ShiF acts as an auxiliary factor of aerobactin secretion in meningitis Escherichia coli strain S88

Abstract

Background: The neonatal meningitis E. coli (NMEC) strain S88 carries a ColV plasmid named pS88 which is involved in meningeal virulence. Transcriptional analysis of pS88 in human serum revealed a strong upregulation of an ORF of unknown function: shiF, which is adjacent to the operon encoding the siderophore aerobactin. The aim of this work is to investigate the role of shiF in aerobactin production in strain S88.

Results: Study of the prevalence of shiF and aerobactin operon in a collection of 100 extra-intestinal pathogenic E. coli strains (ExPEC) and 50 whole genome-sequenced E. coli strains revealed the colocalization of these two genes for 98% of the aerobactin positive strains. We used Datsenko and Wanner's method to delete shiF in two S88 mutants. A cross-feeding assay showed that these mutants were able to excrete aerobactin meaning that shiF is dispensable for aerobactin excretion. Our growth assays revealed that the shiF-deleted mutants grew significantly slower than the wild-type strain S88 in iron-depleted medium with a decrease of maximum growth rates of 23 and 28% (p < 0.05). Using Liquid Chromatography-Mass Spectrometry, we identified and quantified siderophores in the supernatants of S88 and its shiF deleted mutants after growth in iron-depleted medium and found that these mutants secreted significantly less aerobactin than S88 (- 52% and - 49%, p < 0.001).

Conclusions: ShiF is physically and functionally linked to aerobactin. It provides an advantage to E. coli S88 under iron-limiting conditions by increasing aerobactin secretion and may thus act as an auxiliary virulence factor.

Keywords: Aerobactin; Escherichia coli; ShiF; Siderophore.

Fig. 1

Deletion of shiF reduces growth in iron-depleted medium. Strains were grown LB medium (a), in MM9 minimal medium with 20 μM iron (b) and 100 μM of 2,2′-dipyridyl (c). Data presented are average of results from two (a) and five (b and c) independent experiments

Fig. 2

LC-MS/MS siderophore profiles of E. coli S88 in iron-limited medium. Aerobactin (a), salmochelin S1 (b) and S2 (c), enterobactin (d) and yersiniabactin (e) were extracted after addition of 0.1 M ferric chloride and identified with the following specific transitions 565 > 205, 506 > 319, 627 > 224, 670 > 224 and 535 > 303 m/z respectively. Data are from the MassLynx software. ES, electrospray positive mode; MRM, multiple reaction monitoring

Fig. 3

Relative production of aerobactin, enterobactin, salmochelin S2 and yersiniabactin measured by LC-MS/MS. Production of aerobactin, enterobactin, salmochelin S2 and yersiniabactin by the two deleted strains S88∆shiF1 and S88∆shiF2 were compared to that of the wild type strain S88 in MM9 medium with 100 μM of 2,2′-dipyridyl. Results are expressed in relation to the production of the wild type strain which represent 100%. Data presented are means of 8 independent experiments and were compared using Mann-Whitney’s test. Error bars represent the standard derivations. * p < 0.001

Fig. 4

Schematic representation of aerobactin operon and shiF. The large arrows represent the genes of the aerobactin operon and shiF IucA, iucB, iucC and iucD encode the operon allowing aerobactin synthesis and iutA encodes aerobactin receptor. The small arrows annotated P1 and P2 represent the primers described in Table 1 and are located in front of their hybridizing sequences