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Characterization of a dipartite iron uptake system from uropathogenic Escherichia coli strain F11.

ABSTRACT: In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His(44), Met(90), His(97), and His(127), and CuB, a second degenerate octahedral geometry with the addition of Glu(46). The copper ions of each site occupy distinct positions and are separated by ?1.3 ?. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.

SUBMITTER: Koch D

PROVIDER: S-EPMC3137103 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Characterization of a dipartite iron uptake system from uropathogenic Escherichia coli strain F11.

Koch Doreen D Chan Anson C K AC Murphy Michael E P ME Lilie Hauke H Grass Gregor G Nies Dietrich H DH

The Journal of biological chemistry 20110519 28

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enh ...[more]

PMID: 21596746

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Project description:More than half of women will experience a urinary tract infection (UTI), with uropathogenic Escherichia coli (UPEC) causing ~80% of uncomplicated cases. Iron acquisition systems are essential for uropathogenesis, and UPEC strains encode highly diverse iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7, lacks this diversity and instead encodes the synthesis pathway for a sole siderophore, enterobactin. To determine if HM7 possesses unidentified iron acquisition systems, we performed RNA sequencing under iron-limiting conditions and demonstrated that the ferric citrate uptake system (fecABCDE and fecIR) was highly upregulated. Importantly, there are high levels of citrate within urine, some of which is bound to iron, and the fec system is enriched in UPEC isolates compared to fecal strains. Therefore, we hypothesized that HM7 and other similar strains use the fec system to acquire iron in the host. Deletion of both enterobactin biosynthesis and ferric citrate uptake (ΔfecA/ΔentB) abrogates use of ferric citrate as an iron source, and fecA provides an advantage in human urine in the absence of enterobactin. However, in a UTI mouse model, fecA is a fitness factor independent of enterobactin production, likely due to the action of host lipocalin-2 chelating ferrienterobactin. These findings indicate that ferric citrate uptake is used as an iron source when siderophore efficacy is limited, such as in the host during UTI. Defining these novel compensatory mechanisms and understanding the nutritional hierarchy of preferred iron sources within the urinary tract are important in the search for new approaches to combat UTI. IMPORTANCE UPEC, the primary causative agent of uncomplicated UTI, is responsible for five billion dollars in health care costs in the United States each year. Rates of antibiotic resistance are on the rise; therefore, it is vital to understand the mechanisms of UPEC pathogenesis to uncover potential targets for novel therapeutics. Iron acquisition systems used to obtain iron from sequestered host sources are essential for UPEC survival during UTI and have been used as vaccine targets to prevent infection. This study reveals the ferric citrate uptake system is another important iron acquisition system that is highly enriched in UPEC strains. Ferric citrate uptake has not previously been associated with UPEC isolates, underlining the importance of the continued study of these strains to fully understand their mechanisms of pathogenesis.

Project description:UnlabelledThe vacuolating autotransporter toxin (Vat) contributes to uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here, we characterized Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, ST73, and ST95), and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator; we termed this gene vatX The vat-vatX genes were present in the UPEC reference strain CFT073, and reverse transcriptase PCR (RT-PCR) revealed that the two genes are cotranscribed. Overexpression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator histone-like nucleoid structuring protein (H-NS); thus, the hns gene was mutated in CFT073 (to generate CFT073 hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073 hns compared to that in wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating that Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic serine protease autotransporter protein of Enterobacteriaceae (SPATE) secreted by UPEC during infection.ImportanceUropathogenic Escherichia coli (UPEC) is the major cause of hospital- and community-acquired urinary tract infections. The vacuolating autotransporter toxin (Vat) is a cytotoxin known to contribute to UPEC fitness during murine sepsis infection. In this study, Vat was found to be highly conserved and prevalent among a collection of urosepsis clinical isolates and was expressed at human core body temperature. Regulation of vat was demonstrated to be directly repressed by the global transcriptional regulator H-NS and upregulated by the downstream gene vatX (encoding a new MarR-type transcriptional regulator). Additionally, increased Vat-specific IgG titers were detected in plasma from corresponding urosepsis patients infected with vat-positive isolates. Hence, Vat is a highly conserved and tightly regulated urosepsis-associated virulence factor.

Dataset Information

Characterization of a dipartite iron uptake system from uropathogenic Escherichia coli strain F11.

Publications

Characterization of a dipartite iron uptake system from uropathogenic Escherichia coli strain F11.

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