Identification And Characterization Of The Iron Uptake Function Of TBDRs In Avian Pathogenic Escherichia Coli DE205B

Escherichia coli(E.coli)is a kind of Gram-negative bacteria,which can cause colibacillosis in people,some land animals,including poultry,and aquatic animals,leading to great economic losses.Among them,avian pathogenic E.coli(APEC)is one of the pathogens that cause the three most serious infectious diseases in poultry.Studies have shown that APEC shares some virulence factors with Newborn meningitis-causing E.coli and has similar pathogenesis.Therefore,the study on virulence factors of APEC has a great significance to public health.Iron is a microelement essential for bacteria to survive in host tissues and is important for bacterial reproduction and infection.In nature,the available iron for bacteria is very rare.Therefore,most bacterial genes encode various iron uptake factors to meet the needs of growth.Among them,the TonB-dependent receptors(TBDRs)are common ?-barreled proteins in the outer membrane of bacteria.They play an important role in the recognition of iron carriers in the iron uptake process,and promote the adaptation of bacteria to the iron-restricted environment through the TonB-dependent iron uptake system.TBDRs recognize and bind iron vectors to form the TBDRs-iron carrier complex,which binds with TonB to transport iron into the cell.It has been reported in the literature that ireA may playa role of TBDRs in Uropathogenic E.coli,but which has not been experimentally verified.Other studies have suggested that TonB is associated with APEC toxicity,but its regulatory role in APEC iron intake has not been reported.In this study,the whole genome of APEC strain DE205B was analyzed,and four hypothesis TBDRs were predicted,encoded by genes ireA,0007,0008,and 2235,respectively.The role of TBDRs in APEC iron uptake was identified by in vitro protein interaction,fluorescence quantitative PCR,growth curve measurement,and iron uptake detection.In addition,the deletion strain of tonB gene was constructed to study the effect of tonB deletion on TBDRs.1.TBDRs identification and study on its iron uptake functionBy analyzing the whole genome of strain DE205B,four hypothesis TBDRs were predicted,which were encoded by genes ireA,0007,0008 and 2235,respectively.To identify whether the proteins encoded by these four genes were TonB-dependent iron carrier receptors,the GST pull down test(glutathion-s-transferase pull down)was used to detect the interactions between the four hypothesized TBDRs and TonB proteins.The test results showed that IreA,0007,0008 and 2235 could interact directly with TonB.Next,we further studied the role of IreA,0007,0008 and 2235 in DE205B.Strain DE205B was cultured to logarithmic stage under iron-rich and iron-poor conditions,the results of fluorescence quantitative PCR showed that the expression level of four TBDRs in iron poor M9 medium was significantly higher than that in iron rich M9 medium(P<0.01),indicating that these four TBDRs played a role in iron deficient environment.In order to study the role of four receptor genes in iron uptake,a single gene deletion strain of TBDRs was constructed by red homologous recombination technology,and a complemented strain was constructed by plasmid pSTV28.The results of growth curve showed that the growth rate of TBDRs deletion mutants(DE205B?ireA DE205B?0007,DE205B?0008 and DE205B?2235)were lower than that of the wild type in M9 iron poor medium,while the expression level of most of the genes related to iron uptake was significantly increased(P<0.05),and the up-regulation of the genes related to iron uptake was the most obvious in the ireA mutant strain.Finally,the iron uptake assay kit was used to determine the iron content in the cells of wild and absent strains to investigate whether TBDRs were directly involved in iron uptake.The results showed that the iron uptake of ireA mutant strain was significantly higher than that of wild-type strain(P<0.05).In conclusion,ireA,0007,0008 and 2235 do encode TBDR,and ireA plays a key role in iron uptake.2.Preliminary study on the regulation of TBDRsThe uptake of iron by TBDRs requires energy from TonB to trigger the transmembrane process of active ion transport.To investigate whether the expression of TBDRs depends on TonB,the tonB gene deletion and complemented strains were constructed.The results of the growth curve showed that tonB deficient strain grew much slower than the wild strain both in LB and M9 medium.The results of iron uptake test showed that the iron uptake capacity of the tonB mutant strain was significantly reduced compared with that of the wild strain(P<0.05),indicating that TBDRs can not work well without TonB,suggesting that function maintaining of TBDRs were depended on TonB.In addition,we also found that cultured medium of DE205B?tonB turned red compared with wild and complemented strains,which was due to the greatly reduced iron uptake by DE205B ?tonB,resulting in excessive iron in the medium being oxidized and turned red,which further support the ideamentioned above.The results of fluorescent quantitative PCR showed that compared with the wild strain DE205B,the expression level of ireA in the mutant strain DE205B?tonB was up-regulated by 2.6 times(P<0.01)in M9 iron deficient medium,and the expression levels of 0007,0008,2235 gene were all up-regulated;In addition,the expression level of TBDRs were further up-regulated in tonB deficient strain in M9 iron-poor medium compared with in M9 iron-rich medium,which indicated that in the case of tonB deficiency,even though the expression level of many TBDRs were up-regulated compensatory,iron could not be effectively absorbed.It has been reported that the expression level of TBDRs are mainly regulated by Fur,an iron absorption regulating protein,whose main function is to inhibit iron absorption when iron ions are abundant.Therefore,the mRNA expression level of fur was detected.The results showed that the expression level of fur in DE205B?tonB was down-regulated(P<0.01)compared to the wild strain,which explained that the ability of iron uptake of the APEC strainwas greatly reduced without tonB,resulting in the decrease of intracellular iron ions,which led to the down-regulation of fur,followed by the up regulation of TBDRs.In conclusion,the expression level of TBDRs are regulated by fur,but the function maintaining of TBDRs depends on TonB.