Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ...ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer’s, Huntington’s, and Parkinson’s diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.
This Primer reviews the mechanisms, tools, and guidelines to study ferroptosis, a form of cell death that is emerging as a central player in several degenerative diseases.
Mammalian cells synthesize the antioxidant glutathione (GSH) to shield cellular biomolecules from oxidative damage. Certain bacteria, including the gastric pathogen Helicobacter pylori, can perturb ...host GSH homeostasis. H. pylori infection significantly decreases GSH levels in host tissues, which has been attributed to the accumulation of reactive oxygen species in infected cells. However, the precise mechanism of H. pylori-induced GSH depletion remains unknown, and tools for studying this process during infection are limited. We developed an isotope-tracing approach to quantitatively monitor host-derived GSH in H. pylori-infected cells by mass spectrometry. Using this method, we determined that H. pylori catabolizes reduced GSH from gastric cells using γ-glutamyl transpeptidase (gGT), an enzyme that hydrolyzes GSH to glutamate and cysteinylglycine (Cys-Gly). gGT is an established virulence factor with immunomodulatory properties that is required for H. pylori colonization in vivo. We found that H. pylori internalizes Cys-Gly in a gGT-dependent manner and that Cys-Gly production during H. pylori infection is coupled to the depletion of intracellular GSH from infected cells. Consistent with bacterial catabolism of host GSH, levels of oxidized GSH did not increase during H. pylori infection, and exogenous antioxidants were unable to restore the GSH content of infected cells. Altogether, our results indicate that H. pylori-induced GSH depletion proceeds via an oxidation-independent mechanism driven by the bacterial enzyme gGT, which fortifies bacterial acquisition of nutrients from the host. Additionally, our work establishes a method for tracking the metabolic fate of host-derived GSH during infection.
Redox signaling—the regulation of cell signaling by oxidative posttranslational modifications (OxiPTMs)—modulates diverse cellular processes including cell division, metabolism, and antioxidant ...defense 2. ROS produced by bacteria, host epithelial cells, and migratory immune cells can alter cell signaling via the site-specific Ox of proteins containing redox-sensitive cysteines. By using liquid chromatography (LC)–MS to quantify the relative abundance of probe-labeled peptides under different sample conditions (e.g., cells grown in the presence or absence of oxidative stress), chemical proteomic techniques like isotopic tandem orthogonal proteolysis–activity-based protein profiling (isoTOP–ABPP) 12 and OxICAT 13 can resolve precise sites of cysteine oxidation. Following enrichment and trypsin digestion of probe-labeled proteins, the relative abundance of probe-labeled peptides in each sample is quantified by MS. isoTOP–ABPP, isotopic tandem orthogonal proteolysis–activity-based protein profiling; LC, liquid chromatography; MS, mass spectrometry; Ox, oxidation. https://doi.org/10.1371/journal.ppat.1009070.g002 The indirect detection of oxidized cysteines using isoTOP–ABPP or OxICAT offers an unbiased approach to identifying OxiPTMs. Because these methods quantify proteome-wide changes in cysteine reactivity, they can detect a wide range of thiol-based modifications that inhibit probe labeling, irrespective of the type of modification.
Low-molecular-weight (LMW) thiols are an abundant class of cysteine-derived small molecules found in all forms of life that maintain reducing conditions within cells. While their contributions to ...cellular redox homeostasis are well established, LMW thiols can also mediate other aspects of cellular physiology, including intercellular interactions between microbial and host cells. Here we discuss emerging roles for these redox-active metabolites at the host–microbe interface. We begin by providing an overview of chemical and computational approaches to LMW-thiol discovery. Next, we highlight mechanisms of virulence regulation by LMW thiols in infected cells. Finally, we describe how microbial metabolism of these compounds may influence host physiology.
Mycobacterium tuberculosis (Mtb) has evolved into a highly successful human pathogen. It deftly subverts the bactericidal mechanisms of alveolar macrophages, ultimately inducing granuloma formation ...and establishing long-term residence in the host. These hallmarks of Mtb infection are facilitated by the metabolic adaptation of the pathogen to its surrounding environment and the biosynthesis of molecules that mediate its interactions with host immune cells. The sulfate assimilation pathway of Mtb produces a number of sulfur-containing metabolites with important contributions to pathogenesis and survival. This pathway is regulated by diverse environmental cues and regulatory proteins that mediate sulfur transactions in the cell. Here, we discuss the transcriptional and biochemical mechanisms of sulfur metabolism regulation in Mtb and potential small molecule regulators of the sulfate assimilation pathway that are collectively poised to aid this intracellular pathogen in its expert manipulation of the host. From this global analysis, we have identified a subset of sulfur-metabolizing enzymes that are sensitive to multiple regulatory cues and may be strong candidates for therapeutic intervention.
Low-molecular-weight (LMW) thiols are small-molecule antioxidants required for the maintenance of intracellular redox homeostasis. However, many host-associated microbes, including the gastric ...pathogen Helicobacter pylori, unexpectedly lack LMW-thiol biosynthetic pathways. Using reactivity-guided metabolomics, we identified the unusual LMW thiol ergothioneine (EGT) in H. pylori. Dietary EGT accumulates to millimolar levels in human tissues and has been broadly implicated in mitigating disease risk. Although certain microorganisms synthesize EGT, we discovered that H. pylori acquires this LMW thiol from the host environment using a highly selective ATP-binding cassette transporter—EgtUV. EgtUV confers a competitive colonization advantage in vivo and is widely conserved in gastrointestinal microbes. Furthermore, we found that human fecal bacteria metabolize EGT, which may contribute to production of the disease-associated metabolite trimethylamine N-oxide. Collectively, our findings illustrate a previously unappreciated mechanism of microbial redox regulation in the gut and suggest that inter-kingdom competition for dietary EGT may broadly impact human health.
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•Helicobacter pylori imports the human dietary antioxidant ergothioneine (EGT)•ABC transporter EgtUV takes up host-derived EGT and protects against bleach stress•WT H. pylori outcompetes EgtUV-deficient H. pylori in mice•EGT import and metabolism are widespread among human gastrointestinal microbes
Low-molecular-weight thiols are necessary for the maintenance of intracellular redox homeostasis; however, certain clinically important microbial pathogens are not known to synthesize these antioxidants. By analyzing H. pylori, a microbial transporter of ergothioneine was discovered that regulates microbial redox homeostasis in the gut.
Host cells sense and respond to pathogens by dynamically regulating cell signaling. The rapid modulation of signaling pathways is achieved by post‐translational modifications (PTMs) that can alter ...protein structure, function, and/or binding interactions. By using chemical probes to broadly profile changes in enzyme function or side‐chain reactivity, activity‐based protein profiling (ABPP) can reveal PTMs that regulate host–microbe interactions. While ABPP has been widely utilized to uncover microbial mechanisms of pathogenesis, in this review, we focus on more recent applications of this technique to the discovery of host PTMs and enzymes that modulate signaling within infected cells. Collectively, these advances underscore the importance of ABPP as a tool for interrogating the host response to infection and identifying potential targets for host‐directed therapies.
Vibrio cholerae, the causative agent of the human diarrheal disease cholera, exports numerous enzymes that facilitate its adaptation to both intestinal and aquatic niches. These secreted enzymes can ...mediate nutrient acquisition, biofilm assembly, and V. cholerae interactions with its host. We recently identified a V. cholerae-secreted serine protease, IvaP, that is active in V. cholerae-infected rabbits and human choleric stool. IvaP alters the activity of several host and pathogen enzymes in the gut and, along with other secreted V. cholerae proteases, decreases binding of intelectin, an intestinal carbohydrate-binding protein, to V. cholerae in vivo. IvaP bears homology to subtilisin-like enzymes, a large family of serine proteases primarily comprised of secreted endopeptidases. Following secretion, IvaP is cleaved at least three times to yield a truncated enzyme with serine hydrolase activity, yet little is known about the mechanism of extracellular maturation. Here, we show that IvaP maturation requires a series of sequential N- and C-terminal cleavage events congruent with the enzyme’s mosaic protein domain structure. Using a catalytically inactive reporter protein, we determined that IvaP can be partially processed in trans, but intramolecular proteolysis is most likely required to generate the mature enzyme. Unlike many other subtilisin-like enzymes, the IvaP cleavage pattern is consistent with stepwise processing of the N-terminal propeptide, which could temporarily inhibit, and be cleaved by, the purified enzyme. Furthermore, IvaP was able to cleave purified intelectin, which inhibited intelectin binding to V. cholerae. These results suggest that IvaP plays a role in modulating intelectin–V. cholerae interactions.
Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in ...adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative σ-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.
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