Detoxification Of Methylglyoxal By The Glyoxalase System Is Required For Glutathione Availability And Virulence Activation In Listeria Monocytogenes

Author summary Listeria monocytogenes is a facultative intracellular pathogen that dramatically changes gene expression upon infection of host cells by activating its major virulence regulator PrfA. The allosteric activator of PrfA is glutathione (GSH) that is produced by the bacterial glutathione synthase GshF. The requirement for GshF can be bypassed by PrfA mutations (PrfA*) that are locked in their active form. In this study we screened for additional mutations that prevented virulence gene expression but were rescued by PrfA*. One of these mutations was in gloA, which encodes glyoxylase A, which detoxifies methylglyoxal (MG) in a glutathione-dependent fashion. In response to MG, gloA mutants up-regulated gshF mRNA but have lower levels of available GSH. These and other data suggest that MG production by the host activates gshF expression leading to elevated GSH levels and increased virulence gene expression.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.