[Construction of aerobactin and sit operon mutant of avian pathogenic Escherichia coli E058 and evaluation of its pathogenicity].

OBJECTIVE: To elucidate the pathogenesis role of the vir region of APEC 02 strain E058. METHODS: The gene aerobactin/sitABC operon knockout mutants E058deltavir of APEC E058 strain was generated using Red recombination system. A series of pathogenicity tests including chick embryo inoculation, the competition experiment and the colonization and persistence in vivo were used to evaluate the pathogenicity of APEC E058deltavir and the wild-type strain E058. RESULTS: E058deltavir was similar to its parental strain E058 in the growth curves, invasion assays of HD-11 cell and in vitro competition assay. In the colonization and persistence test, the recovery colonies of E058deltavir were significantly decreased in all of the organs tested (P < 0.001). CONCLUSIONS: These results indicate that the virulence factors encoded by aerobactin/sit operon genes were important for the pathogenesis of APEC E058.

Role of the lpxM lipid A biosynthesis pathway gene in pathogenicity of avian pathogenic Escherichia coli strain E058 in a chicken infection model.

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.

Aerobactin synthesis genes iucA and iucC contribute to the pathogenicity of avian pathogenic Escherichia coli O2 strain E058.

Aerobactin genes are known to be present in virulent strains and absent from avirulent strains, but contributions of iucC and iucA, which are involved in aerobactin synthesis, to the pathogenicity of avian pathogenic Escherichia coli (APEC) have not been clarified. In this study, effects of double mutants (iucA/iutA or iucC/iutA) compared to those of single mutants (iucA, iucC or iutA) of aerobactin genes on the virulence of APEC strain E058 were examined both in vitro (aerobactin production, ingestion into HD-11 cells, survival in chicken serum) and in vivo (competitive growth against parental strain, colonization and persistence). In competitive co-infection assays, compared to the E058 parental strain, the E058ΔiucA mutant was significantly reduced in the liver, kidney, spleen (all P<0.01), heart and lung (both P<0.001). The E058ΔiutA mutant also was significantly reduced in the liver, lung, kidney (all P<0.01), heart and spleen (both P<0.001). The E058ΔiucC mutant was significantly attenuated in the heart and kidney (both P<0.05) and showed a remarkable reduction in the liver, spleen and lung (P<0.01); meanwhile, both E058ΔiucAΔiutA and E058ΔiucCΔiutA double mutants were sharply reduced as well (P<0.001). In colonization and persistence assays, compared with E058, recovered colonies of E058ΔiucA were significantly reduced from the lung, liver, spleen and kidney (P<0.01) and significantly reduced in the heart (P<0.001). E058ΔiutA was significantly reduced from the heart, lung, liver, spleen and kidney (P<0.01). E058ΔiucC, E058ΔiucAΔiutA and E058ΔiucCΔiutA were significantly decreased in all organs tested (P<0.001). These results suggest that iutA, iucA and iucC play important roles in the pathogenicity of APEC E058.

Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coli: salmochelin and aerobactin contribute more to virulence than heme in a chicken infection model.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) and uropathogenic E. coli (UPEC) are the two main subsets of extraintestinal pathogenic E. coli (ExPEC). Both types have multiple iron acquisition systems, including heme and siderophores. Although iron transport systems involved in the pathogenesis of APEC or UPEC have been documented individually in corresponding animal models, the contribution of these systems during simultaneous APEC and UPEC infection is not well described. To determine the contribution of each individual iron acquisition system to the virulence of APEC and UPEC, isogenic mutants affecting iron uptake in APEC E058 and UPEC U17 were constructed and compared in a chicken challenge model. RESULTS: Salmochelin-defective mutants E058ΔiroD and U17ΔiroD showed significantly decreased pathogenicity compared to the wild-type strains. Aerobactin defective mutants E058ΔiucD and U17ΔiucD demonstrated reduced colonization in several internal organs, whereas the heme defective mutants E058ΔchuT and U17ΔchuT colonized internal organs to the same extent as their wild-type strains. The triple mutant ΔchuTΔiroDΔiucD in both E058 and U17 showed decreased pathogenicity compared to each of the single mutants. The histopathological lesions in visceral organs of birds challenged with the wild-type strains were more severe than those from birds challenged with ΔiroD, ΔiucD or the triple mutants. Conversely, chickens inoculated with the ΔchuT mutants had lesions comparable to those in chickens inoculated with the wild-type strains. However, no significant differences were observed between the mutants and the wild-type strains in resistance to serum, cellular invasion and intracellular survival in HD-11, and growth in iron-rich or iron-restricted medium. CONCLUSIONS: Results indicated that APEC and UPEC utilize similar iron acquisition mechanisms in chickens. Both salmochelin and aerobactin systems appeared to be important in APEC and UPEC virulence, while salmochelin contributed more to the virulence. Heme bounded by ChuT in the periplasm appeared to be redundant in this model, indicating that other periplasmic binding proteins likely contributed to the observed no phenotype for the heme uptake mutant. No differences were observed between the mutants and their wild-type parents in other phenotypic traits, suggesting that other virulence mechanisms compensate for the effect of the mutations.

Construction of iucB and iucBiutA mutants of avian pathogenic Escherichia coli and evaluation of their pathogenicity.

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.