Infect Immun. 2016 Oct 17;84(11):3220-3231.

Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli.

Kurabayashi K1, Agata T1, Asano H1, Tomita H2,3, Hirakawa H1*

  1. Advanced Scientific Research Leaders Development Unit, Gunma University, Graduate School of Medicine, Showa-machi, Maebashi, Gunma, Japan.
  2. Department of Bacteriology, Gunma University, Graduate School of Medicine, Showa-machi, Maebashi, Gunma, Japan.
  3. Laboratory of Bacterial Drug Resistance, Gunma University, Graduate School of Medicine, Showa-machi, Maebashi, Gunma, Japan.

*Email: hirakawa@gunma-u.ac.jp.

 

Abstract

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD, which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs. Copyright © 2016, American Society for Microbiology.
PMID: 27572332

 

Supplement:

Urinary tract infection (UTI) is one of the most common diseases, and it affects urinary organs involving the bladder and kidneys. Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes uncomplicated UTI [1]. This bacterium is a member of extraintestinal E. coli which does not exhibit any virulence in the intestine but urinary tracts.

 

At an early stage of UTI, UPEC adheres to and enters the epithelial cells in the bladder, where it forms polymicrobial colonies termed intracellular bacterial community (IBC), and it protects UPEC from antimicrobial agents and host immune system [2] (Figure 1). Therefore, bacterial adherence and entry to the bladder epithelial cells followed by IBC formation determine whether infection becomes refractory to treatment.

 

 

Figure 1. Schematic (Top) and fluorescent  images (Bottom) of UPEC infecting to the bladder epithelial cells. At an early stage of infection, UPEC adheres to and  enters the bladder epithelial cells, and forms polymicrobial structure termed intracellular bacterial community (IBC). In fluorescent  images, bacteria and the bladder epithelial cells are colored green and red, respectively.

 

The type I fimbriae [3] and flagella [4] contribute to the virulence of UPEC including adhesion and invasion to the bladder epithelial cells and IBC formation. Although these virulence elements have been well characterized, the regulatory mechanism such as how they are expressed in the bladder is relatively less understood.

 

Our recent study demonstrated that expression of the type I fimbriae and flagella is repressed by Fur, the iron-binding transcriptional repressor which only functions in the presence of ferrous iron, and de-repressed under iron-depleted media, which leads to promotion of UPEC adhesion, invasion IBC formation [5]. Some in vivo studies using mice model suggest that the availability of iron for UPEC is very low in the bladder since hallmark siderophore production of UPEC is promoted [6]. These combined literatures including our study indicate that virulence of UPEC associated with the type I fimbriae and flagella is suppressed by Fur when UPEC is able to utilize relatively enough iron such as in enteric sites, and it is de-repressed when UPEC enters the bladder since Fur is inactivated due to low level of iron (Figure 2). It might explain the reason why UPEC exhibits higher virulence in the urinary tract rather than intestine.

 

Recently, UPEC acquiring resistance to some antimicrobial agents including fluoroquinolones that is primary used for treating uncomplicated UTI is increasing [7]. Our in vitro expression study supports that the type I fimbriae and flagella are particularly important for virulence of UPEC, and it is expected that these virulence elements could be effective drug targets for treating refractory UPEC infections.

 

 

Figure 2. Schematic of Fur-mediated regulation of genes encoding type I fimbriae and flagella in UPEC. When UPEC is able to acquire enough iron such as in enteric sites, Fur and ferrous iron complex binds to the promoter of target genes, then inhibits their transcriptions. When UPEC enters the bladder, Fur is dissociated with ferrous iron due to deletion of iron. Then, Fur is inactivated and transcription of target genes is de-repressed. Consequently, the virulence to the bladder associated with adhesion, invasion and IBC formation is induced.

 

 

References

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[5] K. Kurabayashi, T. Agata, H. Asano, H. Tomita, H. Hirakawa, Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli, Infect. Immun. 84 (2016) 3220-3231.

[6] J.A. Snyder, B.J. Haugen, E.L. Buckles, C.V. Lockatell, D.E. Johnson, M.S. Donnenberg, R.A. Welch, H.L. Mobley, Transcriptome of uropathogenic Escherichia coli during urinary tract infection, Infect. Immun. 72 (2004) 6373–6381.

[7] C. Aypak, A. Altunsoy, N. Düzgün, Empiric antibiotic therapy in acute uncomplicated urinary tract infections and fluoroquinolone resistance: a prospective observational study, Ann. Clin. Microbiol. Antimicrob. 8 (2009) 27.