Construction And Characterization Of IucC And IutA Mutants Of Avian Pathogenic Escherichia Coli

Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) is one of the most common infectious diseases in birds, and is often infected with combination of other pathogens such as other bacteria or viruses. It is responsible for worldwide economic losses to the poultry industry.The density of iron for bacterial growth is10-7mol.L-1, while in the normal body fluid is just10-12mol.L-1. To overcome this natural growth limitation, many pathogenic E. coli strains have adopted aecial iron assimilation system. As a low molecular weight compound, aerobactin is encoded by5genes, all located at one operon. It comprises genes responsible for the synthesis of the hydroxamate siderophore aerobactin (iucABCD) and for ferric aerobactin uptake (iutA). The74-kilodalton protein, the product of gene iutA, is the outer membrane receptor forerric aerobactin, and is tentatively responsible for the first step in the synthetase reaction, iucC is coding for Nε-hydroxylysine acetyltransferase, iucC is putatively for aerobactin synthetase. In this study, the relationship between iucC gene or iutA gene and the pathogenicity of APEC strain E058was studied. Nevertheless, all the actual roles of the known virulence factors of APEC are not fully elucidated and certain steps of the infection process have not been related to previously identified factors. Furthermore, the mechanisms by which APEC cause infection are largely unknown.In this study, genes in APEC serogroup02strain E058chosen for deletion were iucC and iutA. Each knockout mutant was constructed and named as E058iucC and E058iutA, and a knockout mutant of both genes was also constructed and named as E058iucCiutA. Southern blot showed single copy of Kanr gene or Zeor gene was inserted into the plasmid DNA of APEC E058; RT-PCR showed that the insertion didn’t have a polar effect on the upstream or downstream gene of iucC or iutA. None of the mutants deviated from the growth pattern of the wide-type strain E058in M9medium. The mutants but not E058iutA were deprived of producing aerobactin; SDS-PAGE results indicate that the mutant E058iutA still express the IutA protein. The50%lethal doses (LD50) of E058, E058iucC, E058iutA and E058iucCiutA were104.4,105.0,104.6and105.1CFU, respectively. In12-day old embryonated lethality assay, the lethality rates of E058, E058iucC, E058iutA and E058iucCiutA were90.0%,86.7%,83.3%,80%, respectively. The mutants all were slightly out-competed by the wild-type strain both in vivo and in vitro, especially in vivo, E058iutA was highly out-competed by the wild-type strain E058in the liver and spleen of the challenged birds, and moderately out-competed in the heard and kidney. At24hours after inoculation, the number of colony forming units (CFUs) recovered from the liver, spleen, lung, kidney and blood of chickens inoculated with the mutants were less than those of birds inoculated with the parent strain E058, especially, E058iutA and E058iucCiutA were declined obviously in the lung and blood. These results indicated that the iucC but not iutA gene was important for the synthesis of aerobactin; the iutA gene had no effect on the expression of the IutA protein, but compared to the mutant E058iucC, E058iutA seemed to exhibit a stronger effect on the pathogenicity of APEC E058based on the both results of the out-competed in vivo, and the colonization in the tissues of challenged birds; the iucC and iutA genes related virulence factors might contribute to the pathogenicity of APEC E058. even though there was no significances in statistics in certain circumstances.