Metabolic changes that promote adaptation of Listeria monocytogenes to oxidative stress and preserving intracellular redox homeostasis

Project Summary
Listeria monocytogenes (LM) is the causative agent of listeriosis, a fatal foodborne infection in humans and
animals. Invasive form of listeriosis has a mortality rate that ranges from approximately 20%-30% or higher
despite antibiotic treatment. Penicillin or ampicillin are reportedly bacteriostatic for LM infections and require
effective host defense mechanisms for bacterial clearance. New effective strategies for successful treatment of
LM invasive infections are urgently needed. The ability of LM to counteract oxidative stress and reactive
oxygen species (ROS) is essential for survival in phagocytic cells and established infection. However, less is
known regarding how LM manages or detoxifies ROS encountered during systemic infection. The goal of this
proposal is to understand how LM adapts its metabolism and metabolic pathways to alleviate deleterious
effects of oxidative stress and adapt to the host environment. The working hypothesis of this application is
that FruR alters LM metabolism by shifting metabolic flux from glycolysis to PPP, this metabolic switch is
critical for LM’s ability to counteract oxidative stress. This hypothesis will be tested with the following three
specific aims: (Aim 1) Explore whether FruR regulates an LM shift from glycolysis to the PPP to combat
oxidative stress. In this aim, we will use metabolomics analysis to identify glycolysis and PPP metabolites
impacted by FruR upon exposure to oxidative stress. We will also determine the role of FruR in rerouting flux
from glycolysis to PPP using 13C-perturbation. (Aim 2) Determine the role of FruR in generating NADPH and
protecting against host oxidative burst. In this aim, we will verify if the ΔfruR strain could be complemented by
expression of NADPH enzymes. We will investigate the contribution of FruR to LM resistance against ROS
produced by the host respiratory burst. (Aim 3) Decipher the role of the oxidative branch of PPP in resistance
of LM to oxidative stress via regulation of redox status. In this aim, we will determine the role of two NADPH-
producing enzymes in maintaining intracellular redox balance using redox-sensitive green fluorescent protein
(GFP) reporter, roGFP2. This study is innovative because it will test a novel mechanism by which LM
responds to oxidative stress and host respiratory burst. This mechanism involves upregulation of PPP,
focusing on metabolic regulation as a mechanism of LM pathogenesis is a highly innovative approach and
could be widespread among other human pathogens. The proposed project is significant because it will (a)
delineate the role of FruR on protecting LM from oxidative stress and innate host defenses, (b) provide an
opportunity to investigate a new connection between metabolism and virulence, (c) provide a set of metabolic
proteins that could be targeted to develop novel therapeutic agents, and (b) enhance understanding of survival
mechanisms and oxidative stress response of other intracellular pathogens.

Grant Number: 1R15AI180880-01
NIH Institute/Center: NIH
Principal Investigator: Hossam Abdelhamed