTb-_N prokaryotic expression vectors and expression and purification of recombinant protein binary toxin A and B chains（1）The carboxy-terminal（C129-1392）of the A chain and the amino-terminal（N60-765）of the B-chain were amplified by PCR,respectively.The target genes were linked to the p MD^TM19-T vector and transformed into E.coli DH5α,respectively.（2）The p MD^TM19-T-CDTa-_C and p MD^TM19-T-CDTb-_N recombinant plasmids were extracted and double-enzyme digested after sequencing.The CDTa-_C and CDTb-_Ngene fragments were recovered by agarose gel electrophoresis to link with pET-28b.The pET-28b-28b-CDTa-_C and pET-28b-CDTa-_N prokaryotic expression vectors were transformed into E.coli BL21（DE3）.（3）The target proteins were expresed in the desired expression conditions,with the final concentrations of inducer IPTG were 0.4,0.6,0.8,1.0,and 1.2 m M,the induction time was 4,6,8 and 10 h,the induction temperature was at 25,30,and 37°C,respectively,when the culturing bacteria were cultured until OD₆₀₀ was about 0.6.The target proteins were purified using the AKTA protein purification device.Finally,the concentration of the purified protein was determined using a BCA protein quantification kit,and the specify of target protein binary toxin A and B chains were also tested in Western Blotting.Second:Construction of random library and screening of aptamers of recombinant protein binary toxins A and B chains.（1）A random ssDNA library with the length of 80 bp was designed and synthesized.The amount of synthesis was 2000 pmol,and the library capacity of ssDNA reached10¹⁵,which reaches the required screening capacity.（2）The recombinant protein binary toxin A and B chains were used as target molecules to coat on the plate wells,blank wells were set up,and high-affinity ssDNA libraries were enriched by the procedure of binding-dissociation-amplification.（3）The ssDNA library was amplified in a stable binding rate,cloned and sequenced it,to analyze the secondary structure and predict free energy of the ssDNA obtained by sequencing,and found the optimal ssDNA of the A and B chains of the recombinant protein binary toxin.（4）The affinity constants of the enriched aptamers with the recombinant protein were determined by a non-competitive ELISA.Third:Genome bioinformatics analysis of Clostridium difficile（1）Two strains of Clostridium difficile were determination to the minimum inhibitory concentration of metronidazole,vancomycin,ampicillin,clindamycin,meropenem,amoxicillin/clavulanic potassium,piperacillin/tazobactam,penicillin,pipericin,and piperacine by Etest,which was recommended by CLSI in 2017.（2）Two multidrug resistant Clostridium difficile isolates were cultivated in Brucelli broth.The Clostridium difficile isolates were collected for high-throughput de novo sequencing of bacterial genome.（3）A complete contig genome was assembled,and Glimmer software was used to predict the gene result of the assembly after filtering the sequencing raw data.（4）The whole genome was analyzed by prediction and repetitive sequence analysis using the r RNAmmer software and Tandem Repeats Finder software respectively.（5）The whole genome prephage and CRISPR system respectively were analyzed using the PHAST software and CRISPR Finder software.（6）the genome function was analyzed using BLAST software to combine with the characteristics of each database.（the database is GO,KEGG,COG,Swiss-Prot,Trembl,NR,Egg NOG,ARDB,PHI,P450,CAZy,VFDB,T3SS,Transport DB,CARD and db CAN）（7）The comparative genomic analysis of CDT4 and CD161 were compared with C.botulinum.ATCC.3502,C.perfringens.ATCC.13124,.septicum.P1044,C.tetani.E88,P.sordellii.CBA7122,respectively.Results:（1）The 976 bp of the carboxyl terminus of the A chain and the 1629 bp of amino terminus of the B chain in the C.difficile binary Toxin were successfully amplified,respectively,and were ligated to the p MD^TM19-T vector,and no gene mutations were identified by sequencing.（2）After digesting the recombinant plasmids,CDTa-_C and CDTb-_N gene fragments were recovered,and pET-28b-CDTa-_C and pET-28b-CDTb-_N prokaryotic expression vectors were successfully constructed.（3）After induced by IPTG,the target proteins with protein molecular weights of approximately 48.9 KDa and 33.5 KDa were appeared in the supernatants by the SDS-PAGE electrophoresis,which were in agreement with the expected values.（4）The recombinant protein binary toxin A and B chains were purified by the AKTA protein purification device,with a concentration of 29.67μg/m Land 216.33μg/m L,respectively.The recombinant protein binary toxin A chain and B chains were confirmed by Western blot.（5）Using recombinant protein binary toxin A and B chains as targets,SELEX technology was used for aptamer screening.As the number of screening rounds increased,The binding rate of random ssDNA library with recombinant protein binary toxin A chain and B chain respectively were increased from 3.2%to 42.3%and from2.5%to 41.7%,respectively,and the increase rate of the binding rate in the 9th,10th,and 11th rounds was not significant,indicating that the binding rate was approaching the plateau period and the screening was stopped.（6）The 11th round of screened libraries were selected for cloning and sequencing,and performed secondary structure analysis and free energy prediction.The free energies of Apt-A53 and Apt-B31 are-3.8 kcal/mol and-2.6 kcal/mol,respectively.Two aptamers,Apt-A53 and Apt-B31,were selected to detect their affinity constants.（7）The affinity constant of the recombinant protein binary toxin A chain with the Apt-A53 was determined by non-competitive ELISA.The affinity constant value is3.75×10⁶ M^-1;in the same method,the affinity constant value of recombinant protein binary toxin B chain with Apt-B31 is 2.22 x 10⁶ M^-1.（8）The Clostridium difficile CDT4 and CD161 are resistant to clindamycin,moxifloxacin,penicillin,while they were sensitive to other antibiotis such as metronidazole,vancomycin,ampicillin,meropenem,amoxicillin/clavulanate,and piperacillin/tazobactam,piperacillin,which were multi-resistant strains.（9）C.difficile CDT4 was sequenced in Pac Bio platform in depth of 273X,a gene size of 4,289,548 bp,a gene number of 3887,a number of nc RNAs of 145,a repeat number of 1097,an annotation of 99.94%,and the GC content of CDT4 is 28.88%.The CDT4 genome consists of a chromosomal genome and a plasmid genome,all of which are circular structures.（10）C.difficile CD161 was sequenced in Pac Bio platform in depth of 326X,a gene size of 4,474,333 bp,a gene number of 4137,an nc RNA quantity of 146,a repeat number of 1122,a CRISPR quantity of 21,and a prophage The number was 9,the annotation of 99.95%,and the GC content of CD161 is 28.88%.The CD161 genome consists of a chromosomal genome and two plasmid genomes,all of which are circular structures.（11）The genome of C.difficile CDT4 was determined large about resistance-related gene,as followed:AAC（6’）-Ie-APH（2’’）-Ia,ANT（6）-Ib,gyr A（T82I,ACT-ATT）,gyr B（S366A,TCA-GCA）,Llm A,qac H,tet M,tet（D）,tet B（46）,cata16,mdtg,cde A,baca.The genome of C.difficile CD161 have identified a large number of resistance-related genes:AAC（6’）-Ie-APH（2’’）-I,ANT（6）-Ib,gyr A（T82I,ACT-ATT）,gyr B（S366A,TCA-GCA）,erm B,Llm A,qac H,tet M,tet（D）,tet B（46）,cata16,mdtg,cde A,baca.（12）The multidrug-resistant CDT4 and CD161 strains carry multiple virulence factors such as toxin A,toxin B,hemolysin,flagella,and IV pilus.（13）Prophages are found on the genome of CD161 with 3 intact prophages,2incomplete prophage and 1 suspicious prophage on the chromosome.There is one intact prophage in each of plasmid 1 and plasmid 2.（14）The CRISPR system was found on the genome of CD161.There are 18 CRISPR sequences on the chromosome,12 CRISPR sequences have been confirmed,and 6CRISPR sequences need to be confirmed by experiments.Two confirmed CRISPR sequences were found on plasmid1,and one found on plasmid2 required experimental confirmation of the CRISPR sequence.（15）Comparative genomic analysis of toxin-producing multi-drug resistant CDT4 and CD161 strains and five other important pathogenic clostridial strains revealed that the relationship of CDT4 and CD161 strains are the closest to the P.sordellii.CBA7122,the farthest to the C.tetani.E88 in the evolutionary.Conclusion（1）The pET-28b-CDTa-_C and pET-28b-CDTb-_N prokaryotic expression vectors were successfully constructed and induced to obtain purified recombinant protein binary toxin A and B chains.The high-affinity aptamers of the A and B chains of the binary toxin was successfully obtained using SELEX technology.（2）The multiple resistant genes and drug target site mutations in C.difficile CDT4and CD161 play an important role in resistance to multiple antibiotics.The multidrug resistance efflux pump genes also promote drug resistance.The transposase,integrase and other mobile genetic elements can make bacterial resistance spread between species,so that the recipient bacteria may become resistant bacteria.（3）The toxigenic CDT4 and CD161 strains were detected multiple virulence factors such as toxin A,toxin B,hemolysin and IV fimbriae,which play an important role in intestinal adhesion,invasion,lysis of cells and so on in C.difficile,enhancing the compressive and pathogenicity of C.difficile,resulting in the occurrence of C.difficile infection in the host.（4）CD161 was found to carry the CRISPR system and prophage.The cas-specific cas protein of the CRISPR system can recognize,bind,and cleave foreign nucleic acid sequences,thus effectively resisting the invasion of exogenous nucleic acids such as bacteriophage and plasmids.The generation of CD161 CRISPR system may be the immune system passively evolved by the invasion of prophage.（5）Comparative genomic analysis of toxin-producing multi-drug resistant CDT4 and CD161 strains and five other important pathogenic clostridial strains revealed that the relationship of CDT4 and CD161 strains are the closest to the P.sordellii.CBA7122,the farthest to the C.tetani.E88 in the evolutionary.