Effects Of Prophage Fragment Phiv205-3 On Fitness Of Avian Pathogenic Escherichia Coli DE205B

Escherichia coli(E.coli)is a very common prokaryote and pathogen in nature infecting humans and animals.E.coli can be divided into three categories according to clinical pathogenicity and genetic characteristics:commensal group,diarrheal group,and extratestinal group.Among them,Extraintestinal Pathogenic E.coli(ExPEC)can cause infections in humans and farm animals,which usually divided into Avian Pathogenic E.coli(APEC),Uropathogenic E.coli(UPEC,(causing urethral and urinary tract infections UTI),and Newborn meningitic E.coli(NMEC).Local and systemic infections caused by APEC strains in poultry are commonly referred to as avian pathogenic E.coli disease.APEC pathogens cause huge economic losses to the poultry industry each year and is a potential threat to mammals such as humans.Therefore,the prevention and control of APEC is of great significance to the aquaculture industry and public health.Bacteriophages and bacteria are widespread in the world and the number of phages is ten times greater than bacteria.Bacteriophages can be divided into three types:lytic phage,lysogenic phage(prophage)and pseudolysogen.Most bacteria harbore prophage genes in their genomes.The prophages often encode some foreign genes which could reproduce with host bacteria genomo to the next generation,and causing the host bacteria to adapt to the external environment therefor.Our previous study found that a prophage residue existed in the genome of APEC strain DE205B,named as phiv205-3.This study performed a complete and single gene deletion of phiv205-3 to study the effect of this prophage fragment on the environmental adaptability of bacteria.1.Effects of prophage fragment phiv205-3 on fitness and virulence of APEC strain DE205BA prophage fragment deficient strain DE205B?phiv205-3 was constructed by phage? Red recombination technology.The growth curve measurement showed that the growth of the deletion mutant had no significant difference compared with the wild strain.Further tests of the tolerance of wild and deleted strains to acid,alkali,hyperosmotic and oxidative stress showed that compared with wild strains,the survival rate of the mutant strains decreased by 20.6%(P<0.05),20.3%(P<0.05),50.6%(P<0.01)and 33%(P<0.05)in acidic(pH 4.0),alkaline(pH 10.0),osmotic(2.4 M NaCl)and oxidative stress(10 mM H2O2)environments,respectively,indicating that the deletion of the prophage phiv205-3 fragment caused the host bacteria's deficiency in tolerance to the external environment.The chicken pathogenicity test was performed in order to test whether the loss of phiv205-3 would affect the virulence of the host bacteria.The results showed that compared with the wild strain,the LD50 of the deletion mutants were not significantly different,but the colonization ability was significantly reduced in the brain of chicken.The results of cell adhesion and invasion experiments showed that the the copacities of adhesion and invasion to DF-1 cells of the mutant strain were significantly reduced compared to the wild strain,indicating that the loss of phiv205-3 affected the adhesion and invasion to cells and colonization in animals,but did not have a significant impact on the virulence of bacteria.2.Gene nrdF contributes to the tolerance to oxidative stress in APEC strain DE205BThe analysis of genes sequence of DE205B revealed that phiv205-3 encoded four oxidative stress-related genes,nrdE,nrdF,nrdH,and nrdI.In order to investigate whether these genes affected the host's ability to resist oxidative stress,the expression levels of these genes were detected by fluorescent quantitative PCR.The results showed that the transcription level of nrdF was significantly increased under oxidative stress(P<0.05).The phage ? Red recombination method was used to construct gene deletion strains DE205B ?nrdE,DE205B ? nrdF,DE205B ? nrdH,and DE205B ? nrdI.We tested the oxidative stress tolerance of these deletion strains,the results showed that compared with the wild strain,the survival rate of the deletion strain DE205B ? nrdF in oxidative stress environment(10 mM H2O2)decreased by 21%(P<0.05),but the survival rates of the deletion strains DE205B ? nrdE,DE205B ? nrdH and DE205B ? nrdI under oxidative stress did not change significantly.It is suggested that the nrdF gene encoded by the prophage fragment played a rolein the resistance to oxidative stresses of APEC strain DE205B.3.Gene proV contributes to the tolerance to hyperosmotic stresse in APEC strain DE205BThe analysis of genes sequence of DE205B also showed that phiv205-3 encoded two hyperosmotic related genes,pro V and pro W.In order to detect which genes play a role in the host's antihyperosmotic ability,the transcription levels of these genes in a hyperosmotic environment were detected by fluorescent quantitative PCR,and the results showed that the transcription levels of proV and proW increased by 51%(P<0.01)and 39%(P<0.05)repectively in a hyperosmotic environment.The deletion strains DE205B ?proW and DE205B ?proW were further constructed.The results showed that compared with the wild strain,the growth of DE205B ? proV and DE205B ? pro W was significantly inhibited under high osmotic pressure,and growth inhibition of wild strain was effectively alleviated after adding with betaine while growth inhibition of the deletion strains did not change significantly with betaine,suggesting that proV and proW help to increase host bacteria's resistant to hyperosmotic stress.The cytoplasm of the host cells is an adverse environment that exposes bacteria to hyperosmotic stress.Therefore,it is speculated that the genes pro V and proW will affect the intracellular growth of DE205B.The results showed that,compared to the wild strain,the copacity of adhesion and invasion to DF-1 cells of the mutant strain DE205B ? pro V was significantly reduced,suggesting that pro V also enhance the adhesion and invasion ability of DE205B to host cells.