| Avian Pathogenic E.coli(APEC)infection is common in poultry industry worldwide,leading to great economic losses.The possible route of infection is the inhalation of contaminated dust or aerosols.Pericarditis,perihepatitis,salpingitis,and peritonitis are observed in infected chickens.In addition,evidence shows that APEC strains are similar to UPEC and NMEC,indicating that APEC has a zoonotic capability.Most bacterial genomes contain prophage sequence,and prophage can constitute up to 20%of the DNA.It has been reported that lysogenic bacteria can release progeny phage particles through spontaneous induction,thus improving the pathogenic or competitive ability of lysogenic bacteria.While little is known about the influence of prophages in APEC strains.In this study,we screened the APEC strains that could spontaneously release phage particles,and explored the biological effects of releasing phage on lysogenic bacteria themselves.As prophage was reported to encode protein contributing to bacterial virulence,competitiveness and adaptability,we also detected the funtion of prophage in APEC strain DE142.The mechanism of how prophage phiv 142-3 affected colonization ability of DE142 was explained in the study.And through RNA-seq,several genes in phiv 142-3 related to survival ability in serum were indentified.1.The screening for APEC strains with spontaneous prophage induction and the study of biological characteristics of the released phage.With the application of genome sequencing technology,many prophage sequences are found in bacterial genomes,which can constitute up to 20%of the bacterial genome.It has been reproted that prophages can affect the virulence and competitiveness of bacteria,but the role of prophages in APEC has not been reported in detail.It is found that the life cycle of prophages can be interconverted,and it has some biological effects on lysogenic bacteria when the prophage turns into lytic life cycle.We found that prophages could be spontaneously induced from APEC strain DE456 and DE458.The spontaneous induction rate were tested under different conditions.The results showed that spontaneous induction rate of the prophages increased to 10-100 times at 42? or incubated with DF-1 cells compared with that in LB media.The competitive assays showed that the DE456 and DE458 were in a competitive advantage position when growing with other strains,suggesting that spontaneous induction of phage in the strains may play a role in the competition.Meanwhile,the released phage456 and phage458 were isolated and purified,and their biological characteristics were studied.2.The effects of prophage phiv142-3 on the colonization ablitiy and resistance ability to environmental stresses of APEC DE142Some prophage could turn into lytic life cycle and released phage particles after being induced,while a number of prophage in lysogenic bacteria couldn't.Though DE142 couldn't released phage particles,previous studies showed that the prophages in DE142 may encode proteins and affect the virulence and adaptability of bacteria.Thus,in this study,the genome of APEC strain DE142 was sequenced,which was predicted to contain 7 prophages,named as phiv 142-1 to phiv 142-7,respectively,by online software Phast.The role of prophage in APEC was detected.We found the prophage deletion mutant DE142?phiv 142-3 colonies on plates were much smaller than those of the wild-type,but the mutant was elongated than wild-type under microscopy.The mutant showed reduced adherence to DF-1 cells(87.4%reduction)and decreased resistance to chicken serum compared to that of wild-type(P<0.001).The mutant demonstrated 95.6%,71.6%,and 99.6%reduced survival rate under acid,alkaline,and oxidative stress,respectively.In vitro competition assays showed that the cell number of the mutant was about one-tenth of the wild-type(competitive index(CI)value,0.12).In vivo,the mutant showed significantly decreased colonization of chicken tissues compared with that of wild-type.The results showed that phiv 142-3 helps DE142 to cope with adverse environments and aids bacterial colonization.3.The molecular mechanism of prophage phiv142-3 contributes to the adhesion and colonization ability of APEC strain DE142In order to further investigate the mechanism of prophage phiv 142-3 enhances bacterial colonization ability,the phiv 142-3 sequence was divided into 13 sections and transferred into DE142?phiv 142-3.The adhesion ability of DE142?phiv142-3 was increased by the plasmid carrying orf20-orf24.Next,we knocked out each orf in the fragment and assessed the adhesion ability of each mutant.The adhesion assays showed that orf20 and orf24 had a significant effect on the bacterial adhesion and colonization abilities.As the adhesion ability of DE142?orf20 decreased most(85.2%reduction)among 5 orf KO,orf20 was selected as the object of subsequent study.Avian infection assays showed that bacterial loads in lungs and hearts of chickens challenged with the mutant are decreased to 7%and 8.3%compared with those challenged with the WT.The number of flagella and I fimbriae of the mutant were decreased and the mutant exhibited filamentation.The mRNA leves of flhD,fliA,fliZ,flgM,fliC and flgE in DE142?orf20 showed no significant defference to those in WT.Meanwhile there was no obvious difference in the expression levels of FliC between DE142?orf20 and WT by Western-blot.In the view of the fact that flagella of DE142?orf20 was decreased and the mRNA level of nine genes associated with the exportation of flagella were down-regulated 2-4 fold in DE142?orf20,it revealed that orf20 in prophage phiv 142-3 played a role in flagellar exportation.Compared with the WT,the transcription level of the cell-division-associated genes minC and minD was increased 1.4-fold and 2.5-fold in mutant DE142?orf20,the expression levels of I-fimbriae-related fimA and fimH was decreased 3-fold and 2.1-fold.The adherence inhibition experiments showed that protein ORF20 had no adhesion ability to DF-1 cells.Thus,our study revealed that orf20 in prophage phiv 142-3 played a role in flagellar exporting,maintenanced of normal cell morphology,influenced I fimbriae synthesis,and attributed to the adhesion and colonization ability of APEC.4.Transcriptome analysis of DE142 and identification of genes in phiv142-3 related to serum resistance.The survival ability in chicken serum is an important pathogenic factor for APEC.In order to further detected the role of prophage in DE142,the transcriptome was used to analysis the differentially expressed genes of DE142 between cultured in chicken serum and in LB.When cultured DE142 in chicken serum,RNA-seq showed that the number of up-regulated genes in prophage was far more than that of down-regulated genes compared with in LB,indicating that prophage in DE142 may play an role in the bacteria survival abilities in chicken serum.More than 31%(16/52)genes in phivl42-3 were significantly up-regulated,meanwhile phiv 142-3 was attributed to the survival abilities in chicken serum according to our previous research.So we chose orf6,orf1 0 and orf24 which were up-regulated most in phiv 142-3 for further study.We constructed the deletion mutant DE142?orf6?DE142Aorf10 and DE142?orf24,and found that the survival abilities in chicken serum of DE142?orf10 and DE142?orf24 were decreased by 37%and 46%in vitro,respectively.In vivo,the colonization abilities of DE142?orf10 and DE142?orf24 were decreased 10-20 folds compared to that of wild-type.It revealed that orf10 and orf24 might play an role in resistance to chicken serum. |
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