Effect of Intracellular Expression of Antimicrobial Peptide LL-37 on Growth of Escherichia coli Strain TOP10 under Aerobic and Anaerobic Conditions

Abstract

Antimicrobial peptides (AMPs) can cause lysis of target bacteria by directly inserting themselves into the lipid bilayer. This killing mechanism confounds the identification of the intracellular targets of AMPs. To circumvent this, we used a shuttle vector containing the inducible expression of a human cathelicidin-related AMP, LL-37, to examine its effect on Escherichia coli TOP10 under aerobic and anaerobic growth conditions. Induction of LL-37 caused growth inhibition and alteration in cell morphology to a filamentous phenotype. Further examination of the E. coli cell division protein FtsZ revealed that LL-37 did not interact with FtsZ. Moreover, intracellular expression of LL-37 results in the enhanced production of reactive oxygen species (ROS), causing lethal membrane depolarization under aerobic conditions. Additionally, the membrane permeability was increased after intracellular expression of LL37 under both aerobic and anaerobic conditions. Transcriptomic analysis revealed that intracellular LL-37 mainly affected the expression of genes related to energy production and carbohydrate metabolism. More specifically, genes related to oxidative phosphorylation under both aerobic and anaerobic growth conditions were affected. Collectively, our current study demonstrates that intracellular expression of LL-37 in E. coli can inhibit growth under aerobic and anaerobic conditions. While we confirmed that the generation of ROS is a bactericidal mechanism for LL-37 under aerobic growth conditions, we also found that the intracellular accumulation of cationic LL-37 influences the redox and ion status of the cells under both growth conditions. These data suggest that there is a new AMP-mediated bacterial killing mechanism that targets energy metabolism.

Combined Systems Approaches Reveal a Multistage Mode of Action of a Marine Antimicrobial Peptide against Pathogenic Escherichia coli and Its Protective Effect against Bacterial Peritonitis and Endotoxemia

“…However, in our study, downregulated NADH dehydrogenase may significantly contribute to the change in ΔΨ and reduced respiration ( ttrB ) may lead to a decline in energy consumption (Fig. 4c and 6) (54). …”

“…N4 might trigger disruption of the plasma membrane directly or indirectly by inducing apoptosis-like cell death via intracellular ROS accumulation (Fig. 6), which may cause lethal membrane depolarization (54). …”

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15

“…Expression of LL-37 from a plasmid in the E. coli cytoplasm led to enhanced fluorescence from the ROS reporter dye DCFH-DA, detected on a 2-h timescale (30). In both Bacillus subtilis and E. coli, the natural antibacterial agent peptidoglycan recognition protein (PGRP) enhanced fluorescence of the •OH reporter dye HPF, detected on a 1-h timescale (31).…”

TheC. difficile clnRABoperon initiates adaptations to the host environment in response to LL-37

“…3 ). The regulation of metabolic pathways in response to LL-37 was previously observed in other pathogens, including S. pyogenes , E. coli, P. aeruginosa , and S. pneumonia e, but their role in the bacterial response to LL-37 has not been clear (43, 46-48). Our data indicate that the regulation of genes by LL-37/ClnRAB in vivo has notable effects on C. difficile colonization and virulence ( Fig.…”