The Relation Between The Iron Uptake Function Of HPI And The Virulence Of Avian Pathogenic E,coli Strains

Avian colibacillosis is a variety of birds of acute, chronic infectious diseasecaused by avian pathogenic Escherichia coli(APEC), and is often mixed with otherrespiratory or concurrent infection, have became one of the most important poultrybacterial diseases. As one of important factor for E. coli virulence and pathogenicity,Yersinia high pathogenicity island(HPI) is widely distributed in a variety of intestinalbacteria, carrying yersiniabactin synthesesis, regulation and transit-related irp2-fyuAgene cluster.In order to study the relation between the virulence of APEC and the iron uptakefunction of HPI, this experiment identified from whether seven APEC(A-F) andCVCC1565carried the HPI irp2, irp1and fyuA gene, then detected the expression ofHMWPs high molecular weight proteins of different APEC strains and siderophoresynthesis, combined with half lethal dose test(LD50) to analysis the virulence ofAPEC strains, on this groundwork, we successfully constructed an irp2gene deletionstrain.Then we preliminary discussed the pathogenic tests of APEC were associatedwith the iron uptake function of HPI, and lay the foundation for following study of theirp2virulence contribution in HPI. The specific content and results are as follows:1. The distribution of the HPI genes irp1, irp2, fyuA in APECExtracting the genomic DNA of APEC strains as a template, three pairs ofoligonucleotide primers were designed and synthesized, the HPI genes of irp2, irp1and fyuA of seven APEC strains were detected by the established PCR. The resultsshowed that the sequences of segment of each gene of seven APEC were consistentwith the expected, with detection rate were as high as100%. It can be speculated thatthe poultry E.coli disease is becoming increasingly serious with APEC high carryingrate of the HPI.2. Expression of HMWPs and siderophore synthesis of APEC HPI positivestrainsSDS-PAGE method was used to detect the expression of HMWPs of HPIpositive APEC strains under the condition of iron deficiency, and siderophoresynthesesis of APEC was detected by universal CAS assay.The results showed onlytwo strains expressed two high molecular weight proteins HMWP1and HMWP2with2-2’-bipyridine final concentration0.2,0.3,0.4mmol/L. The remaining five strainsexpressed different proteins without HMWPs. All APEC strains can make the CASsolid flat and liquid detection occurred obvious color change from blue to orange, but the ability of siderophore production were different. Maybe the APEC which harboredHPI has the same iron uptake function with the Yersinia HPI.3. Deletion of irp2iron-regulated gene ofAPEC HPI positive strainThe chloramphenicol-resistant gene flanked by homologues of target genes wasamplified by PCR and electro-transformed into APEC strain cells containing pKD46and get the irp2deleted mutant. SDS-PAGE method was used to detect and comparethe proteins expression of irp2deletion mutant strain and irp2original strain, viewfrom the protein expression profiles, irp2original strain carrying irp2, irp1, fyuAtessenial genes ofYbt synthesis, but it did not express HMWPs; irp2deletion mutantstrain growed slowly, however there is no growth inhibition phenotype under irondeficiency condition, and proteins expression did not have obvious difference. Theremay be another iron uptake system to perform the task of the intake of iron inAPEC.4. The relation between iron uptake function of HPI and theAPEC pathogenicityThe median lethal dose (LD50) determination of the APEC strains pathogenicity.Configuration of seven APEC and control of non-pathogenic strains into3×104to3×108cfu/mL five dose groups. Experimental animals were divided into eight groups,each group of five chicks have been given0.2ml intraperitoneal injection. Onceobserved after vaccination every12h, continuous observation7d. The results showedthat the test chickens had a performance of the typical E.coli poisoning diseasesymptoms and had different degrees of death. Control non-pathogenic strainsE.coli(CMCC44102) had no symptoms and death. According to Reed-Muench,determine three high-pathogenic strains, CVCC1565、APEC(E) and APEC(F); twomiddle-pathogenic strains, APEC(B) and APEC(C); the toxicity of APEC(A) andAPEC(D) were weaker than other strains, LD50were respectively4.967×108and5.836×108cfu·mL-1. Study found that the high-pathogenic CVCC1565, APEC(E) andAPEC(F) produced siderophore capacity is the strongest, and under the condition ofiron deficiency, CVCC1565and APEC(F) can express high molecular weight proteins,HMWP1and HMWP2. The low-pathogenic APEC(A) and APEC(D) producedsiderophore capacity is also the weakest. There is a certain relationship between thepathogenicity of APEC and HPI encoding siderophore Ybt mediated iron uptakefunction.Above all, this study demonstrated the relation between the virulence of APECand the HPI iron uptake function, and successfully constructed a irp2deletion mutantstrain of APEC, confirmed the presence of another iron uptake system besides irp2to perform the task of iron intake in APEC. Whether the iron uptake function of HPI isalso related with the increased and prevalent pathogenicity of poultry E.coli, this needdemonstrate in further study.