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The increasing resistance of human pathogens severely limits the efficacy of antibiotics in medicine, yet many animals, including solitary beewolf wasps, successfully engage in defensive alliances with antibiotic-producing bacteria for millions of years. Here, we report on the in situ production of 49 derivatives belonging to three antibiotic compound classes (45 piericidin derivatives, 3 streptochlorin derivatives, and nigericin) by the symbionts of 25 beewolf host species and subspecies, spanning 68 million years of evolution. Despite a high degree of qualitative stability in the antibiotic mixture, we found consistent quantitative differences between species and across geographic localities, presumably reflecting adaptations to combat local pathogen communities. Antimicrobial bioassays with the three main components and in silico predictions based on the structure and specificity in polyketide synthase domains of the piericidin biosynthesis gene cluster yield insights into the mechanistic basis and ecoevolutionary implications of producing a complex mixture of antimicrobial compounds in a natural setting.

Primary effusion lymphoma (PEL) is classified as a rare non-Hodgkin's B-cell lymphoma that is caused by Kaposi's sarcoma-associated herpesvirus (KSHV); PEL cells are latently infected with KSHV. PEL is frequently resistant to conventional chemotherapies. Therefore, the development of novel therapeutic agents is urgently required. Nigericin, a H and K ionophore, possesses unique pharmacological effects. However, the effects of nigericin on PEL cells remain unknown. We examined the cytotoxic effects of the K ionophores, nigericin, nonactin, and valinomycin, on various B-lymphoma cells including PEL. We also evaluated ionophore-induced changes in signaling pathways involved in KSHV-induced oncogenesis. Moreover, the effects of nigericin on mitochondrial membrane potential and viral reactivation in PEL were analyzed. Although the three tested ionophores inhibited the proliferation of several B-lymphoma cell lines, nigericin inhibited the proliferation of PEL cells compared to KSHV-negative cells. In PEL cells, nigericin disrupted the mitochondrial membrane potential and caused the release of cytochrome c, which triggered caspase-9-mediated apoptosis. Nigericin also induced both an increase in phosphorylated p38 MAPK and proteasomal degradation of β-catenin. Combination treatment of nigericin with the p38 MAPK inhibitor SB203580 potentiated the cytotoxic effects towards PEL cells, compared to either compound alone. Meanwhile, nigericin did not influence viral replication in PEL cells. Nigericin induces apoptosis in PEL cells by mitochondrial dysfunction and down-regulation of Wnt/β-catenin signaling. Thus, nigericin is a novel drug candidate for treating PEL without the risk of de novo KSHV infection.

Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, Candida albicans and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA-induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, C. albicans and GBS use a related pathway for NET induction, whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling mechanisms, suggesting that extrusion of NETs is important in host defence. The immune system protects the body against microorganisms that can cause infections and diseases. Neutrophils are a type of immune cell that patrol the blood in search of germs. Once they encounter potentially harmful microbes, neutrophils eradicate them in different ways. One way to catch the germs is by using ‘neutrophil extracellular traps’, or NETs for short, to confine and kill the invaders. NETs are web-like structures made up of anti-microbial proteins and the neutrophil’s own DNA. The process of making NETs kills the neutrophil, as it eventually explodes to release the NETs. NETs play a key role in disease prevention, but producing too many NETs or producing them at the wrong time or in the wrong place can actually make certain diseases worse. Therefore, it is important to fully understand the signaling pathways and molecules the neutrophils use to make NETs. Kenny et al. exposed neutrophils from healthy people to five different compounds known to cause the cells to make NETs, including some harmful molecules, a fungus and a bacterium. Microscopy was then used to count how many neutrophils made NETs in response to each of the five stimuli. Further experiments showed that neutrophils from patients with an immune system disorder produced fewer NETs when stimulated with some of the compounds, while the other stimuli caused neutrophils to produce the same levels of NETs as healthy individuals. Kenny et al. also revealed that neutrophils use several different ways to produce and release NETs, depending on the stimulus used. Some of the ways required reactive oxygen species, such as hydrogen peroxide and enzymes, while others produced NETs without the need for these molecules. Lastly, Kenny et al. showed that the way the cells die after the NET is released is unique from other pathways that are known to kill cells. Future work will aim to identify a single molecule that can block neutrophils from releasing NETs at the wrong time and place, without affecting the important role NETs play in fighting germs. Such a molecule could be developed into a drug for people with diseases like lupus or rheumatoid arthritis, where the release of NETs makes the disease worse not better.

Caspase-1 activated in inflammasomes triggers a programmed necrosis called pyroptosis, which is mediated by gasdermin D (GSDMD). However, GSDMD-deficient cells are still susceptible to caspase-1-mediated cell death. Therefore, here, we investigate the mechanism of caspase-1-initiated cell death in GSDMD-deficient cells. Inflammasome stimuli induce apoptosis accompanied by caspase-3 activation in GSDMD-deficient macrophages, which largely relies on caspase-1. Chemical dimerization of caspase-1 induces pyroptosis in GSDMD-sufficient cells, but apoptosis in GSDMD-deficient cells. Caspase-1-induced apoptosis involves the Bid-caspase-9-caspase-3 axis, which can be followed by GSDME-dependent secondary necrosis/pyroptosis. However, Bid ablation does not completely abolish the cell death, suggesting the existence of an additional mechanism. Furthermore, cortical neurons and mast cells exhibit little or low GSDMD expression and undergo apoptosis after oxygen glucose deprivation and nigericin stimulation, respectively, in a caspase-1- and Bid-dependent manner. This study clarifies the molecular mechanism and biological roles of caspase-1-induced apoptosis in GSDMD-low/null cell types. In inflammasomes, caspase-1 activation leads to pyroptosis mediated by gasdermin D, but cells lacking gasdermin-D still initiate caspase-dependent cell death. Here, Tsuchiya et al. show that these cells undergo Bid- and caspase-3-dependent apoptosis.

Photosystem I (PSI) photoinhibition suppresses plant photosynthesis and growth. However, the mechanism underlying PSI photoinhibition has not been fully clarified. In this study, in order to investigate the mechanism of PSI photoinhibition in higher plants, we applied repetitive short-pulse (rSP) illumination, which causes PSI-specific photoinhibition in chloroplasts isolated from spinach leaves. We found that rSP treatment caused PSI photoinhibition, but not PSII photoinhibition in isolated chloroplasts in the presence of O₂. However, chloroplastic superoxide dismutase and ascorbate peroxidase activities failed to protect PSI from its photoinhibition. Importantly, PSI photoinhibition was largely alleviated in the presence of methyl viologen, which stimulates the production of reactive oxygen species (ROS) at the stromal region by accepting electrons from PSI, even under the conditions where CuZn-superoxide dismutase and ascorbate peroxidase activities were inactivated by KCN. These results suggest that the ROS production site, but not the ROS production rate, is critical for PSI photoinhibition. Furthermore, we found that not only superoxide (O₂⁻) but also singlet oxygen (¹O₂) is involved in PSI photoinhibition induced by rSP treatment. From these results, we suggest that PSI photoinhibition is caused by both O₂⁻ and ¹O2 produced within the thylakoid membranes when electron carriers in PSI become highly reduced. Here, we show, to our knowledge, new insight into the PSI photoinhibition in higher plants.

Interleukin (IL)-18 and IL-1β are potent pro-inflammatory cytokines that contribute to inflammatory conditions such as rheumatoid arthritis and Alzheimer’s disease. They are produced as inactive precursors that are activated by large macromolecular complexes called inflammasomes upon sensing damage or pathogenic signals. NLRP3 inflammasome activation is regarded to require a priming step that causes NLRP3 and IL-1β gene upregulation, and also NLRP3 post-translational licencing. A subsequent activation step leads to the assembly of the complex and the cleavage of pro-IL-18 and pro-IL-1β by caspase-1 into their mature forms, allowing their release. Here we show that human monocytes, but not monocyte derived macrophages, are able to form canonical NLRP3 inflammasomes in the absence of priming. NLRP3 activator nigericin caused the processing and release of constitutively expressed IL-18 in an unprimed setting. This was mediated by the canonical NLRP3 inflammasome that was dependent on K + and Cl − efflux and led to ASC oligomerization, caspase-1 and Gasdermin-D (GSDMD) cleavage. IL-18 release was impaired by the NLRP3 inhibitor MCC950 and by the absence of NLRP3, but also by deficiency of GSDMD, suggesting that pyroptosis is the mechanism of release. This work highlights the readiness of the NLRP3 inflammasome to assemble in the absence of priming in human monocytes and hence contribute to the very early stages of the inflammatory response when IL-1β has not yet been produced. It is important to consider the unprimed setting when researching the mechanisms of NLRP3 activation, as to not overshadow the pathways that occur in the absence of priming stimuli, which might only enhance this response.

Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins’ distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.

Within the present study we proposed a novel approach for senolysis based on the simultaneous disturbance of the several homeostasis-maintaining systems in senescent cells including intracellular ionic balance, energy production and intracellular utilization of damaged products. Of note, we could not induce senolysis by applying ouabain, amiloride, valinomycin or NH4Cl—compounds that modify each of these systems solely. However, we found that ionophore nigericin can disturb plasma membrane potential, intracellular pH, mitochondrial membrane potential and autophagy at once. By affecting all of the tested homeostasis-maintaining systems, nigericin induced senolytic action towards stromal and epithelial senescent cells of different origins. Moreover, the senolytic effect of nigericin was independent of the senescence-inducing stimuli. We uncovered that K+ efflux caused by nigericin initiated pyroptosis in senescent cells. According to our data, the higher sensitivity of senescent cells compared to the control ones towards nigericin-induced death was partially mediated by the lower intracellular K+ content in senescent cells and by their predisposition towards pyroptosis. Finally, we proposed an interval dosing strategy to minimize the negative effects of nigericin on the control cells and to achieve maximal senolytic effect. Hence, our data suggest ionophore nigericin as a new senotherapeutic compound for testing against age-related diseases.

The adaptor ASC is required for caspase-1 activation via the NLRP3 and AIM2 inflammasomes. Mitsuyama and colleagues show that signaling dependent on the kinases Syk and Jnk controls ASC speck formation through ASC phosphorylation. The inflammasome adaptor ASC contributes to innate immunity through the activation of caspase-1. Here we found that signaling pathways dependent on the kinases Syk and Jnk were required for the activation of caspase-1 via the ASC-dependent inflammasomes NLRP3 and AIM2. Inhibition of Syk or Jnk abolished the formation of ASC specks without affecting the interaction of ASC with NLRP3. ASC was phosphorylated during inflammasome activation in a Syk- and Jnk-dependent manner, which suggested that Syk and Jnk are upstream of ASC phosphorylation. Moreover, phosphorylation of Tyr144 in mouse ASC was critical for speck formation and caspase-1 activation. Our results suggest that phosphorylation of ASC controls inflammasome activity through the formation of ASC specks.

Cyclic electron flow is increasingly recognized as being essential in plant growth, generating a pH gradient across thylakoid membrane (ΔpH) that contributes to ATP synthesis and triggers the protective process of nonphotochemical quenching (NPQ) under stress conditions. Here, we report experiments demonstrating the importance of that ΔpH in protecting plants from stress and relating to the regulation of cyclic relative to linear flow. In leaves infiltrated with low concentrations of nigericin, which dissipates the ΔpH without significantly affecting the potential gradient, thereby maintaining ATP synthesis, the extent of NPQ was markedly lower, reflecting the lower ΔpH. At the same time, the photosystem (PS) I primary donor P700 was largely reduced in the light, in contrast to control conditions where increasing light progressively oxidized P700, due to down-regulation of the cytochrome bf complex. Illumination of nigericin-infiltrated leaves resulted in photoinhibition of PSII but also, more markedly, of PSI. Plants lacking ferredoxin (Fd) NADP oxidoreductase (FNR) or the polypeptide proton gradient regulation 5 (PGR5) also show reduction of P700 in the light and increased sensitivity to PSI photoinhibition, demonstrating that the regulation of the cytochrome bf complex (cyt bf) is essential for protection of PSI from light stress. The formation of a ΔpH is concluded to be essential to that regulation, with cyclic electron flow playing a vital, previously poorly appreciated role in this protective process. Examination of cyclic electron flow in plants with a reduced content of FNR shows that these antisense plants are less able to maintain a steady rate of this pathway. This reduction is suggested to reflect a change in the distribution of FNR from cyclic to linear flow, likely reflecting the formation or disassembly of FNR–cytochrome bf complex.