Specific Roles of the iroBCDEN Genes in Virulence of an Avian Pathogenic Escherichia coli O78 Strain and in Production of Salmochelins

Abstract

Avian pathogenic Escherichia coli (APEC) strains are a subset of extraintestinal pathogenic E. coli (ExPEC) strains associated with respiratory infections and septicemia in poultry. The iroBCDEN genes encode the salmochelin siderophore system present in Salmonella enterica and some ExPEC strains. Roles of the iro genes for virulence in chickens and production of salmochelins were assessed by introducing plasmids carrying different combinations of iro genes into an attenuated salmochelin-and aerobactin-negative mutant of O78 strain 7122. Complementation with the iroBCDEN genes resulted in a regaining of virulence, whereas the absence of iroC, iroDE, or iroN abrogated restoration of virulence. The iroE gene was not required for virulence, since introduction of iroBCDN restored the capacity to cause lesions and colonize extraintestinal tissues. Prevalence studies indicated that iro sequences were associated with virulent APEC strains. Liquid chromatography-mass spectrometry analysis of supernatants of APEC 7122 and the complemented mutants indicated that (i) for 7122, salmochelins comprised 14 to 27% of the siderophores present in iron-limited medium or infected tissues; (ii) complementation of the mutant with the iro locus increased levels of glucosylated dimers (S1 and S5) and monomer (SX) compared to APEC strain 7122; (iii) the iroDE genes were important for generation of S1, S5, and SX; (iv) iroC was required for export of salmochelin trimers and dimers; and (v) iroB was required for generation of salmochelins. Overall, efficient glucosylation (IroB), transport (IroC and IroN), and processing (IroD and IroE) of salmochelins are required for APEC virulence, although IroE appears to serve an ancillary role.

The Small RNA RyhB Contributes to Siderophore Production and Virulence of Uropathogenic Escherichia coli

“…For production and detection of siderophores, 6-h cultures grown in LB medium were diluted 50-fold in M63-glycerol minimal medium, and strains were cultured at 37°C with agitation. Siderophores from culture supernatants were obtained as described by Caza et al (47). Briefly, supernatants of 17-h cultures were obtained following centrifugation of bacterial cells at 3,200 ϫ g for 15 min and filtration of the supernatants on 0.2-m membranes.…”

“…Samples were injected onto a Kinetex 2.6-m C 8 4.6-by 100-mm column at a flow rate of 400 l min Ϫ1 , with a linear gradient of water-acetonitrile with 1% acetic acid. The analyses were performed in positive electrospray ionization mode with a cone voltage of 30 V as described by Caza et al (47).…”

“…Salmochelins are glycosylated molecules of enterobactin that are synthesized by the iroBCDE gene products, while aerobactin is synthesized by the iucABCD gene products (39)(40)(41)(42). Interestingly, it has been reported that salmochelins and aerobactin are often associated with ExPEC strains and contribute to their virulence (38,(43)(44)(45)(46)(47)(48)(49)(50)(51).…”

Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms

“…Analysis of the same APEC O2: K1:H5 mutant library in a low-dose model of chicken lung colonization identified a novel fimbrial locus, encoding ExPEC adhesin I (yqi), which influences virulence (20,21). A number of other virulence factors have been identified by using defined mutants in poultry models, including the autotransported proteins AatA (22), Tsh (23,24), and Vat (25), fimbrial gene clusters pap (26), fim (27,28), stg (29), and csg (28), a degenerate inv/mxi-spa-like type III secretion locus termed ETT2 (30), and factors mediating iron uptake (31)(32)(33) and phosphate transport (34). Flagellin contributes to persistence and invasion after oral dosing of chicks with APEC O78 (28), and ExPEC invasins (Ibe proteins) also facilitate APEC invasion (35,36).…”

Strong Concordance Between Transcriptomic Patterns of Spleen and Peripheral Blood Leukocytes in Response to Avian Pathogenic Escherichia coli Infection

“…Time-course experiments of other bacterial infections have shown increasing rates of colonization over the first 48 hr after infection (16). Many reports of spleen colonization of APEC are measured 48 hr or more after infection (4,6,17), whereas APEC has been shown to gain access to the bloodstream within hours after intra-air sac inoculation (20,24). At least one study demonstrated early splenic colonization at 6 hr after infection, but data comparing bacterial counts in the spleen to those found in blood were not reported (20).…”