Part:BBa_K2500000

Heme-Deleted Bacterioferritin (M52H)

Bacterioferritins are bacterial iron storage proteins. It has been shown that overexpression of bacterioferritin in E. coli Nissle 1917 can lead to a visible contrast change in MRI, which allows for visualization of the bacteria. We used a heme-deletion mutant as it does not decrease contrast change.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Ferritins are iron storage proteins found in different species. Through ferroxidase, they convert iron from its toxic, ferrous state (Fe2+) into a non-toxic, ferric state (Fe3+). When present in its ferric state, iron acts as a paramagnetic agent and causes a shortening of the transversal relaxation (T2) in MRI. Depending on the concentration of iron, this can lead to a visible change in contrast.¹

Bacterioferritin is one of the three forms of ferritin-like proteins found in bacteria. It has been shown that overexpression of bacterioferritin in E. coli Nissle 1917 can lead to a visible contrast change in MRI, which allows for visualization of the bacteria. Additionally, by site directed mutagenesis, resulting in a heme-deleted version of bacterioferritin, it was shown that the absence of heme does not influence the contrast change.²

For this part, a decrease in signal intensity was observed for all bacteria grown in iron-supplemented medium, independent of bacterioferritin, probably due to presence of various inherent bacterial iron-storage proteins. However, an additional significant drop in the signal was observed in samples where bacterioferritin overexpression was induced as expected. This result proves the usability of bacterioferritin as an MRI contrast agent in vitro and confirms the potential to use it as an in vivo reporter of tumor sensing.

Design

Bacterioferritin was integrated into our system to serve as an MRI reporter, able to point out locations inside the body colonized by bacteria. When iron is present in the medium, overexpression of bacterioferritin leads to accumulation of iron, which causes a shortening of the T2 relaxation time, seen as a drop in the signal intensity (i.e. darker area on an MRI T2 scan).¹

To test the consequences of bacterioferritin overexpression in an MRI scanner, we transformed E. coli Nissle 1917 with a plasmid containing an AHL-inducible promoter (pLux) that controls the expression of both a green fluorescent protein (GFP) and bacterioferritin (Figure 1).

Figure 1: AHL diffuses into the cell and binds to LuxR. The AHL/LuxR complex activates pLux, which results in transcription of both GFP and bacterioferritin.

Characterization

AHL-induced bacterioferrtin production
First, we determined the concentration of AHL needed for full induction of the system. To do this, we measured changes in fluorescence caused by different concentrations of AHL used for induction (Figure 2). Second, we performed an SDS-PAGE analysis that confirmed that bacterioferritin was indeed expressed along with GFP after induction with the appropriate concentration of AHL (Figure 2).

Figure 2: (Left) AHL dose-response curve obtained by measuring fluorescence. (Right) SDS-PAGE analysis of bacterioferritin expression upon induction with different concentrations of AHL. (Bfr = sample from bacterioferritin-overexpressing bacteria, NC = negative control)

MRI imaging of bacterioferritin-expressing E. coli Nissle
Bound to bacteriferritin, iron is in its paramagnetic ferric state which shortens the T2 relaxation time in the area, and result in a visible signal drop in the T2-weighted image.² Both of these effects, shortening of the T2 relaxation time (Table 1, Figure 3 (Left)) and a visible change in the T2-weighted image (Figure 7) were experienced by the bacteria in this experiment that were grown in the presence of iron and induced to overexpress bacterioferritin.

Table 1: T2 relaxation times for different samples. Iron (+) indicates growth in an iron-supplemented medium, while Induction (+) indicates induction of bacterioferritin expression by AHL or IPTG. pLux-bfr M52H represents our E. coli Nissle transformed with bacterioferritin under the control of an AHL-responsive promoter, while T7lacO-bfr has bacterioferritin expression controlled by an IPTG-inducible promoter. T2 relaxation times are significantly lower for samples where bacterioferritin expression is induced and iron is supplemented to the growth medium.

Figure 3: (Left) Influence of bacterioferritin on the T2 relaxation time. T7lacO-bfr has bacterioferritin expression controlled by an IPTG-inducible promoter, while pLux-bfr M52H represents our E. coli Nissle transformed with bacterioferritin under the control of an AHL-responsive promoter. Both strains show a significant drop in the signal caused by bacterioferritin overexpression. (Right) MRI T2-weighted image of six samples in 0.5 mL microcentrifuge tubes, transversal section. Samples 4 and 5 (both of bacteria grown in iron-supplemented medium and induced with AHL, T2 = 175 ms and T2 = 155 ms respectively) appear visibly darker than the rest. Sample 1 is pure PBS (T2 = 332 ms), sample 2 is PBS supplemented with ferric citrate (T2 = 316 ms), sample 3 is a bfr-knockout grown without iron supplementation (T2 = 343 ms), while sample 6 is T7lacO-bfr, also grown without iron supplementation (T2 = 326 ms).

All the bacteria were resuspended and imaged in PBS, after washing of the culture medium. To test if any unwashed iron could mask the signal, the T2 relaxation time of pure PBS was compared to PBS supplemented with iron. The results showed that the free iron in the medium only slightly changes the signal (T2 = 332 ms in PBS and T2 = 316 ms in iron-supplemented PBS) and should not interfere with the measurements (Figure 4).

As observed with other strains (Table 1), the bfr-knockout strain experienced a change in the signal when grown in an iron-supplemented medium, suggesting that even in the absence of bacterioferritin, other iron-storage systems present in the bacteria contribute to iron binding and background noise (Table 1, Figure 4).

Figure 4: Influence of iron on the T2 relaxation time. PBS supplemented with 150 µM of ferric citrate showed an insignificant change in the signal when compared to pure PBS. bfr-knockout experienced a drop in the signal when grown in an iron-supplemented medium.

Summary

We believe the results demonstrate the usability of bacterioferritin as an MRI contrast agent in vitro and confirms the potential to use it as an in vivo reporter of tumor sensing. All samples where bacterioferrition overexpression was induced and iron supplementation was provided showed a significant change in the signal, as expected. There was, however, a more modest level of signal change experienced by all bacteria grown in an iron-supplemented medium, which can probably be contributed to presence of various inherent bacterial iron-storage proteins, as the knockout experiments suggest.

• We characterized the expression of a genetically encoded MRI contrast agent bacterioferritin in E. coli Nissle 1917.
• We contributed to parameter fitting of the model.
• In an MRI imaging session, we showed that bacterioferritin expressed in our strain indeed leads to a marked decrease in the MRI signal which demonstrates its usability as an MRI contrast agent in vitro and confirms the potential to use it as an in vivo reporter of tumor sensing.

References

1. Cohen, Batya et al. “Ferritin as an Endogenous MRI Reporter for Noninvasive Imaging of Gene Expression in C6 Glioma Tumors.” Neoplasia (New York, N.Y.) 7.2 (2005): 109–117. Print.
2. Hill, Philip J., et al. "Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli." PLoS One 6.10 (2011): e25409.