Genome-wide transcriptional profiling of the Escherichia coli response to a proline-rich antimicrobial peptide

Abstract

Most antimicrobial peptides (AMPs) impair the viability of target bacteria by permeabilizing bacterial membranes. However, the proline-rich AMPs have been shown to kill susceptible organisms without causing significant membrane perturbation and may act by inhibiting the activity of bacterial targets. To gain initial insight into the events that follow interaction of a proline-rich peptide with bacterial cells, we used DNA macroarray technology to monitor transcriptional alterations of Escherichia coli in response to challenge with a subinhibitory concentration of the proline-rich Bac7(1-35). Substantial changes in the expression levels of 70 bacterial genes from various functional categories were detected. Among these, 26 genes showed decreased expression, while 44 genes, including genes that are potentially involved in bacterial resistance to antimicrobials, showed increased expression. The generation of a transcriptional response under the experimental conditions used is consistent with the ability of Bac7(1-35) to interact with bacterial components and affect biological processes in this organism.

FIG. 1.

Growth rate of E. coli MG1655 in the absence (control) or presence of 1 or 2.5 μM Bac7(1-35) or 2.5 μM Bac7(5-35). Data are means ± standard deviations of at least three experiments.

FIG. 2.

Northern blot analyses of total RNA extracted from E. coli MG1655 after 30 min of incubation in the absence (−) or presence (+) of 2.5 μM Bac7(1-35). Blots were hybridized with gene-specific probes, as indicated. Hybridization with the rrlH probe served as a control for the amount and quality of RNA loaded in each lane.

FIG. 3.

Northern blot analyses of basS and ompT gene expression. Total RNA was extracted from E. coli MG1655 after incubation of bacteria in the presence (+) or absence (−) of 2.5 μM peptide for the indicated times. Blots were hybridized with basS-, ompT-, or rrlH-specific cDNA probes.

FIG. 4.

Northern blot analysis of RNA extracted from E. coli MG1655 exposed to 1 μM (open bars) and 2.5 μM (filled bars) Bac7(1-35) for 30 min. The expression levels of the indicated genes were determined as described in Materials and Methods and are reported as levels of induction (n-fold) in peptide-treated cultures relative to that in untreated bacteria. Data representative of two independent experiments with similar results are shown.

FIG. 5.

(A) Induction of basS and ompT gene expression in response to LL-37, PG-1, and PB. Total RNA was extracted from E. coli MG1655 exposed for 15 min to each peptide at the indicated concentrations. Blots were hybridized with basS-, ompT-, and rrlH-specific cDNA probes. The expression levels of basS (open bars) and ompT (filled bars) were determined as described in Materials and Methods and reported as levels of induction (n-fold) in peptide-treated cultures relative to those in cultures of untreated bacteria. Data representative of two independent experiments with similar results are shown. (B) Activity of LL-37, PG-1, and PB against E. coli MG1655. Bacterial cultures were incubated for 15 min at 37°C in the presence of increasing amounts of each peptide, and the numbers of CFU per milliliter were determined. Data are means ± standard deviations of at least three experiments.