Construction And Characterization Of IucA, IucAiutA, Sit And Aerobactin Operon Mutants In Avian Pathogenic E. Coli E058

Avian colibacillosis is one of the most common infectious diseases caused entirely or partly by avian pathogenic Escherichia coli (APEC) in birds, and is often infected with other pathogens such as bacteria or viruses. So far, this disease has been an important bacteriosis and it is responsible for worldwide economic losses to the poultry industry.APEC has a specific iron assimilation system that consists of the synthesis of the hydroxa siderophore aerobactin (iucABCD) and for ferric aerobactin uptake (iutA) and this system, originally was found in E. coli by Williams. Both components are strongly induced under iron starvation. Lafont et al. stated that aerobactin genes were present in virulent strains and absent from avirulent strains. The five genes encoded aerobactin are localized to an operon and this operon was present in ColV plasmid. Genes responsible for the synthesis of the aerobactin was iucA, iucB, iucC and iucD, and iutA encoded an out-membrane protein receptor. In order to demonstrate the relationship between iucA, iutA gene, sit operon or aerobactin operon and the pathogenicity of APEC strain E058and to reveal the pathogenic mechanism and the biological characteristics of the mutants, the iucA, iutA, aerobactin operon and sit operon were chosen and mutated.In this study, the mutants E058AiucA, E058AiucAAiutA, E058Avir were developed, through the technology of allelic recombination, vir was a15kb fragment including sit operon and aerobactin operon. The plasmid pGEX-6P-1-iucA was electroporated into E058AiucA to generate the revertant of E058AiucA. Gene iucA was amplificated and subcloned into pBluescript II SK (-), and then the Zeorgene was cloned into iucA to form pS-iucA-Zeo. And the amplificated fragment iucA-Zeo was electroporated into E058to form mutant E058AiucA. The amplificated fragment iutA-Kan constructed by the similar way was electroporated into E058AiucA to form mutant E058AiucAAiutA. A designed PCR amplification of a Kanr cassette in pUC4K was electroporated into E058to form mutant EO58Avir.The results of the Southern blot showed zeocin resistance gene or kanamycin gene was singly inserted in pAPEC-O2-ColV-like plasmid of the mutants. The results of RT-PCR demonstrated that the deletion of the gene iucA or iutA was disrupted in the transcription of the target gene or genes, while the upstream gene and the downstream gene of the target genes were expressed normally. E058AiucA, E058AiucAAiutA and E058Avir were unable to produce aerobactin, whereas E058and Re-E058AiucA were able to do. The growth curves of E058AiucA, E058AiucAAiutA and E058Avir were similar to that of their parental strain E058in LB broth. The50%lethal doses (LD50) of E058, E058AiucA, E058AiucAAiutA and E058Avir were1044,105.0,104.8and105.0CFU, respectively. The result of invasion assays of HD-11cell showed an inconspicuous difference of mutant strains compared with that of the parent strain E058. The mutants were slightly out-competed by the wild-type strain in vitro. After24h post-challenge in vivo competition assay, the mutant E058ΔiucA and E058AiucAAiutA showed a significant attenuation growth in blood, liver, spleen, lung and kidney. The capacity of the wild-type strain and its mutants to colonize internal organs was studied by determining colony numbers in selected organs24h after infection. At24h after infection, the maximum colonization of the wild-type E058was observed in the lung (1.3×105CFU.g-1), minimal colony counts were obtained in heart (8.2×102CFU. ml-1), besides, the colony numbers in kidney (3.7×104CFU.g-1), liver (4.2×104CFU.g-1.) and spleen (8.8×104CFU.g-1) were intermediate. Compared to those of wild-type strain E058, the mutant E058AiucA colony numbers were significantly decreased in the lung (7.8×103CFU.g-1)(P<0.001), spleen (7.1×103CFU.g-1)(p<0.01), kidney (9.4×102CFU.g-1)(p<0.001), liver (6.3×102CFU.g-1)(p<.001) and heart (12CFU.ml-1)(p<0.001), and E058AiucAAiutA in the lung (3.6×103CFU.g-1)(p<0.001), heart (15CFU.ml-1)(p<0.001), liver (94CFU.g-1)(p<0.001), spleen (9.1×102CFU.g-1)(p<0.001) and kidney (1.4×102CFU.g-1)(p<0.001), while E058Avir in the lung (6.8×103CFU.g-1)(p<0.001), heart (15CFU.ml-1)(p<0.001), liver (1.05×102CFU.g-1)(p<0.001), spleen (3.2×103CFU.g-1)(p<0.001) and kidney (96CFU.g-1)(p<0.001).