Inhibition of Bacterial Gene Transcription with an RpoN-Based Stapled Peptide

Abstract

In response to environmental and other stresses, the σ⁵⁴ subunit of bacterial RNA polymerase (RNAP) controls expression of several genes that play a significant role in the virulence of both plant and animal pathogens. Recruitment of σ⁵⁴ to RNAP initiates promoter-specific transcription via the double-stranded DNA denaturation mechanism of the cofactor. The RpoN box, a recognition helix found in the C-terminal region of σ⁵⁴, has been identified as the component necessary for major groove insertion at the -24 position of the promoter. We employed the hydrocarbon stapled peptide methodology to design and synthesize stapled σ⁵⁴ peptides capable of penetrating Gram-negative bacteria, binding the σ⁵⁴ promoter, and blocking the interaction between endogenous σ⁵⁴ and its target DNA sequence, thereby reducing transcription and activation of σ⁵⁴ response genes.

Keywords: DNA-binding proteins; antibiotic resistance; bacterial transcription; cell-penetrating peptides; nitrogen starvation; stapled peptides; σ factor 54.

Figure 1

(A) NMR model of the C-terminal domain of σ⁵⁴ interacting with DNA, with the RpoN box highlighted in red, and the −24 and −12 promoter sequences highlighted in blue. Residues essential for binding which also make key contacts with DNA are highlighted in red on the RpoN sequence. (B) Helical wheel of the RpoN box with staple positions indicated. (C) Linear peptide sequence of Sσ⁵⁴. X = (S)-2-(4′-pentenyl)-alanine, Z = (R)-2-(7′-octenyl)-alanine, B = norleucine. All peptides contain β-alanine in the first position as a spacer (Supplementary Table 1). (D) Circular dichroism spectra of Sσ⁵⁴ peptides.

Figure 2

(A) Fluorescence polarization binding analysis of Sσ⁵⁴ peptides with FITC-glnAp2 30-mer. (B) Gel shift assay showing the association of Sσ⁵⁴-2 with FITC-glnAp2 and the disruption of σ⁵⁴(338-398) bound to FITC-glnAp2. (C) Polyacrylamide gel showing digestion of a 5′-6-FAM glnAp2 218-mer with DNase I. V = vehicle. (D) Densitometric quantification of remaining DNA relative to starting material (lane 3) from (B). Data are represented as mean ± SEM.

Figure 3

(A) Flow cytometry of BW25113 E. coli and PA01 P. aeruginosa treated with 4 μM FITC-Sσ⁵⁴ peptides. Data are represented as mean ± SD. (B) Fluorescence microscopy of BW25113 E. coli. FM 4-64: plasma membrane stain, FITC: Sσ⁵⁴. The scale bars represent 10 μm.

Figure 4

(A) Relative normalized expression of σ⁵⁴-controlled genes after treatment with Sσ⁵⁴. Data are represented as mean ± SEM. N+ denotes nitrogen-rich media while N- indicates nitrogen-deficient media. (B) Glutamine synthetase transferase assay with A₅₄₀ values normalized to OD₆₀₀. Data are represented as mean ± SEM. (C) RNA-Seq heat map for transcripts that are differentially expressed in MG1655 E. coli in response to nitrogen starvation and treatment with peptides. RNA of E. coli grown under nitrogen-rich conditions (N+) was used as a control.