Evolution of Polymyxin Resistance Regulates Colibactin Production in Escherichia coli

Abstract

The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can promote colorectal cancer through DNA double stranded breaks and interstrand cross-links. While the structure and biosynthesis of colibactin have been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by E. coli are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC, NC101; the probiotic strain, Nissle 1917; and the antibiotic testing strain, ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb's by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with a heightened tolerance for polymyxin induced greater mammalian DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.

Figure 1.

Differentiation of WT and polymyxin B evolved E. coli cultures. a) Schematic of in vitro E. coli evolution. From a parent culture, three lineages of E. coli were evolved with increasing pressure of polymyxin B (PmB). Lineages were first passaged into 0.5 μg mL⁻¹ PmB, and once significant growth was observed, cultures were passaged into fresh media containing a 100% increase in PmB concentration. This was repeated until the three lineages were able to grow in 8 μg mL⁻¹ PmB. These strains were denoted as NC101 PmB008R-[1–3] b) Antibiotic activity assays of NC101 WT and NC101 PmB008R-[1–3] cultures (n = 3) after growth in varying concentrations of PmB (in μg mL⁻¹). % activity represents the efficacy of PmB in each concentration compared to ampicillin and water controls, and is calculated based on formula (1). Error bars represent +/− 1 standard deviation away from the mean value. ‡ represents activity of −66 ± 43%. Brackets represent estimated the expected range of the minimum inhibitory concentration for each of the isolates. c) Volcano plots of proteomic changes in NC101 PmB008R-[1–3] compared to NC101 WT (n = 4). Vertical lines represent the cutoff used to determine significant proteomic changes (|log₂(FC)| > 1). Horizontal lines represent the threshold used to determine significant false discovery rates determined by an FDR-corrected two-sided t-test (−log₁₀(q-value) > 1.3). d) Principal component analysis of proteomic replicates of each strain analyzed.

Figure 2.

Heatmap showing cellular pathways with significantly changing proteins across evolved strains. Colors depict log₂(FC) relative to NC101 WT, as represented in the figure key. Bolded pathways depict proteins important protein FC values determined through manual annotation. A complete list of protein accessions in this table is supplied in Table S2.

Figure 3.

Regulation of the pks island in evolved E. coli lineages. a) Layout of the pks island in the E. coli NC101 genome. Transcription start sites for clbB and clbA are shown with arrows. The variable numbers of tandem repeats (VNTR) region is depicted in pink. b) Relative abundance of pks island gene products that were significantly changing in at least one evolved strain, each dot represents a single replicate (n = 4). Data points in red represent conditions with a significant difference compared to the WT condition (blue). Data points in gray represent conditions that were not significantly changing based on two criteria: log₂(FC) > 1 and −log₁₀(q-value) > 1.3 based on an FDR-corrected two-sided t-test. Center lines in the box plots represent mean values for the respective conditions, surrounded by the first and third quartiles. Whiskers extend from the hinge to the highest and lowers values that is within 1.5 * the inner quartiles. c) qPCR data representing transcriptional regulation of clb genes of the WT and evolved strains in the absence of polymyxin B. Each dot represents a single biological replicate (n = 3). Lines are at mean +/− SEM. Significance was noted at p < 0.05 by one-way ANOVA d) Changes in the colibactin prodrug cleavage motif (red) upon colibactin activation by ClbP in the WT and evolved strains in the absence of polymyxin B. Each dot represents a single biological replicate (n = 3). Significance was noted at p < 0.05 by a two-sided t-test. Center lines in the box plots represent mean values for the respective conditions, surrounded by the first and third quartiles. Whiskers extend from the hinge to the highest and lowers values that is within 1.5 * the inner quartiles.

Figure 4.

Genotoxicity of E. coli NC101 and evolved lineages. a) Representative microscopy images from IEC-6 rat epithelial cells infected with the following strains of NC101 at a multiplicity of infection (MOI) = 100: NC101 WT, NC101 Δpks (colibactin-deficient), PmB evolved isolates PmB008R-[1–3]. Uninfected samples show baseline staining, while Staurosprine treated samples serve as a positive control for pan-nuclear γH2AX staining indicating apoptosis, rather than punctate staining from DNA damage. Nuclei (Blue, DAPI channel) and γH2AX (Red, TxRed channel) are shown for each group. Scale bars = 25 μM. b) Density plots of quantified γH2AX signal intensity. Significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001), denoted by lines to the right of density plots, was determined via t-test with Bonferonni correction. Center lines in the box plots represent mean values for the respective conditions, surrounded by the first and third quartiles. Whiskers extend from the hinge to the highest and lowers values that is within 1.5 * the inner quartiles. Outlier data beyond the end of the whiskers are plotted as points.

Figure 5.

Quantitative proteomics of Clb enzymes in PmB treated pks+ E. coli strains. Data depict log₂(FC) of Clb enzymes from NC101 (n = 4), Nissle 1917 (n = 4), and ATCC 25922 (n = 3) relative to untreated cultures of each strain, respectively. Proteins highlighted in red and labelled were in significantly higher abundance in PmB treated cultures compared to WT cultures meeting the significance criteria of log₂(FC) > 1 and −log₁₀(q-value) > 1.3 based on an FDR-corrected two-sided t-test.