Detoxification of methylglyoxal by the glyoxalase system is required for glutathione availability and virulence activation in Listeria monocytogenes

Abstract

Listeria monocytogenes is a Gram-positive, food-borne pathogen that lives a biphasic lifestyle, cycling between the environment and as a facultative intracellular pathogen of mammals. Upon entry into host cells, L. monocytogenes upregulates expression of glutathione synthase (GshF) and its product, glutathione (GSH), which is an allosteric activator of the master virulence regulator PrfA. Although gshF mutants are highly attenuated for virulence in mice and form very small plaques in host cell monolayers, these virulence defects can be fully rescued by mutations that lock PrfA in its active conformation, referred to as PrfA*. While PrfA activation can be recapitulated in vitro by the addition of reducing agents, the precise biological cue(s) experienced by L. monocytogenes that lead to PrfA activation are not known. Here we performed a genetic screen to identify additional small-plaque mutants that were rescued by PrfA* and identified gloA, which encodes glyoxalase A, a component of a GSH-dependent methylglyoxal (MG) detoxification system. MG is a toxic byproduct of metabolism produced by both the host and pathogen, which if accumulated, causes DNA damage and protein glycation. As a facultative intracellular pathogen, L. monocytogenes must protect itself from MG produced by its own metabolic processes and that of its host. We report that gloA mutants grow normally in broth, are sensitive to exogenous MG and severely attenuated upon IV infection in mice, but are fully rescued for virulence in a PrfA* background. We demonstrate that transcriptional activation of gshF increased upon MG challenge in vitro, and while this resulted in higher levels of GSH for wild-type L. monocytogenes, the glyoxalase mutants had decreased levels of GSH, presumably due to the accumulation of the GSH-MG hemithioacetal adduct. These data suggest that MG acts as a host cue that leads to GSH production and activation of PrfA.

The aldehyde hypothesis: metabolic intermediates as antimicrobial effectors

“…Furthermore, depletion of glucose from cell culture media reverses the sensitivity of a gloA mutant in these neutrophils, emphasizing the importance of glycolysis in the production of one or more toxic metabolites. More recently, Portnoy and co-workers [ 44 ] showed that mutants of Listeria monocytogenes lacking GloA are highly attenuated in mice. Because this phenotype is rescued by constitutive activation of PrfA, a bacterial transcription factor activated upon entry into host cell cytosol, the authors speculate that host-derived MG is a cue that bacteria sense to activate transcription of virulence genes.…”

Aldehyde accumulation inMycobacterium tuberculosiswith defective proteasomal degradation results in copper sensitivity

“…More broadly, aldehydes may also have a role in the defense against other microbes. For example, a recent report by the Portnoy lab showed that in Listeria monocytogenes (L. monocytogenes ), MG activates transcription of glutathione (GSH) synthase-encoding gene gshF , leading to increased GSH production and thereby activating the master virulence regulator PrfA (52). A L. monocytogenes mutant that lacks a gene encoding glyoxalase A, a key enzyme in methylglyoxal detoxification, has decreased GSH levels in vitro and is attenuated for infection.…”

Destroying glutathione peroxidase improves the oxidative stress resistance and pathogenicity of Listeria monocytogenes

“…The amount of GSH could be calculated by subtracting the amount of GSSG from the total glutathione (GSSG + GSH). GSH concentrations of the parental and mutant strains were measured as described above ( Anaya-Sanchez et al, 2021 ). A 96-well plate was used for measurement, and the standards and samples were added in sequence.…”