Construction And Function Of Enterohemorrhagic Escherichia Coli O157:H7 Ppk-deleted Strain

1.Background and ObjectiveEnterohemorrhagic Escherichia coli 0157:H7(EHEC 0157:H7)is a very diverse species of bacteria found naturally in the intestinal tract of many animal species.A subset of E.coli is capable of causing enteric/diarrhoeal disease.A different subset cause extra-intestinal disease,including urinary tract infection(UTI),haemorrhagic eolitis(HC),sepsis and neonatal meningitis,which are classified as extra intestinal pathogenic E.coli.EHEC causes diseases by Stxl and Stx2 toxin encoded by stx gene,so this type of bacteria is also called shiga toxin-producing escherichia coli(Shiga-toxin producing E.coli,STEC).In1983,the primary outbreak of the haemorrhagic colitis caused by EHEC 0157:H7 happened in the USA.American scholar Riley isolated 0157:H7 firstly from the patients’ waste with severe hemorrhagic diarrhea caused by fast food and identified it as a new kind of diarrhea of E.coli.After this,sporadic cases and smal outbreaks caused by EHEC 0157:H7 continue to occur throughout the world.In 1999,there was an outbreak of EHEC 0157:H7 infection in Washington for food poisoning that involves 116 people.From 1982 to 2002,a total of 350 outbreaks were reported from 49 states,accounting for 8598 cases of EHEC 0157:H7 infection,including 1493(17.4%)hospitaliza-tions,354(4.1%)cases of HUS,and 40(0.5%)deaths.In 1996,from May to August,the Osaka 堺 city of Japan had a large outbreak of enterohaemorrhagic Escherichia coli(EHEC)0157:H7 infection among school-age children,that lasted up to 3 months and spreaded to more than 40 prefectures.About 9000 people were infected and 11 people dead.It created the record of highest incidence and aroused the attention all over the world.In 2006,more than 200 people in 25 states of the United States suffered from the food poisoning incident caused by eating contaminated fresh spinach,the culprit also being 0157:H7.From 1986,in our country,we isolated the 0157:H7 for the first time,and then,it had several outbreaks across the country which big one is happened in parts of places of Jiangsu and Northern Huaihe Area and the neighbouring parts of Anhui some places.Patients with more than 20 thousand people,177 people died,popular up to seven months,is considered the largest and most deaths and complex,langest in the world so far away.Currently,out breaks and spread of EHEC0157:H7 continue to occur,posing a great threat to human health and a global public health challenge.The pathogenicity of EHEC 0157:H7 mainly embodied in two aspects of adhesion and toxins.Adhesion mainly depends on its adhesion molecule-tight adhesion(Intimin),by eae gene encoding,settled in the gut,caused adhesion and erase(A/E)damage.Shiga toxin(shigatoxin,Stx),a kind of mainly virulence factors which determines the characteristics of EHEC and contains Stx1 and Stx2,has neurotoxic,cytotoxic,and intestinal toxicity of three active.Stx,on one hand directly kill the intestinal villus epithelial cells;On the other hand,caused by Stx intestinal epithelial damage and LPS,inflammatory cytokines can promote Stx across intestinal epithelial cells in blood circulation which leads to organs such as kidney damage.Research shows that the Stx is the main pathogenic factor resulting in HC and HUS.Stx can cause serious ultrastructural morphological changes of kidney cells and lead to apoptosis,and apoptosis can lead to HUS occurs.A large number of evidence suggests that,Stx toxin is the base of HC and HUS occur.Shiga toxin also has the function of platelet aggregation which damages endothelial cells which may be related to TTP.Stationary phase is the critical period for virulence gene expression associated with the invasion in E.coli and most invasive E.coli are stationary phase bacteria.Therefore polyP which closely related to the bacterial activity in the stationary phase and ppk which is the key synthesis enzymes genes caused people’s attention.PPK,the Polyphosphate Kinase,encoded by the ppk gene,is a homotetramer of subunits of 687 amino acids and each 80-kDa monomer contains four structural domains and one of the major enzymes to reversibly catalyze the synthesis of polyphosphate(polyP)from the terminal phosphate of ATP.PPK contains PPK1 and PPK2,and PPK1 exists only in part of microorganism,and there are many microorganisms not only PPK1,also PPK2.PPK2 has two significantly different characteristics with PPK1.One is can use polyP to synthetic GTP,and mainly synthetic GTP,the other one is that their selective to guanylic acid and adenosine:PPK2 has the same activity to use GTP or ATP synthesis polyP,but PPK1 can only use ATP to synthesis polyP;PPK2 can use polyP to syntheisi GTP or ATP,but the ability PPK1 using polyP synthesis ATP is equivalent to more than 30 times than synthesis GTP.In E.coli,there is only PPK1,so in this article PPK means to PPK1.Autophosphorylation is the first step for PPK1 to synthesis polyphosphate.The PPK1 active site is located in a tunnel that contains a unique ATP-binding pocket which reversibly transfers the y phosphate of ATP into the terminal of polyphosphate.PolyP is a polymer of hundreds of phosphate residues linked by high-energy phosphonhydride bonds and is found in every cell in nature:bacterial,fungal,plant and animal.PolyP has many biological functions,including:involved in energy metabolism,maintain the cell morphology,regulating insurance within the pH and osmotic pressure,and the formation of cell phospholipids membrane channel,etc.In E.coli,polyP is needed for the induction of rpoS.RpoS is the specificity of RNA polymerase sigma factor in stationary phase.It involved in the stabilization of more than 500 genes expression regulation,involving the virulence of pathogenic bacteria and oxidative stress,high permeability,thermal,near ultraviolet radiation,ethanol,acidic pH and other stresses adaptations regulation which is not yet clear.E.coli with a mutation in PPK,fails in stationary-phase adaptations for stringencies and stresses and fails to survive.The studies indicated that polyp has an essential regulatory role in E.coli in responses in the stationary-phase.Study shows that the polyP can affect the expression of stationary phase regulating factor RpoS through cAMP receptor proteins and participate in the regulation of virulence.The PPK gene deletion,polyP synthesis is reduced,and the expression of RpoS also declined obviously,and some gene expression related to RpoS regulation will decline.E.coli 0157:H7 have been found more than 30 years as pathogenic bacteria,we still not find a suitable treatment method.Although E.coli 0157:H7 is sensitive to antibiotics,but the antibiotics against bacteria will release shiga toxin,leads to the deterioration of the hemolytic urinary tract syndrome(HUS).The therapeutic effect of dynamic resistance is poor,because it can make the E.coli O157:H7 persistent exist and constantly express toxicity.Oral immunization antigens are easily destroyed by digestive juices during their passage through the gastrointestinal tract,and mucosal vaccine is not enough for the protection of the body.The scientists who study PPK1 structure suggest that the binding pocket could be a target for developing PPK1 inhibitors,and that new inhibitors could disrupt the PPK1 dimer interface because intact PPK1 is essential for activity.Once disrupted,PPK1 will lose activity,and thus affect the synthesis of polyphosphate and some related gene expression,further affect the biological function of E.coli and infection ability,so as to achieve the purpose of treatment of E.coli infection.In rats,mice and human genome sequence analysis,and with eukaryotes involves phosphate metabolism enzyme sequence blast ratio,higher eukaryotes had not been found ppk gene or the PPK homologous sequences in the body,so the ppk gene or PPK1 as targets of antibiotics may be a good E.coli 0157:H7 treatment.Although the PPK has been reported in other parts of the pathogen function,such as possible involvement of polyP and PPK in nucleotide metabolism,and applications of polyP as an ATP substitute and energy source,involvement of polyP in SOS regulated genes,affection of polyP to rpoS expression etc,but if has the same function in 0157 is still unknown.Concider the threat of 0157 to human health,it has a great significance to study pathogenic mechanism and drug treatment of 0157 in studying the role of PPK1.This study use of Red recombination system constructed EHEC 0157:H7 EDL933w ppk gene deletion strains(EHEC 0157:H7 EDL933w△ppk),at the same time compared their biological characteristics between wild strains and deleted strains.we compared the mRNA expression level of stabilization regulating gene rpoS,virulence gene stxl and stx2 in wild strains,compensation strains and deleted strains using real-time fluorescent quantitative PCR(real time RT-PCR),and preliminary explored the role of polyP and PPK in E.coli which laid a foundation for further research.2.Methods(1)Construction of enterohemorrhagic Escherichia coli 0157:H7ppk-deleted strains and compensation strainsAccording to the ppk gene sequence in GenBank(Accession NO NC 002655)and the DNA sequences of its upstream and downstream,designed and synthesized three pairs of primers:among them,H1-K1,H2-K2 contain homology arms primers,of which the 5’end is homologous with the ppk gene and the 3’end is homologous with the kanamycin resistant gene.PPK-P1,PPK-P2,Nl-F,N2-R were crossing identifying primer of ppk gene in EHEC 0157:H7;Kana-F,Kana-R were internal identifying primer within the plasmid pKD4 of kana resistance gene.Kanamycin resistance gene was amplified using Hl-Kl,H2-K2 as primers and pKD4 plasmid as a template,then EHEC 0157:H7 EDL933w competent strains with plasmid pKD46 were prepared and transformed with the amplification products using electroporation.Using Red homologous recombination system,the ppk gene was replaced by kanamycin resistance gene through pKD46-mediated Red recombination system.Screened positive strains by kanamycin plates,the recombinant strain was confirmed by PCR and sequencing.Using pMD19T plasmid as vectors,ppk external primers P1,P2 as primers amplified gene fragments which covered,to gel extraction and ligate with the vectors,transfer it into ppk gene deletion strains and screened positive ampicillin-resistant strains through flat,through PCR and sequencing confirmed it.(2)Biological characteristics compare of wild and mutant strainsGram staining:take a ring of wild strain and mutant strain fungus liquid and coat a even thin film on separate glass slides,then dry,fix and stain it following the Gram staining procedure:primary dye-mordant-destain-restain,using 100 X objective lens of the optical microscope to observe.Bacterial growth curve:single colonies were picked and cultureed overnight statically with fresh LB medium,after the first overnight growth in LB,subcultured the bacteria into fresh LB medium by 1:100 culturing at 190r/min in 37 ℃.Then to determinate OD600 value at Oh,2h,4h,6h,8h,10h,12h,14h,drawing growth curve with time as the abscissa and logarithmic OD600 as ordinate.Bacterial adhesion assay:after digested the Lovo cells in Asian fusion state using trypsin,adjusted the concentration to 1.5 × 105cells/well,inoculated to 6-well cell culture plate within treated coverslip culturing in 37 ℃,5%CO2 incubator for 24h untill growning to monolayers.Added the overnight cultured bacteria suspension of EHEC 0157:H7 EDL933w wild and mutant strains into the cells with 100:1 infection times and repeated three holes,while the orifice plate without bacteria cells as a negative control culturing in 37 ℃,5%CO2 incubator and incubated 2h.The cells were washed 3 times by 1×PBS and fixed 20min by 4%paraformaldehyde.Gimsa stained for 15min,washed 2-3 times,observed them using inverted microscope after dried.(3)The effects PPK/polyP have on the expression levels of the stabilization regulating gene rpoS and important virulence genes stxl and stx2 of hemorrhagic Escherichia coli O157:H7 EDL933 strains.To test whether the ppk gene knockout have an impact on the stability of the regulation gene rpoS,virulence gene stx1 and stx2 expression,we compared the relative gene expression levels of rpoS,stx1,stx2 of wild strains,mutant strains and compensative strains by real-time quantitative RT-PCR method which mainly uses the sybrgreen fluorescent dye method.3.Results(1)Constructed EHECO157:H7 ppk gene deletion strains and compension strainsKana resistance fragments amplified using primers H1-K1,H2-K2,which is approximately 1577bp,was electroporated into EHEC O157:H7 EDL933w competent strains with plasmid pKD46.Positive strains were screened by kanamycin plates and identified by ppk internal primers.430bp fragment can be seen in wild strains,no fragments were found in mutant strains.The EHEC O157:H7 ppk gene(2067bp)was completely deleted,confirmed by sequencing.(2)Biological characteristics comparison of wild strains and mutant strainsIn Gram staining,the wild strains and mutant strains are gram-negative corynebacterium parvum.According to the growth curve plotted by the OD600 values,the trend of the growth curve of wild strains and mutant strains are basically the same.By using Giemsa staining to relatively observe the adhesion of EHEC 0157:H7 strains to Lovo cells,we can see that the cell adhesion of wild strains is less than that of mutant strains.(3)After standardizing by reference gene GAPDH,analyzing to the results of fluorescence quantitative PCR(relative quantification)and comparing to the wild strains and compension strains,the 2-△△Ct of rpoS,stx1 and stx2 genes of ppk gene mutant strains is obviously lower than them.4.Conclusion(1)Constructed ppk gene-deleted strains successfully by using Red recombinati-on system,and constructed compension strains on the basis of ppk gene-deleted strains which laid the foundation for the subsequent experiments.(2)Ppk gene deletion has no influence on the morphology and growth rhythm of EHEC 0157:H7.Bacterial adhesion results showed that it’s different of the ability to the cell adhesion of mutant strains and wild strains and it’s weaker of ppk gene deletion strains which suggesting that ppk gene is related to the adhesion of bacterias.(3)Compared the relative gene expression levels of rpoS,stxl,stx2 by fluorescence quantitative PCR(relative quantification)of wild strains,mutant strains and compensative strains which showed that it led to the reduction of polyP,then the expression levels of regulating gene rpoS and important virulence genes stxl and stx2 of wild strains,mutant strains and compensative strains.