Listeria monocytogenes GlmR is an accessory uridyltransferase essential for cytosolic survival and virulence

Abstract

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions for these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell wall stress, cytosolic survival, inflammasome avoidance and ultimately virulence in vivo. A genetic suppressor screen revealed that blocking utilization of UDP-GlcNAc by a non-essential wall teichoic acid decoration pathway restored resistance to cell wall stress and partially restored virulence of ΔglmR mutants. In parallel, metabolomics revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggest that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell wall stress responses and virulence in vivo. Taken together these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes.ImportanceBacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK) to survive in the host cytosol. GlmR is important for resistance to some cell wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell wall metabolite, UDP-GlcNAc, and suppressors which increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc and this enzymatic activity is conserved in pathogens from Firmicutes and Actinobacteria phyla. These results highlight the importance accessory cell wall metabolism enzymes in responding to cell wall stress in a variety of bacterial pathogens.

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

“…Using a previously described luciferase-based assay to quantify cytosolic plasmid release, we confirmed that the Δ menB strain exhibited increased intracellular bacteriolysis ( Figure 5D ; Sauer et al, 2010 ). We further found that NOX expression rescued Δ menB bacteriolysis, but not a comparable bacteriolysis phenotype in a Δ glmR strain that was previously shown to result from unrelated deficiencies in cell wall biosynthesis ( Figure 5D ; Pensinger et al, 2021 ). These studies thus show that efficient NAD + regeneration is essential for limiting cytosolic bacteriolysis and suggest a model whereby respiration-mediated NAD + regeneration promotes virulence, in part, by maintaining cell viability and facilitating evasion of innate immunity ( Figure 6 ).…”

Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

“…42 We further found that NOX expression rescued ΔmenB bacteriolysis, but not a comparable bacteriolysis phenotype in a ΔglmR strain that was previously shown to result from unrelated deficiencies in cell wall biosynthesis (Fig 5d). 52 These studies thus show that efficient NAD + regeneration is essential for limiting cytosolic bacteriolysis and suggest a model whereby respiration-mediated NAD + regeneration promotes virulence, in part, by maintaining cell viability and facilitating evasion of innate immunity (Fig 5e).…”

Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes

“…Compared to the wildtype, the eslB mutant was more sensitive towards t -Cin and fosfomycin (Fig. 2), which target the UDP-Glc N Ac biosynthetic pathway and MurA, respectively (Marquardt et al, 1994; Pensinger et al, 2021; Sun et al, 2021). In contrast, the resistance towards D-cycloserine, nisin, vancomycin, moenomycin, penicillin, ampicillin and bacitracin (Fig.…”

“…UDP-Glc N Ac-consuming enzymes are labelled in red. Antibiotics targeting different steps of the UDP-Glc N Ac, peptidoglycan and WTA biosynthesis pathways or degrade peptidoglycan are depicted in bold (Campbell et al, 2011; Pensinger et al, 2021; Sarkar et al, 2017).…”

“…Recent findings suggest that the UDP-Glc N Ac biosynthetic pathway can be inhibited by trans -cinnamaldehyde ( t -Cin) (Fig. 1)(Pensinger et al, 2021; Sun et al, 2021). UDP-Glc N Ac is also consumed by TarO/TagO, which catalyzes the first committed step for the synthesis of WTA (Soldo et al, 2002).…”