Peptidoglycan Recognition Proteins Kill Bacteria by Inducing Oxidative, Thiol, and Metal Stress

Abstract

Mammalian Peptidoglycan Recognition Proteins (PGRPs) are a family of evolutionary conserved bactericidal innate immunity proteins, but the mechanism through which they kill bacteria is unclear. We previously proposed that PGRPs are bactericidal due to induction of reactive oxygen species (ROS), a mechanism of killing that was also postulated, and later refuted, for several bactericidal antibiotics. Here, using whole genome expression arrays, qRT-PCR, and biochemical tests we show that in both Escherichia coli and Bacillus subtilis PGRPs induce a transcriptomic signature characteristic of oxidative stress, as well as correlated biochemical changes. However, induction of ROS was required, but not sufficient for PGRP killing. PGRPs also induced depletion of intracellular thiols and increased cytosolic concentrations of zinc and copper, as evidenced by transcriptome changes and supported by direct measurements. Depletion of thiols and elevated concentrations of metals were also required, but by themselves not sufficient, for bacterial killing. Chemical treatment studies demonstrated that efficient bacterial killing can be recapitulated only by the simultaneous addition of agents leading to production of ROS, depletion of thiols, and elevation of intracellular metal concentrations. These results identify a novel mechanism of bacterial killing by innate immunity proteins, which depends on synergistic effect of oxidative, thiol, and metal stress and differs from bacterial killing by antibiotics. These results offer potential targets for developing new antibacterial agents that would kill antibiotic-resistant bacteria.

Ca 2+ -Induced Two-Component System CvsSR Regulates the Type III Secretion System and the Extracytoplasmic Function Sigma Factor AlgU in Pseudomonas syringae pv. tomato DC3000

“…The ΔcvsR strain demonstrated increased expression of catalase and genes related to methionine uptake as well as decreased expression of some thiol biosynthesis-related genes (Table 1). Similar gene expression patterns were seen in Escherichia coli during treatment with toxic levels of homocysteine and in E. coli treated with antimicrobial peptidoglycan recognition proteins (51,52). This suggests that the bacteria are under distress when grown in a Ca 2ϩ -supplemented medium.…”

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). In B. subtilis, treatment with the synthetic peptide MP196 was shown to dislodge the electron carrier protein cytochrome-c (which plays the role of the E. coli ubiquinones) from the cytoplasmic membrane (4).…”

Generation of reactive oxygen species by lethal attacks from competing microbes

“…1D), indicating the toxicity of this single T6SS effector is sufficient to induce ROS. Interestingly, certain mammalian innate immunity proteins that also target bacterial peptidoglycan have recently been shown to induce oxidative stress based on transcriptomic analysis (32).…”