Abstract: Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.
Abstract: The polyphenolic flavonoid Baicalein has been shown to trigger suicidal death or apoptosis of tumor cells and is thus considered for the prevention and treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide. The present study explored whether Baicalein stimulates eryptosis. To this end, forward scatter was taken for measurement of cell volume, annexin-V-binding for phosphatidylserine-exposure, Fluo3 fluorescence for [Ca2+]i and fluorescent antibodies for ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Baicalein was followed by significant decrease of forward scatter (≥10 µM), significant increase of the percentage of annexin-V-binding cells (≥25 µM), significant increase of [Ca2+]i (50 µM) and significant increase of ceramide abundance (50 µM). The effect of Baicalein (50 µM) on annexin-V-binding was significantly blunted but not abrogated by removal of extracellular Ca2+. In conclusion, at the concentrations employed, Baicalein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to the combined effects of Ca2+ entry and ceramide formation.
Abstract: Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively.
Abstract: The activity of serine proteases is influenced by their substrate specificity as well as by the physicochemical conditions. Here, we present the characterization of key biochemical features of the two SPATE members EspPα and EspI from Shiga-toxin producing Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC). Both proteases show high activity at conditions mimicking the human blood stream. Optimal activities were observed at slightly alkaline pH and low millimolar concentrations of the divalent cations Ca2+ and Mg2+ at physiological temperatures indicating a function in the human host. Furthermore, we provide the first cleavage profile for EspI demonstrating pronounced specificity of this protease.
Abstract: Isolated from Dictamnus dasycarpus Turcz., fraxinellone exhibited multiple bioactivities against insects. In the present paper, the changes of digestive enzymes and detoxification enzymes of Mythimna separata Walker (5th instar larvae), treated with fraxinellone, were investigated. Compared with those of the control, the α-amylase activity of the fraxinellone-treated 5th instar larvae was inhibited, whereas the level of their protease activity was increased. Based upon further studies on the specific proteases, the levels of the active alkaline trypsin-like enzyme (BApNA as the substrate) and the chymotrypsin-like enzyme (BTEE as the substrate) activities of the treated larvae were declined; however, the level of activity of the weak alkaline trypsin-like enzyme (TAME as the substrate) of the treated ones was increased. Meanwhile, the activities of two detoxification enzymes, such as carboxylesterase (CarE) and glutathione S-transferase (GST), of the treated larvae were increased to some extent, but the activities of NADPH-P450 reductase and O-demethylase of the treated ones declined. Therefore, protease (especially the weak alkaline trypsin-like enzyme), CarE and GST played important roles in the metabolism of fraxinellone in the midgut of Mythimna separata (M. separata).
Abstract: Two colonies of Asian corn borer, Ostrinia furnacalis (Guenée), artificially selected from a Bt-susceptible colony (ACB-BtS) for resistance to Cry1Ab (ACB-AbR) and Cry1Ac (ACB-AcR) toxins, were used to analyze inheritance patterns of resistance to Cry1 toxins. ACB-AbR and ACB-AcR evolved significant levels of resistance, with resistance ratios (RR) of 39-fold and 78.8-fold to Cry1Ab and Cry1Ac, respectively. The susceptibility of ACB-AbR larvae to Cry1Ac and Cry1F toxins, which had not previously been exposed, were significantly reduced, being >113-fold and 48-fold, respectively. Similarly, susceptibility of ACB-AcR larvae to Cry1Ab and Cry1F were also significantly reduced (RR > nine-fold, RR > 18-fold, respectively), indicating cross-resistance among Cry1Ab, Cry1Ac, and Cry1F toxins. However, ACB-AbR and ACB-AcR larvae were equally susceptible to Cry1Ie as were ACB-BtS larvae, indicating no cross-resistance between Cry1Ie and Cry1Ab or Cry1Ac toxins; this may provide considerable benefits in preventing or delaying the evolution of resistance in ACB to Cry1Ab and Cry1Ac toxins. Backcrossing studies indicated that resistance to Cry1Ab toxin was polygenic in ACB-AbR, but monogenic in ACB-AcR, whilst resistance to Cry1Ac toxin was primarily monogenic in both ACB-AbR and ACB-AcR, but polygenic as resistance increased.