Peptidoglycan recognition proteins kill bacteria by inducing oxidative, thiol, and metal stress.

Des Raj Kashyap,Annemarie Rompca,John D Helmann,Jefferson Chan,Ahmed Gaballa,Roman Dziarski,Dipika Gupta,Christopher J Chang,Iztok Hozo,Dana J Philpott

doi:10.1371/journal.ppat.1004280

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.

Highlights

Mammalian Peptidoglycan Recognition Proteins (PGRPs) are a family of four evolutionary conserved antibacterial innate immunity proteins [1,2,3]
New targets for developing new approaches to antibacterial therapy are needed, because discovering new or improving current antibiotics have become increasingly difficult. One such approach is developing new antibacterial agents based on the antibacterial mechanisms of bactericidal innate immunity proteins, such as human peptidoglycan recognition proteins (PGRPs)
We previously proposed that PGRPs kill bacteria by inducing toxic oxygen by-products (‘‘reactive oxygen species’’, ROS) in bacteria

Summary

Introduction

Mammalian Peptidoglycan Recognition Proteins (PGRPs) are a family of four evolutionary conserved antibacterial innate immunity proteins [1,2,3]. PGRPs activate envelope stress responses in bacteria, which results in membrane depolarization and intracellular production of toxic hydroxyl radicals (HON), which leads to energy depletion and inhibition of intracellular synthesis of peptidoglycan, proteins, RNA, and DNA, and cell death [7]. Bactericidal PGRPs do not inhibit extracellular peptidoglycan synthesis, do not hydrolyze the cell wall, and do not kill by permeabilizing bacterial membranes, or by osmotic lysis [4,5,7]. The induction of envelope stress by PGRPs in two model Grampositive and Gram-negative bacteria is to a large extent dependent on the inappropriate over-activation of two-component systems that normally function to detect and dispose of misfolded proteins in bacteria, CssRS in Bacillus subtilis, and CpxRA in Escherichia coli [7]. The exact nature of the signal that activates CssRS and CpxRA is not known, because these two-component systems respond to many other types of stress besides misfolded proteins, including pH, osmolarity, Cu, and Zn [8]

Objectives

Methods

Results

Discussion

Conclusion