Identification and characterization of Cronobacter iron acquisition systems

Abstract

Cronobacter spp. are emerging pathogens that cause severe infantile meningitis, septicemia, or necrotizing enterocolitis. Contaminated powdered infant formula has been implicated as the source of Cronobacter spp. in most cases, but questions still remain regarding the natural habitat and virulence potential for each strain. The iron acquisition systems in 231 Cronobacter strains isolated from different sources were identified and characterized. All Cronobacter spp. have both the Feo and Efe systems for acquisition of ferrous iron, and all plasmid-harboring strains (98%) have the aerobactin-like siderophore, cronobactin, for transport of ferric iron. All Cronobacter spp. have the genes encoding an enterobactin-like siderophore, although it was not functional under the conditions tested. Furthermore, all Cronobacter spp. have genes encoding five receptors for heterologous siderophores. A ferric dicitrate transport system (fec system) is encoded specifically by a subset of Cronobacter sakazakii and C. malonaticus strains, of which a high percentage were isolated from clinical samples. Phylogenetic analysis confirmed that the fec system is most closely related to orthologous genes present in human-pathogenic bacterial strains. Moreover, all strains of C. dublinensis and C. muytjensii encode two receptors, FcuA and Fct, for heterologous siderophores produced by plant pathogens. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed which genes and operons are components of the Fur regulon. Taken together, these results support the proposition that C. sakazakii and C. malonaticus may be more associated with the human host and C. dublinensis and C. muytjensii with plants.

Fig 1

Ferric iron transporters encoded by Cronobacter spp. (A) Cronobactin siderophore; (B) enterobactin-like siderophore; (C) hydroxamate ABC transporter encoded by fhuACDB; (D) ferric iron/siderophore/heme ABC transporter encoded by eitCBAD; (E) ferric dicitrate transport system. Arrows show the direction of transcription, and arrow fills identify genes encoding synthesis of siderophores (black), TonB-dependent outer membrane receptors (diagonal lines), ABC transporters (vertical lines), export of enterobactin (horizontal lines), intracellular release of the iron from siderophore-iron complex (gray), sigma factor (small grids), transmembrane signal transducer (dots), IS transposases (white), and unknown function (diamonds). Numbers in boxes shown in the enterobactin-like siderophore diagrams show locations of the three putative bidirectional promoter-operator regions. The small filled boxes upstream of some genes or operons show locations of putative Fur boxes.

Fig 2

Genes for ferrous iron transporters, feoABC and efeUOB, carried by the chromosome of Cronobacter spp. Arrows show the direction of transcription, and arrow fills identify genes encoding GTP-binding protein (probably permease) (diagonal lines), Fe-S-dependent transcriptional regulator of FeoABC expression (square), high-affinity iron permease (horizontal lines), transporter periplasmic protein (vertical lines), periplasmatic peroxidase protein (dots), and unknown function (white). The small filled boxes upstream of the operons show location of putative Fur boxes.

Fig 3

Evolutionary history of iron acquisition system genes. (A) Enterobactin gene cluster in Cronobacter spp., entHABEC-fepB and entS-fepDGC; (B) the TonB receptor-encoding gene foxA; (C) the TonB receptor-encoding gene fcuA; (D) the ferric reductase gene, viuB.

Fig 4

Representative RT-PCR of Cronobacter iron acquisition systems under iron-replete (even-numbered lanes) and iron-depleted (odd-numbered lanes) conditions. (A) Cronobactin and shiF-viuB operons. Lane 1, 1 kb plus DNA ladder; lanes 2 and 3, 16S rRNA; lanes 4 and 5, viuB; lanes 6 and 7, shiF; lanes 8 and 9, iucA; lanes 10 and 11, iucB; lanes 12 and 13, iucC; lanes 14 and 15, iucD; lanes 16 and 17, iutA. (B) Enterobactin genes. Lane 1, 1 kb plus DNA ladder; lanes 2 and 3, fepA; lanes 4 and 5, entF; lanes 6 and 7, fepE; lanes 8 and 9, entC; lanes 10 and 11, fepB; lanes 12 and 13, entS; lanes 14 and 15, fepG; lanes 16 and 17, 16S rRNA. (C) TonB-dependent iron receptors. Lane 1, 1 kb plus DNA ladder; lanes 2 and 3, pfeA; lanes 4 and 5, fhuE; lanes 6 and 7, foxA; lanes 8 and 9, yncD; lanes 10 and 11, btuB; lanes 12 and 13, fcuA; lanes 14 and 15, fct; lanes 16 and 17, 16S rRNA.

Fig 5

Siderophore activity using the CASAD assay. Wells were filled with cell-free culture supernatants of wild-type C. sakazakii BAA-894 (1), plasmid-cured derivative BAA-894.3 (2), fosmid clone ESA-C01 containing the cronobactin genes (3), and fosmid clone ESA-M04 lacking the cronobactin genes (4).

Fig 6

Growth of wild-type C. turicensis z3032 harboring pCTU1 and its plasmid-cured derivative, 3032.2A, in LB broth and iron-depleted LB broth containing 300 μM DIP. The data were obtained from 3 independent experiments. *, P < 0.001.

Fig 7

Results of the Csáky (A) and Arnow (B) tests used to identify hydroxamate-type and catechol-type siderophore activity, respectively, using cell-free culture supernatants of wild-type C. turicensis z3032 harboring pCTU1[P(+)] and its plasmid-cured derivative, 3032.2A [P(−)]. Cell-free culture supernatants of Vibrio vulnificus UNCC913 (C) and catechol (6 μg) were used as positive controls in the Csáky and Arnow tests, respectively.