Azure Imagers used to better understand the inhibitory mechanism of gut-derived colibactin production

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The gut microbiota is made up of the trillions of microorganisms that colonize the human gut and have a significant impact on human health through their secreted products.  These microbes can be commensal or pathogenic, and some have been connected to the development of colorectal cancer. Colibactin is a common genotoxin produced in the gut by bacteria, such as E. coli, and colibactin-mediated DNA damage appears to play a role in colorectal cancer development. The Azure Sapphire and the Azure 300 imagers helped identify a small molecule inhibitor that prevents bacterial genotoxin production in a recent study by Volpe et. al1 at Harvard University.

Sapphire Helps Assess Inhibitor’s Specificity

Colibactin is a known genotoxic bacterial product produced by a non-ribosomal peptide synthetase called polyketide synthase and encoded by the gene pks. This pks gene is carried by many Escherichia coli strains (pks+ E. coli).

To determine the inhibitors’ mechanism of action, the researchers initially examined the structure of ClbP and found the inhibitors mimicked some intermediates in the hydrolysis of precolibactin.

The researchers tested the effectiveness of Inhibitor 3, the most potent of the four inhibitors, against pks+ E. coli. Using metabolomics, this inhibitor was observed to be able to block the colibactin biosynthesis while only minimally disrupting other metabolic functions.

Volpe et. al used an activity-based protein profile (ABPP) to find additional targets of their four inhibitors. In this gel-based assay, small molecules that bind to a target protein are detected by the target protein’s decreased ability to bind to a nonspecific fluorophosphonate (FP) probe compound. Using the Azure Sapphire to image the gels, no measurable differences were found (Image 4). This demonstrates a lack of additional inhibitor targets and their specificity to ClbP.

Western blots from Azure Sapphire used by Volpe et al
Image 4 from Volpe et. al. (2022) examining the efficiency of their ClbP inhibitors. The Azure Sapphire Biomolecular Imager was used in Figure 4C to examine secondary targets of the inhibitors via a ABPP assay.

Examining the Impact of Inhibitor 3 on Genotoxic Effects of Colibactin using the Azure 300

The researchers assessed whether Inhibitor 3 could inhibit the genotoxic effects of colibactin on human cells. They exposed HeLa cells to a pks+ strain of E. coli, and treated the cells with Inhibitor 3. The results indicated Inhibitor 3 is able to inhibit the genotoxic effects of colibactin in human cells, as determined by the number of cells experiencing cell-cycle arrest post treatment. It suppressed the DNA alkylating activity caused by colibactin comparable to what is observed with a genetic deletion of clbP.

Volpe et al also looked at the effects on the colibactin-induced DNA damage response. FANCD2 is a protein known to be monoubiquinated (FANCD2-Ub) in response to stalled replication forks. Previous studies show when cells are missing FANCD2, there is an increased sensitivity to colibactin3. When HeLa cells exposed to pks+ E. coli were treated with Inhibitor 3, the ubiquitination of FANCD2 was prevented, which was observed using Western blot imaged on the Azure 300.

Western blot from Volpe et al imaged with Azure 300 imager
Summary image from the abstract of Volpe et. al. representing the basic dynamic between E. coli, colibactin, and DNA damage, as well as where the created ClbP inhibitors function.

This same effect on the DNA damage response was not seen in the presence of other DNA damaging agent. This indicates Inhibitor 3 is specific to the colibactin biosynthetic pathway and not merely the DNA damage response; it ultimately inhibits the genotoxicity caused by colibactin.

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Designing and Selecting Potential Inhibitors

Considering the proposed structure of colibactin and knowing colibactin-activating peptidase ClbP is involved in its biosynthesis, the team designed and characterized a series of inhibitors that target ClbP2.

ClbP recognizes a motif not commonly found in substrates other than colibactin. so choosing it as a target reduces the change of secondary, off-target effects from its inhibition. The researchers found four inhibitors with potential using enzymatic activity assays.

Genotoxic Bacterial Products and the Gut Microbiome

Colibactin-mediated DNA damage appears to play a role in colorectal cancer development. Additionally, colorectal cancer patients are more frequently reported to have pks+ E.coli and mouse models show an increased tumor load when colonized with pks+ E. coli. While there is a strong correlation between colibactin and colorectal cancer, markers of colibactin mutations have been observed in normal patient samples as well. This indicates the exact timing and duration of exposure to colibactin is likely an important factor influencing colorectal cancer risk and is still poorly understood.

It would be ideal to study the effects of colibactin in the natural gut environment; however, the complexity of such an environment would make it difficult to pinpoint colibactin-specific effects. Genetic manipulations of colibactin could affect the expression or functions of other genes, including structural components, and could confound the results. Since timing and exposure to colibactin is likely important in colorectal cancer development, these variables need to be accounted for in a study of this relationship.

Volpe et. al. reasoned a compound that could specifically inhibit colibactin production would allow for the complex microbiota to remain intact while assessing the specific effects of colibactin itself.

Effects of the Inhibitor on a Complex Microbial Community

Considering the inhibitors would be used in the context of the gut microbiota, the researchers examined how Inhibitor 3 would affect other members of this complex microbe community and if these conditions affected the inhibitor’s effectiveness.

These results showed in a complex microbe environment, Inhibitor 3 would likely be able to maintain its efficacy and not damage other key members of the gut microbiota.

The Findings

By targeting the biosynthetic pathway, Inhibitor 3 abrogates colibactin and completely blocks the genotoxic effects, usually observed when mammalian cells are exposed to colibactin in culture. This precise control presents an opportunity to study natural products secreted in complex microbial communities and determine potential therapeutic strategies.

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SOURCES

  1. Volpe, M.R., Velilla, J.A., Daniel-Ivad, M. et al. A small molecule inhibitor prevents gut bacterial genotoxin production. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01147-8
  2. Dubois, D., Baron, O., Cougnoux, A. al. ClbP Is a Prototype of a Peptidase Subgroup Involved in Biosynthesis of Nonribosomal Peptides. Journal of Biological Chemistry (2011).
  3. Bossuet-Greif, N., Vignard, J., Taieb, F., Mirey, G., Dubois, D., Petit, C., Oswald, E., & Nougayrède, J.-P. . The colibactin genotoxin generates DNA Interstrand Cross-Links in infected cells. MBio (2018). https://doi.org/10.1128/mbio.02393-17
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