# Identification and Characterization of Genes Required for Listeria monocytogenes Cytosolic Survival

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $716,794

## Abstract

Abstract:
Bacterial pathogens require specific adaptations to survive and thrive in their replicative niche. A subset of
important human pathogens, including Shigella Spp. Rickettsia spp and Burkholderia spp., utilize the cytosol of
host cells as their primary replication niche. Although the host cell cytosol is an inhospitable environment for
bacteria, the stresses encountered in this environment and the mechanisms pathogens utilize to overcome these
stresses are not well understood. The goal of this proposal is to utilize the cytosolic pathogen Listeria
monocytogenes (Lm) to understand how host cells recognize and respond to bacteria in the cytosol and
in turn how cytosolic pathogens have adapted to life in the cytosol.
Lm is a Gram positive foodborne pathogen that causes the rare but often fatal disease Listeriosis. To identify
mechanisms by which Lm has adapted to survive and thrive in the mammalian cytosol, we have utilized a series
of forward genetic screens to identify genes required for Lm cytosolic survival. These screens highlighted robust
cell wall stress responses and specific metabolic adaptations as essential for cytosolic survival and virulence.
Specifically, we demonstrated that the PASTA kinase PrkA is a master regulator of cell wall stress responses
essential for cytosolic survival and virulence in vivo. We defined the phosphoproteome of PrkA and defined the
role of one phosphosubstrate, ReoM, in regulating peptidoglycan synthesis in response to cell wall stress during
infection. GlmR is another PrkA substrate required for cell wall stress responses, cytosolic survival and virulence
in vivo and we defined its role as an accessory uridyltransferase. Finally, an additional gene of unknown function,
yvcJ, was identified through our genetic screens and shown to be involved in cell wall stress responses, cytosolic
survival and virulence. How the remaining PrkA dependent phosphoproteins coordinate cell wall stress
responses, how phosphorylation of GlmR regulates its function, how YvcJ contributes to cell wall stress
responses and finally the mechanism by which the host imparts cell wall stress on bacteria in the cytosol are all
unknown. These questions are the focus of Aim 1.
Our cytosolic survival screens also revealed multiple genes involved in central metabolism, notably the pyruvate
dehydrogenase complex and genes required for DHNA biosynthesis. Mutants lacking these pathways secrete
lactate instead of acetate as the major metabolic byproduct and interventions that restore acetate production
rescue cytosolic survival of DHNA deficient strains. Additionally, we created a series of mutants missing genes
essential for Lm glycerol utilization, hexose phosphate utilization, or both. As the only described carbon sources
utilized by Lm during infection we were surprised to find minimal defects in ex vivo virulence and only 1-2 log
reductions in virulence in mice. How increased concentrations of cytosolic lactate during Lm infection alter...

## Key facts

- **NIH application ID:** 10936212
- **Project number:** 1R01AI184369-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** JOHN-DEMIAN SAUER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $716,794
- **Award type:** 1
- **Project period:** 2024-06-12 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10936212

## Citation

> US National Institutes of Health, RePORTER application 10936212, Identification and Characterization of Genes Required for Listeria monocytogenes Cytosolic Survival (1R01AI184369-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10936212. Licensed CC0.

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