<i>Listeria monocytogenes</i>utilizes glutathione and limited inorganic sulfur compounds as a source of essential L-cysteine

Berude, John C.; Kennouche, Paul; Reniere, Michelle L.; Portnoy, Daniel A.

doi:10.1101/2023.10.16.562582

Cited by 2 publications

(2 citation statements)

References 66 publications

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“…L. monocytogenes synthesizes GSH by the glutathione synthase GshF (25), and also imports GSH via CtaP and OppDF (26). The ΔPDE strain could fully induce actA expression to the activated level in WT, but only upon both GSH supplementation and gshF over-expression ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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C-di-AMP accumulation disrupts glutathione metabolism and inhibits virulence program expression inListeria monocytogenes

Siletti,

Freeman,

et al. 2024

Preprint

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C-di-AMP is an essential second messenger in many bacteria but its levels must be regulated. Unregulated c-di-AMP accumulation attenuates the virulence of many bacterial pathogens, including those that do not require c-di-AMP for growth. However, the mechanisms by which c-di-AMP regulates bacterial pathogenesis remain poorly understood. In Listeria monocytogenes, a mutant lacking both c-di-AMP phosphodiesterases, denoted as the DPDE mutant, accumulates a high c-di-AMP level and is significantly attenuated in the mouse model of systemic infection. All key L. monocytogenes virulence genes are transcriptionally upregulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Our transcriptomic analysis revealed that the DPDE mutant is significantly impaired for the expression of virulence genes within the PrfA core regulon. Subsequent quantitative gene expression analyses validated this phenotype both at the basal level and upon PrfA activation by GSH. A constitutively active PrfA* variant, PrfA G145S, which mimics the GSH-bound conformation, restores virulence gene expression in DPDE but only partially rescues virulence defect. Through GSH quantification and uptake assays, we found that the DPDE strain is significantly depleted for GSH, and that c-di-AMP inhibits GSH uptake. Constitutive expression of gshF (encoding a GSH synthetase) does not restore GSH levels in the DPDE strain, suggesting that c-di-AMP inhibits GSH synthesis activity or promotes GSH catabolism. Taken together, our data reveals GSH metabolism as another pathway that is regulated by c-di-AMP. C-di-AMP accumulation depletes cytoplasmic GSH levels within L. monocytogenes that leads to impaired virulence program expression.

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Section: Resultsmentioning

confidence: 99%

“…In addition to recognizing GSH as a virulence signal, L. monocytogenes also metabolizes GSH as a source of essential cysteine and uses GSH as antioxidant in broth cultures (26, 44). If these functions are relevant in the mammalian host, they can also explain why the ΔPDE prfA * strain is not fully restored for virulence.…”

Section: Discussionmentioning

confidence: 99%

C-di-AMP accumulation disrupts glutathione metabolism and inhibits virulence program expression inListeria monocytogenes

Siletti,

Freeman,

et al. 2024

Preprint

View full text Add to dashboard Cite

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C-di-AMP accumulation disrupts glutathione metabolism in Listeria monocytogenes

Siletti,

Freeman,

Dang

et al. 2024

Infect Immun

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C-di-AMP homeostasis is critical for bacterial stress response, cell wall integrity, and virulence. Except for osmotic stress response, the molecular mechanisms underlying other processes are not well defined. A Listeria monocytogenes mutant lacking both c-di-AMP phosphodiesterases, denoted as the ΔPDE mutant, is significantly attenuated in the mouse model of systemic infection. We utilized the ΔPDE mutant to define the molecular functions of c-di-AMP. RNAseq revealed that the ΔPDE mutant is significantly impaired for the expression of virulence genes regulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Subsequent quantitative gene expression analyses revealed that the ΔPDE strain is defective for PrfA-regulated gene expression both at the basal level and upon activation by GSH. We further found the ΔPDE strain to be significantly depleted for cytoplasmic GSH and impaired for GSH uptake. The ΔPDE strain was also deficient in GSH under conditions that activate GSH synthesis by the synthase GshF and upon constitutive expression of gshF , suggesting that c-di-AMP accumulation inhibits GSH synthesis activity or promotes GSH catabolism. A constitutively active PrfA* variant restored virulence gene expression in ΔPDE in broth cultures supplemented with GSH but did not rescue virulence defect in vivo . Therefore, virulence attenuation at high c-di-AMP is likely associated with defects outside of the PrfA regulon. For instance, the ΔPDE strain was sensitive to oxidative stress, a phenotype exacerbated in the absence of GshF. Our data reveal GSH metabolism as another pathway that is regulated by c-di-AMP. IMPORTANCE C-di-AMP regulates both bacterial pathogenesis and interactions with the host. Although c-di-AMP is essential in many bacteria, its accumulation also attenuates the virulence of many bacterial pathogens. Therefore, disrupting c-di-AMP homeostasis is a promising antibacterial treatment strategy and has inspired several studies that screened for chemical inhibitors of c-di-AMP phosphodiesterases. However, the molecular functions of c-di-AMP are still not fully defined, and the underlying mechanisms for attenuated virulence at high c-di-AMP levels are unclear. Our analyses in Listeria monocytogenes indicate that virulence-related defects are likely outside of the virulence gene regulon. We found c-di-AMP accumulation to impair L. monocytogenes virulence gene expression and disrupt GSH metabolism. Further studies are necessary to establish the relative contributions of these regulations to virulence and host adaptation.

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Listeria monocytogenesutilizes glutathione and limited inorganic sulfur compounds as a source of essential L-cysteine

Cited by 2 publications

References 66 publications

C-di-AMP accumulation disrupts glutathione metabolism and inhibits virulence program expression inListeria monocytogenes

C-di-AMP accumulation disrupts glutathione metabolism and inhibits virulence program expression inListeria monocytogenes

C-di-AMP accumulation disrupts glutathione metabolism in Listeria monocytogenes

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