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Lipopolysaccharide (LPS) is a major surface component of avian pathogenic Escherichia coli (APEC), and is a possible virulence factor in avian infections caused by this organism. The contribution of the lpxM gene, which encodes a myristoyl transferase that catalyzes the final step in lipid A biosynthesis, to the pathogenicity of APEC has not previously been assessed. In this study, an isogenic lpxM mutant, E058ΔlpxM, was constructed in APEC O2 strain E058 and then characterized. Structural analysis of lipid A from the parental strain and derived mutant showed that E058ΔlpxM lacked one myristoyl (C14:0) on its lipid A molecules. No differences were observed between the mutant and wild-type in a series of tests including growth rate in different broths and ability to survive in specific-pathogen-free chicken serum. However, the mutant showed significantly reduced invasion and intracellular survival in the avian macrophage HD11 cell line (P<0.05). Nitric oxide production reduction (P<0.05) and cytokine gene expression downregulation (P<0.05 or P<0.01) also showed in HD11 treated with E058ΔlpxM-derived LPS compared with that in cells treated with E058-derived LPS at different times. Compared to the parental strain E058, E058ΔlpxM had a significant reduction in bacterial load in heart (P<0.01), liver (P<0.01), spleen (P<0.01), lung (P<0.05), and kidney (P<0.05) tissues. The histopathological lesions in visceral organs of birds challenged with the wild-type strain were more severe than in birds infected with the mutant. However, the E058ΔlpxM mutant showed a similar sensitivity pattern to the parental strain following exposure to several hydrophobic reagents. These results indicate that the lpxM gene is important for the pathogenicity and biological activity of APEC strain E058., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Aerobactin counts for much to the pathogenesis of avian pathogenic Escherichia coli (APEC), iutA is responsible for the expression of a specific outer membrane receptor protein for ferric aerobactin, and iucB is involved in the aerobactin synthesis. To our knowledge, the contribution of iucB to the pathogenicity of APEC has not been assessed till now. In this study, the mutants E058ΔiucB and E058ΔiucBΔiutA were constructed and characterized. There were no differences observed in a series of tests including the embryo lethality, invasion assay in HD11 cells and the ability to survive in SPF chicken serum. Meanwhile, the mutants showed decreased pathogenicity as compared with the wild-type strain through a series of experiments in vivo. The mutants E058ΔiucB and E058ΔiucBΔiutA greatly reduced in all of the tested tissues in vivo persistence (p<0.001). In the meantime, the mutants had no ability to produce aerobactin. Reintroduction of the iucB gene on a multicopy expression plasmid pGEX-6p-1 restored the capacity to produce aerobactin as similar to that of wild-type strain E058. The results indicated that the iucB gene related virulence factors including the iron assimilation system were important for the pathogenesis of APEC E058. As showed in the in vivo competition assay, compared to the parental strain E058, E058ΔiucB had a significant reduction of bacterial loads in heart (p<0.01), liver (p<0.01), kidney (p<0.01), spleen (p<0.05) and lung (p<0.05), respectively, while E058ΔiucBΔiutA had a sharp reduction in all of the five tissues to be tested (p<0.001). These results suggested that the single gene either iucB or iutA was likely to be involved directly or indirectly in iron uptake for the pathogenicity of APEC E058, and there was an obviously synergistic effect between iucB and iutA genes on the pathogenicity of APEC E058., (Copyright © 2012 Elsevier B.V. All rights reserved.)