Study Of The Regulation Mechanism Of Pts? In Avain Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli?APEC?is one of the most common pathogens in poultry industry,which infected with chickens,ducks,turkeys and other birds,and caused severe respiratory and systemic infections in poultry.It also resulted in significant losses of poultry industry.APEC serious drug resistance and complex serotypes,whcih seriously restrict the prevention of the disease.Quorum sensing is mediated by small diffusible molecules termed autoinducers that are synthesized intracellularly to coordinate group behavior.The LuxS/AI-2 quorum sensing is widely found in Gram-positive and negative bacteria,which regulates many of the physiological functions of bacteria through the small molecule autoinducers-2?AI-2?.The signal molecule AI-2 is transported from the extracellular to the intracellular with AI-2 receptors or associated transporter.The previous studies have shown that Escherichia coli MG1655 can internalized AI-2 through lsrB gene.However,avian pathogenic Escherichia coli DE17?O2 serotype?is different from E.coli MG1655,which can also internalized AI-2,but no lsrB-like gene.It is presumed that DE17 may have other genes that are different from the lsrB gene involved in APEC's internalization of AI-2.The present study selected the ptsI?phosphoenolpyruvate-protein phosphotransferase?gene in the phosphoenolpyruvate phosphate transfer system?PTS?and the 1633?ribose ABC transporter permease?gene in the ABC operon by biological information analysis,which may involve in AI-2 internalization.In this study,the effects on AI-2 internalization,APEC pathogenicity and biofilm formation of ptsI and1633 was performed for the prevention and control of avian pathogenic Escherichia coli from the perspective of quorum sensing.1.Construction pts I mutant of APEC and analysis of the impact on AI-2 internalizationThe mutant strains DE17?ptsI and DE17?1633 were constructed using the lambda Red recombinase method.The AI-2 internalization of DE17,DE17?pts I and DE17?1633 was detected by reporter strain BB170,respectively.The results showed that the AI-2 of DE17?ptsI was internalized 4hours earlier than wild strain DE17,while DE17?1633 was no different compared with wild strain DE17.In order to verify whether Pts I has AI-2 binding activity,the pET28a-PtsI recombinant expression vector was constructed,and the Pts I recombinant protein?63.5 KD?was expressed in E.coli BL21?luxS?DE3?.The AI-2 binding and release experiment was performed with recombinant protein PtsI?final concentration of 1 mg/ml?incubation with AI-2?final concentration of 33?m?at 37?for 1hour.The results showed that the Pts I could not bind AI-2.This study suggests that the ptsI gene in the PTS system may be involved in the internalization of AI-2,but has no AI-2 binding activity.2.The ptsI gene effects on the pathogenicity of avian pathogenic Escherichia coliThe biological characteristics of DE17?ptsI and DE17 showed that the deletion of ptsI gene did not effect its growth characteristics.The LD₅₀ of the wild strain DE17 was 1.24×10³ CFU and the DE17?pts I was 2.21×10⁴ CFU,respectively,which was descended by 17.8 times.The amount of bacteria in the blood and organs was calculated after 24 h post injection with the dose of 2.0×10⁶ CFU each 7-day-old cherry valley duck.The results showed that the amount of bacteria of DE17?4.5×10⁸CFU/g?was 13600 times?P<0.05?,compared with DE17?ptsI(3.3×10⁴ CFU/g).The wild strain DE17(2.4×10⁸ CFU/g)in the spleen was 68 times?P<0.05?of the deletion strain DE17?ptsI(3.5×10⁶ CFU/g).The wild strain DE17(3.8×10⁸ CFU/g)in the kidney was 131 times?2.9×106 CFU/g?of the deletion strain DE17?ptsI(2.9×10⁶ CFU/g)?P<0.05?,while the wild strain DE17?2.5×108CFU/ml?in the blood was 362 times?P<0.05?of DE17?ptsI?6.9×104 CFU/ml?.After 24 h post injection of strains with the dose of 2.0×10⁶ CFU each 7-day-old cherry valley ducks,ducks were anatomied and took their hearts,livers,spleens,kidneys and then maked pathological sections.The results showed that part of heart cells showed myocardial necrosisliver;liver cells showed degeneration;Spleen lymphocytes decreased;renal tubular epithelial cells showed swelling in DE17.Compared with the wild strain DE17,symptoms of the deletion strain DE17?ptsI in the heart,liver,spleen and kidney are lighter.The adhesion ability of the strain DE17?ptsI was 2 times(3.4×10⁴ CFU/well)of the wild strain DE17(6.9×10⁶ CFU/well),while the invision ability of DE17?ptsI compared with the wild strain DE17 has no difference.The results showed that ptsI gene could regulate the pathogenicity of avian pathogenic Escherichia coli.3.The regulation of ptsI gene on biofilmBacterial biofilm is a substance adhesion to the bacterial surface,and a tightly structured multi-cell population morphology,which is wrapped with the extracellular matrix.Bacterial biofilm formation is associated with its pathogenicity and motility.In this study,the biofilm formation ability and the motility of DE17?pts I and DE17?1633 was evaluated,respectively.The results showed that the biofilm formation ability of DE17?ptsI was lower than that of wild strain DE17?P<0.0001?,while no difference in DE17?1633,the motility of DE17?ptsI and DE17?1633 was significantly decreased compared with that of the wild strain DE17?p<0.05?.The changes in bacterial morphology after ptsI deletion were observed.The results showed that the deletion of ptsI gene resulted in the colony color of DE17 changing from white to deeply red and the morphology became smaller.Furthermore,the biofilm of the wild strain and the mutant strain was stained by the SYTO/PI dye to study the ratio of dead/live bacteria during the process of biofilm formation.The results showed that the biofilm formation ability was the strongest at 18 hours.In order to further study the morphological structure of biofilm,the DE17and DE17?ptsI were cultured at 37?for 18 h,and the biofilm was observed by scanning electron microscopy.The results showed that the of DE17 biofilm structure of DE17 contained a variety of channel structure,and the surface contained a large number of extracellular matrix.However,the biofilm of mutant strain DE17?ptsI became sparse,extracellular interstitial reduction and damage channel structure compared with the DE17 biofilm.The results showed that the ptsI gene was not the receptor gene of AI-2.The ptsI decreased the APEC virulence,affected the formation of biofilm.The present study provided new ideas for further prevention and control of APEC.