Bongkrekic acid (BKA) produced by pseudomonas cocovenenans is a deadly toxin, and is mainly found in spoiled or fermented foods. However, less is known on its immunotoxicity. Neutrophil extracellular traps (NETs) are a novel effector mechanism of neutrophils against invading pathogens, but excessive NETs also contribute to tissue damage. This study aimed to investigate NET formation triggered by BKA in murine neutrophils, and describe its characteristics and potential mechanisms. Our results showed that BKA triggered NET formation via co-localization of DNA and histone or MPO by immunostaining. Moreover, BKA-triggered NET formation was dose- and time-dependent via NET quantification based on Picogreen-derived fluorescence intensities. Furthermore, BKA increased ROS production in neutrophils. Pharmacological inhibition indicated that BKA-triggered NET formation was associated with ROS-p38 and -ERK signaling pathways, but independent on NADPH oxidase. Besides, PAD4 and P2X1 receptor also mediated BKA-triggered NET formation. To our knowledge, all these findings provide for the first time an initial understanding of BKA on innate immunity, which might be helpful for further investigation on BKA immunotoxicity.

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.

Bongkrekic acid (BKA) inhibits adenine nucleotide translocator (ANT) and suppresses ADP/ATP exchange in the mitochondrial inner membrane. Previously, we demonstrated that BKA exhibited cytotoxic effects on 4T1 tumor cells, depending on the cell number in the culture, but not on NIH3T3 cells. However, the cause of this differential sensitivity was unelucidated. Here we demonstrate that BKA reduced the O2 consumption in both cell lines and increased the mitochondrial membrane potential, thereby facilitating glucose consumption. BKA reduced cellular ATP in 4T1 cells in a dose-dependent manner but not in NIH3T3 cells. The cellular ATP of 4T1 cells was decreased with a reduced glucose concentration in the media, but that of NIH3T3 cells remained constant. We also demonstrated that BKA-induced cell death in both cell lines in low glucose media; however, the susceptibility to the reduced glucose concentration was slightly higher in 4T1 cells, which may be attributed to the difference in the dependency on glycolysis as their energy source. These results indicate that 4T1 tumor cells rely heavily on glucose for energy production. Our data demonstrate that BKA disturbs ATP production in mitochondria and increases the susceptibility to a low glucose condition.

The environmental bacterium Burkholderia gladioli pv. cocovenenans (B. cocovenenans) has been linked to fatal food poisoning cases in Asia and Africa. Bongkrekic acid (BA), a mitochondrial toxin produced by B. cocovenenans, is thought to be responsible for these outbreaks. While there are over 80 species in the Burkholderia genus, B. cocovenenans is the only pathovar capable of producing BA and causing human death. However, the genomic features of B. gladioli and the evolution of the BA biosynthesis gene cluster, bon, in B. cocovenenans remain elusive. In this study, 239 whole genome sequences (WGSs) of B. gladioli, isolated from 12 countries collected over 100 years, were used to analyze the intra-species genomic diversity and phylogenetic relationships of B. gladioli and to explore the origin and evolution of the bon gene cluster. Our results showed that the genome-wide average nucleotide identity (ANI) values were above 97.29% for pairs of B. gladioli genomes. Thirty-six of the 239 (15.06%) B. gladioli genomes, isolated from corn, rice, fruits, soil, and patients from Asia, Europe, North America, and South America, contained the bon gene cluster and formed three clades within the phylogenetic tree. Pan- and core-genome analysis suggested that the BA biosynthesis genes were recently acquired. Comparative genome analysis of the bon gene cluster showed that complex recombination events contributed to this toxin biosynthesis gene cluster's evolution and formation. This study suggests that a better understanding of the genomic diversity and evolution of this lethal foodborne pathovar will potentially contribute to B. cocovenenans food poisoning outbreak prevention.

Burkholderia gladioli pv. cocovenenans is a serious safety issue in black fungus due to the deadly toxin, bongkrekic acid. This has triggered the demand for an efficient toxigenic phenotype recognition method. The objective of this study is to develop an efficient method for the recognition of toxin-producing B. gladioli strains. The potential of multilocus sequence typing and a back propagation neural network for the recognition of toxigenic B. cocovenenans was explored for the first time. The virulent strains were isolated from a black fungus cultivation environment in Qinba Mountain area, Shaanxi, China. A comprehensive evaluation of toxigenic capability of 26 isolates were conducted using Ultra Performance Liquid Chromatography for determination of bongkrekic acid and toxoflavin production in different culturing conditions and foods. The isolates produced bongkrekic acid in the range of 0.05-6.24 mg/L in black fungus and a highly toxin-producing strain generated 201.86 mg/L bongkrekic acid and 45.26 mg/L toxoflavin in co-cultivation with Rhizopus oryzae on PDA medium. Multilocus sequence typing phylogeny (MLST) analysis showed that housekeeping gene sequences have a certain relationship with a strain toxigenic phenotype. We developed a well-trained, back-propagation neutral network for prediction of toxigenic phenotype in B. gladioli based on MLST sequences with an accuracy of 100% in the training set and an accuracy of 86.7% in external test set strains. The BP neutral network offers a highly efficient approach to predict toxigenic phenotype of strains and contributes to hazard detection and safety surveillance.

Most organs use fatty acids (FAs) as a key nutrient, but little is known of how blood-borne FAs traverse the endothelium to reach underlying tissues. We conducted a small-molecule screen and identified niclosamide as a suppressor of endothelial FA uptake and transport. Structure/activity relationship studies demonstrated that niclosamide acts through mitochondrial uncoupling. Inhibitors of oxidative phosphorylation and the ATP/ADP translocase also suppressed FA uptake, pointing principally to ATP production. Decreasing total cellular ATP by blocking glycolysis did not decrease uptake, indicating that specifically mitochondrial ATP is required. Endothelial FA uptake is promoted by fatty acid transport protein 4 (FATP4) via its ATP-dependent acyl-CoA synthetase activity. Confocal microscopy revealed that FATP4 resides in the endoplasmic reticulum (ER), and that endothelial ER is intimately juxtaposed with mitochondria. Together, these data indicate that mitochondrial ATP production, but not total ATP levels, drives endothelial FA uptake and transport via acyl-CoA formation in mitochondrial/ER microdomains.

Fenamates like flufenamic acid (FFA) are anti-inflammatory drugs known to alter ion fluxes through the plasma membrane. They are for instance potent blockers of cation and anion channels, and FFA is now commonly used to block currents through TRP channels and receptor-operated channels. However, FFA exerts complex and multifaceted actions on ion transport systems and, in most instances, a molecular understanding of these FFA-dependent modulations is lacking. In addition, FFA is also to known to perturb the homeostasis of Ca2+. In the present report, we investigated whether the FFA-induced alterations of the Ca2+ homeostasis could play a role in the FFA-dependent modulation of transmembrane ion fluxes. Experiments performed with the Ca2+ indicator Fluo-4 on cultured cortical neurons and HEK-293 cells showed that FFA increased the cytosolic concentration of Ca2+ even in cells kept in a Ca2+-free medium or when the endoplasmic reticulum was depleted with thapsigargin. The FFA-dependent Ca2+ responses were, however, strongly reduced by bongkrekic acid, a specific ligand of the mitochondrial ADP/ATP carrier which, in addition, inhibits the permeability transition pore. Like FCCP, FFA released Ca2+ from isolated brain mitochondria and indirectly modulates store-operated Ca2+ channels. We suggest that some of the effects of FFA on plasma membrane ion channels could be explained, at least partially, by its ability to modulate the mitochondrial Ca2+ homeostasis.

Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade-specific. Strikingly, 13 % of CF infection strains characterized were bongkrekic acid-positive, uniquely linking this food-poisoning toxin to this aspect of B. gladioli disease. Mapping the population biology and metabolite production of B. gladioli has shed light on its diverse ecology, and by demonstrating that the antibiotic trimethoprim suppresses bongkrekic acid production, a potential therapeutic strategy to minimize poisoning risk in CF has been identified.

Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca(2+)-induced permeability transition in the presence of a profound Ca(2+) uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca(2+)-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca(2+)-induced permeability transition. Ca(2+) uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca(2+)-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca(2+)-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.

Galectin-1 and galectin-3 are the most ubiquitously expressed members of the galectin family and more importantly, these two molecules are shown to have opposite effects on pro-inflammatory responses and/or apoptosis depending on the cell type. Herein, we demonstrate for the first time that galectin-3 induces mast cell apoptosis. Mast cells expressed substantial levels of galectin-3 and galectin-1 and to a lesser extent the receptor for advanced glycation end products (RAGE) on their surfaces. Treatment of cells with galectin-3 at concentrations of > or =100 nM for 18-44 h resulted in cell death by apoptosis. Galectin-3-induced apoptosis was completely prevented by lactose, neutralizing antibody to RAGE, and the caspase-3 inhibitor z-DEVD-fmk. Galectin-3-induced apoptosis was also completely abolished by dithiothreitol and superoxide dismutase, but not inhibited by catalase. Moreover, galectin-3 but not galectin-1 induced the release of superoxide, which was blocked by lactose, anti-RAGE, and dithiothreitol. Finally, galectin-3-induced apoptosis was blocked by bongkrekic acid, an antagonist of the mitochondrial permeability transition pore (PTP), while atractyloside, an agonist of the PTP, greatly facilitated galectin-1-induced apoptosis. These data suggest that galectin-3 induces oxidative stress, PTP opening, and the caspase-dependent death pathway by binding to putative surface receptors including RAGE via the carbohydrate recognition domain.

A zinc-induced signaling pathway leading to extracellular signal-regulated kinase 1/2 (ERK1/2) activation and subsequent neuronal death has been investigated. We find that an extracellular zinc application stimulates biphasic phosphorylation of ERK1/2 and p38 MAPK in rat cultured neurons. The activation of ERK1/2, but not p38, is responsible for zinc neurotoxicity as only U0126, a MEK inhibitor that blocks ERK1/2 phosphorylation, significantly protects cortical neurons from zinc exposure. Over-expression of a dominant negative Ras mutant blocks zinc-induced Elk1-dependent gene expression in neurons, indicating the involvement of Ras activation in the zinc pathway leading to ERK phosphorylation and Elk1 signaling. We also find that zinc treatment results in neuronal mitochondrial hyperpolarization. Importantly, both U0126 and bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, effectively reduce zinc-triggered mitochondrial changes. As bongkrekic acid also prevents zinc-triggered neuronal death but not ERK1/2 phosphorylation, activation of MAPK signaling precedes and is required for mitochondrial dysfunction and cell death. These results provide new insight on the mechanism of extracellular zinc-induced toxicity in which the regulation of mitochondrial function by the Ras/MEK/ERK pathway is closely associated with neuronal viability.

Ca(2+)-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca(2+) concentrations (about 30--100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca(2+); 20 microm Ca(2+) was required to depolarize liver mitochondria. Ca(2+) did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca(2+)-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.

The tumor suppressor p53 can translocate into mitochondria and activate apoptosis. Here we studied whether p53 mitochondrial translocation and subsequent apoptosis were affected by blocking mitochondrial permeability transition pore using cyclosporine A (CsA) and bongkrekic acid (BA) in skin epidermal JB6 cells and skin tissues. Our results demonstrated that CsA and BA blocked TPA-induced p53 translocation, leading to protection against the loss of mitochondrial membrane potential and Complex I activity, and eventually suppression of apoptosis. Thus, our results suggest that mitochondrial permeability transition is required for p53 mitochondrial translocation.

Recent pharmacological and molecular genetic approaches have revealed the existence of functional L-type Ca2+ channels (LTCCs) in a variety of hematopoietic cells. We previously reported that Ca(v)1.2 LTCCs are expressed on mast cell surfaces, activated by the high-affinity IgE receptor (FcvarepsilonRI) engagement and protect mast cells against activation-induced cell death (AICD). We also demonstrated that FcvarepsilonRI engagement evokes nitric oxide (NO) generation in a phosphatidylinositol-3-kinase- and NO synthase (NOS)-dependent manner, which is also required for mast cell survival. Here we demonstrate that this endogenous NO mediates Ca(v)1.2 LTCC activation. FcvarepsilonRI engagement but not thapsigargin, a potent Ca2+ release-activated Ca2+ (CRAC) channel agonist, induced Ca2+ influx via NOS-dependent NO generation. RT-PCR analyses revealed predominant expression of eNOS in mast cells. Subsequent experiments involving siRNA-mediated gene silencing of eNOS or Ca(v)1.2 LTCC revealed that eNOS was essential for NOS-dependent NO generation and Ca(v)1.2 LTCC activation but not CRAC channel activation. Similar to Ca(v)1.2 LTCCs, eNOS prevented the dissipation of the mitochondrial membrane potential and mitochondrial integrity collapse, thereby protecting mast cells against AICD. Taken together, the present findings demonstrate the key roles of the eNOS-NO-LTCC axis in mast cell survival after FcvarepsilonRI engagement.

Amino acid sequence from 65th to 76th residue of the N-terminus of Chromogranin A (CGA-N12) is an antimicrobial peptide (AMP). Our previous studies showed that CGA-N12 reduces Candida tropicalis mitochondrial membrane potential. Here, we explored the mechanism that CGA-N12 collapsed the mitochondrial membrane potential by investigations of its action on the mitochondrial permeability transition pore (mPTP) complex of C. tropicalis. The results showed that CGA-N12 induced cytochrome c (Cyt c) leakage, mitochondria swelling and led to polyethylene glycol (PEG) of molecular weight 1000 Da penetrate mitochondria. mPTP opening inhibitors bongkrekic acid (BA) could contract the mitochondrial swelling induced by CGA-N12, but cyclosporin A (CsA) could not. Therefore, we speculated that CGA-N12 could induce C. tropicolis mPTP opening by preventing the matrix-facing (m) conformation of adenine nucleotide transporter (ANT), thereby increasing the permeability of the mitochondrial membrane and resulted in the mitochondrial potential dissipation.

Mineralocorticoid receptor (MR) antagonists decrease morbidity and mortality in heart failure patients for whom oxidative stress is usual; however, the underlying mechanism for this protection is unclear. Since aldosterone stimulates reactive oxygen species (ROS) production in several tissues, we explored its effect and the intracellular pathway involved in the rat myocardium. Aldosterone dose-dependently increased O2(-) production in myocardial slices. At 10 nmol/L, aldosterone increased O2(-) to 165 ± 8.8% of control, an effect prevented not only by the MR antagonists eplerenone and spironolactone (107 ± 7.8 and 103 ± 5.3%, respectively) but also by AG1478 (105 ± 8.0%), antagonist of the EGF receptor (EGFR). Similar results were obtained by silencing MR expression through the direct intramyocardial injection of a lentivirus coding for a siRNA against the MR. The aldosterone effect on O2(-) production was mimicked by the mKATP channel opener diazoxide and blocked by preventing its opening with 5-HD and glibenclamide, implicating the mitochondria as the source of O2(-). Inhibiting the respiratory chain with rotenone or mitochondrial permeability transition (MPT) with cyclosporine A or bongkrekic acid also canceled aldosterone-induced O2(-) production. In addition, aldosterone effect depended on NADPH oxidase and phosphoinositide 3-kinase activation, as apocynin and wortmannin, respectively, inhibited it. EGF (0.1 μg/mL) similarly increased O2(-), although in this case MR antagonists had no effect, suggesting that EGFR transactivation occurred downstream from MR activation. Inhibition of mKATP channels, the respiratory chain, or MPT did not prevent Akt phosphorylation, supporting that it happened upstream of the mitochondria. Importantly, cardiomyocytes were confirmed as a source of aldosterone induced mitochondrial ROS production in experiments performed in isolated cardiac myocytes. These results allow us to speculate that the beneficial effects of MR antagonists in heart failure may be related to a decrease in oxidative stress.

Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT.

This study tested the anti-head and neck squamous cell carcinoma (HNSCC) cell activity by GSK1059615, a novel PI3K and mTOR dual inhibitor. GSK1059615 inhibited survival and proliferation of established (SCC-9, SQ20B and A253 lines) and primary human HNSCC cells. GSK1059615 blocked PI3K-AKT-mTOR activation in HNSCC cells. Intriguingly, GSK1059615 treatment in HNSCC cells failed to provoke apoptosis, but induced programmed necrosis. The latter was tested by mitochondria depolarization, ANT-1-cyclophilin-D mitochondrial association and lactate dehydrogenase (LDH) release. Reversely, mPTP blockers (sanglifehrin A, cyclosporin A and bongkrekic acid) or cyclophilin-D shRNA dramatically alleviated GSK1059615-induced SCC-9 cell death. Further studies demonstrated that GSK1059615 i.p. injection suppressed SCC-9 tumor growth in nude mice, which was compromised with co-administration with cyclosporin A. Thus, targeting PI3K-AKT-mTOR pathway by GSK1059615 possibly provokes programmed necrosis pathway to kill HNSCC cells.

Early in programmed cell death (apoptosis), mitochondrial membrane permeability increases. This is at least in part due to opening of the permeability transition (PT) pore, a multiprotein complex built up at the contact site between the inner and the outer mitochondrial membranes. The PT pore has been previously implicated in clinically relevant massive cell death induced by toxins, anoxia, reactive oxygen species, and calcium overload. Here we show that PT pore complexes reconstituted in liposomes exhibit a functional behavior comparable with that of the natural PT pore present in intact mitochondria. The PT pore complex is regulated by thiol-reactive agents, calcium, cyclophilin D ligands (cyclosporin A and a nonimmunosuppressive cyclosporin A derivative), ligands of the adenine nucleotide translocator, apoptosis-related endoproteases (caspases), and Bcl-2-like proteins. Although calcium, prooxidants, and several recombinant caspases (caspases 1, 2, 3, 4, and 6) enhance the permeability of PT pore-containing liposomes, recombinant Bcl-2 or Bcl-XL augment the resistance of the reconstituted PT pore complex to pore opening. Mutated Bcl-2 proteins that have lost their cytoprotective potential also lose their PT modulatory capacity. In conclusion, the PT pore complex may constitute a crossroad of apoptosis regulation by caspases and members of the Bcl-2 family.

Cigarette smoke is one of major risk factors in the pathogenesis of chronic obstructive pulmonary disease (COPD). It is generally believed that cigarette smoke induces mitochondrial damage in the alveolar epithelial cells to contribute to COPD. However, the exact molecular mechanism remains unknown for the mitochondrial damage. In this study, cigarette smoke extract (CSE) was found to induce the mitochondrial membrane permeability (MMP), which promoted proton leakage leading to the reduction in mitochondrial potential and ATP production. ANT in the mitochondrial inner membrane was activated by CSE for the alteration of MMP. The activation was observed without an alteration in the protein level of ANT. Inhibition of the ANT activity with ADP or bongkrekic acid prevented the MMP alteration and potential drop upon CSE exposure. The ANT activation was observed with a rise in ROS production, inhibition of the mitochondrial respiration, decrease in the complex III protein and rise in mitophagy activity. The results suggest that ANT may mediate the toxic effect of cigarette smoke on mitochondria and control of ANT activity is a potential strategy in intervention of the toxicity.