Functional Study Of Iron-uptake Factors IreA And Fur In Pathogenic Escherichia Coli

Pathogenic Escherichia coli includes the intestinal pathogenic E.coli and extraintestinal Pathogenic E.coli,which can cause colibacillosis,harm to human and animal health.The intestinal pathogenic E.coli is an important zoonosis pathogens;It is reported that APEC and human extraintestinal Pathogenic E.coli also have similar pathogenic mechanism.They share and exchange some virulence factors.There are various virulence factors in Pathogenic E.coli,including the adhesion,invasion,iron uptake factor,toxin etc.The iron-uptake factor is critical to the survival and infection of bacteria.Pathogen usually take the iron in host through siderophores system and heme uptake system,and the whole iron-uptake process was controlled by Ferric uptake regulator(Fur).A wide variety of iron transport systems played key roles in the iron uptake mechanismin E.coli.Recent research suggests that many iron uptake factors not only have the function of iron-uptake,but also participate in physiological metabolic regulation and pathogenic mechanism.ireA is an iron uptake factor discovered in recent years,which coding iron outer membrane receptor IreA.Studies found that ireA may be associated with the virulence of UPEC,but its function in APEC remains unknown.Fur was encoded by fur gene,which inhibit the absorption of iron to maintain the balance of iron bacteria.In recent years,fur has proved to take part in the regulation of various genes.In this study,an ireA deletion mutant and fur deletion mutant was constructed in APEC strain DE205B and EHEC strain SH13,respectively.Fluorescence quantitative PCR,growth curve,adhesion assay,environment resistance test,and animal infection experiment was proceeded to explore the role of these iron uptake factor in pathogenic E.coli.1.Study of ireA gene's function in Avian pathogenic Escherichia coliThe pathogenesis of avian pathogenic E.coli strains is not well defined.Here,the function of an outer membrane protein encoded by the ireA gene of avian pathogenic E.coli train DE205B was investigated.The ireA gene was distributed in 32.9%(46/140)of tested E.coli strains,with high percentages in the phylogenetic ECOR groups B2(58.8%,10/17)and D(55.9%,19/34).ireA was expressed in laboratory conditions,and tested by Immunoblotting.The gene expression level of ireA of DE205B in low Fe M9 media was up to 1.8 times of(P<0.05)that in high Fe M9 media.An ireA deletion mutant and complementary strain were constructed.Compared with the wild-type strain DE205B,the expression of most ferric uptake genes in the ireA deletion mutant were significantly upregulated(P<0.05).The adhesion ability of the ireA deletion mutant to DF-1 cells was significantly decreased.The survival rate of ireA deletion mutant was reduced 21.17%(P<0.01),25.42(P<0.05)and 70.0%(P<0.01)under alkali,high osmolarity,and low temperature(4?)conditions,respectively,compared with the wild-type strain.The results suggested that the protein encoded by the iron-regulated gene ireA has roles in adhesion and stress resistance in avian pathogenic E.coli.2.The biological characteristics research of fur gene in EHEC strain SH13EHEC strain SH13 was selected to explore the function of fur gene in E.coli.fur was expressed,and tested by Immunoblotting.The fur gene expression level of SH13 in low Fe M9 media was 1.4 times higher(P<0.01)than that in high Fe M9 media.An fur deletion mutant and complementary strain were constructed.Compared with the wild-type strain SH13,the survival rate of fur deletion mutant was reduced under acidic condition(pH4.0)(P<0.05).There was no significant difference of adhesion to HEp-2 cells between the wild-type,mutant and complementary strains(P>0.05).The survival rate of different strains has no significance under alkali(pH10.0)and high osmotic pressure condition(2.4M)(P>0.05).Animals infected assay showed that the knock-out of fur gene had no significant effect on the pathogenicity of mice(P>0.05).