Listeria monocytogenes TcyKLMN cystine/cysteine transporter facilitates glutathione synthesis and virulence gene expression

Listeria monocytogenes (Lm) is a saprophyte and a human intracellular pathogen. Upon invasion into mammalian cells, it senses multiple metabolic and environmental signals that collectively trigger its transition to the pathogenic state. One of these signals is the tripeptide glutathione, which acts as an allosteric activator of Lm’s master virulence regulator, PrfA. While glutathione synthesis by Lm was shown to be critical for PrfA activation and virulence gene expression, it remains unclear how this tripeptide is synthesized under changing environments, especially in light of the observation that Lm is auxotrophic to one of its precursors, cysteine. Here, we show that the ABC transporter TcyKLMN is a cystine/cysteine importer that supplies cysteine for glutathione synthesis, hence mediating the induction of the virulence genes. Further, we demonstrate that this transporter is negatively regulated by three metabolic regulators: CodY, CymR and CysK, which sense and respond to changing concentrations of branched chain amino acids (BCAA) and cysteine. The data indicate that under low concentrations of BCAA, TcyKLMN is up-regulated, driving the production of glutathione by supplying cysteine, thereby facilitating PrfA activation. These findings provide molecular insight into the coupling of Lm metabolism and virulence, connecting BCAA sensing to cysteine uptake and glutathione biosynthesis, as a mechanism that controls virulence gene expression. This study exemplifies how bacterial pathogens sense their intracellular environment and exploit essential metabolites as effectors of virulence.ImportanceBacterial pathogens sense the repertoire of metabolites in the mammalian niche and use this information to shift into a pathogenic state to accomplish successful infection. Glutathione is a virulence-activating signal that is synthesized by L. monocytogenes during infection of mammalian cells. In this study, we show that cysteine uptake via TcyKLMN drives glutathione synthesis and virulence gene expression. The data emphasize the intimate cross-regulation between metabolism and virulence in bacterial pathogens.

MYCN mediates cysteine addiction and sensitizes to ferroptosis

Aberrant expression of MYC family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYC induces massive lipid peroxidation upon depletion of cysteine, the rate-limiting amino acid for glutathione biosynthesis and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. In MYCN-amplified childhood neuroblastoma, MYCN mediates resistance to ferroptosis by activating transsulfuration of methionine to cysteine. MYCN may contribute to spontaneous tumor regression in low-risk neuroblastomas by promoting ferroptosis in cells with epigenetically silenced cystathionine-beta-synthase, the rate-limiting enzyme for transsulfuration. We identified enzymes and antiporter proteins crucial to ferroptotic escape, providing multiple previously unknown sites that may be acted on therapeutically.

CREB1 and ATF1 Negatively Regulate Glutathione Biosynthesis Sensitizing Cells to Oxidative Stress

The cAMP response element binding protein (CREB) family activating transcription factor 1 (ATF1) and cAMP response element binding protein 1 (CREB1) have been reported in a diverse group of tumors, however, the mechanistic basis for this remains unclear. Here we found that CREB1 and ATF1 unexpectedly regulate glutathione (GSH) biosynthesis by suppressing the expression of glutamate-cysteine ligase modifier subunit (GCLM) and glutathione synthase (GSS), two key enzymes of GSH biosynthesis pathway. Mechanistic studies reveal that GCLM and GSS are direct transcriptional targets of CREB1 and ATF1. Through repressing the expression of these two enzymes, CREB1 and ATF1 reduce the GSH biosynthesis and the capability of cells to detoxicate reactive oxygen species (ROS), thereby increasing cellular susceptibility to oxidative stress. Therefore, our findings link CREB1 family to cellular metabolism, and uncover a potential therapeutic approach by targeting GCLM or oxidative stress for the treatment of tumors with relatively high expression of CREB1 family proteins.

Functional prediction of environmental variables using metabolic networks

In this manuscript, we propose a novel approach to assess relationships between environment and metabolic networks. We used a comprehensive dataset of more than 5000 prokaryotic species from which we derived the metabolic networks. We compute the scope from the reconstructed graphs, which is the set of all metabolites and reactions that can potentially be synthesized when provided with external metabolites. We show using machine learning techniques that the scope is an excellent predictor of taxonomic and environmental variables, namely growth temperature, oxygen tolerance, and habitat. In the literature, metabolites and pathways are rarely used to discriminate species. We make use of the scope underlying structure—metabolites and pathways—to construct the predictive models, giving additional information on the important metabolic pathways needed to discriminate the species, which is often absent in other metabolic network properties. For example, in the particular case of growth temperature, glutathione biosynthesis pathways are specific to species growing in cold environments, whereas tungsten metabolism is specific to species in warm environments, as was hinted in current literature. From a machine learning perspective, the scope is able to reduce the dimension of our data, and can thus be considered as an interpretable graph embedding.

Intrinsic and Chemotherapeutic Stressors Modulate ABCC-Like Transport in Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent for Chagas disease, which affects 6–7 million people worldwide. The biological diversity of the parasite reflects on inefficiency of benznidazole, which is a first choice chemotherapy, on chronic patients. ABC transporters that extrude xenobiotics, metabolites, and mediators are overexpressed in resistant cells and contribute to chemotherapy failure. An ABCC-like transport was identified in the Y strain and extrudes thiol-conjugated compounds. As thiols represent a line of defense towards reactive species, we aimed to verify whether ABCC-like transport could participate in the regulation of responses to stressor stimuli. In order to achieve this, ABCC-like activity was measured by flow cytometry using fluorescent substrates. The present study reveals the participation of glutathione and ceramides on ABCC-like transport, which are both implicated in stress. Hemin modulated the ABCC-like efflux which suggests that this protein might be involved in cellular detoxification. Additionally, all strains evaluated exhibited ABCC-like activity, while no ABCB1-like activity was detected. Results suggest that ABCC-like efflux is not associated with natural resistance to benznidazole, since sensitive strains showed higher activity than the resistant ones. Although benznidazole is not a direct substrate, ABCC-like efflux increased after prolonged drug exposure and this indicates that the ABCC-like efflux mediated protection against cell stress depends on the glutathione biosynthesis pathway.