Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

spores germinate in the presence of a single germinant, inosine, yet with a significant lag period. spores. spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination

In Vitro Assessment of Methylene Blue on Chloroquine-Sensitive and -Resistant Plasmodium falciparum Strains Reveals Synergistic Action with Artemisinins

Methylene blue (MB) represents a promising antimalarial drug candidate for combination therapies against drug-resistant parasite strains. To support and facilitate the application of MB in future field trials, we studied its antiparasitic effects in vitro. MB is active against all blood stages of both chloroquine (CQ)-sensitive and CQ-resistant P. falciparum strains with 50% inhibitory concentration (IC(50)) values in the lower nanomolar range. Ring stages showed the highest susceptibility. As demonstrated by high-performance liquid chromatography-tandem mass spectrometry on different cell culture compartments, MB is accumulated in malarial parasites. In drug combination assays, MB was found to be antagonistic with CQ and other quinoline antimalarials like piperaquine and amodiaquine; with mefloquine and quinine, MB showed additive effects. In contrast, we observed synergistic effects of MB with artemisinin, artesunate, and artemether for all tested parasite strains. Artemisinin/MB combination concentration ratios of 3:1 were found to be advantageous, demonstrating that the combination of artemisinin with a smaller amount of MB can be recommended for reaching maximal therapeutic effects. Our in vitro data indicate that combinations of MB with artemisinin and related endoperoxides might be a promising option for treating drug-resistant malaria and should be studied in future field trials. Resistance development under this drug combination is unlikely to occur

Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues from Plasmodium falciparum▿

Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k = 4 M−1 s−1), thioredoxins (k = 8.5 to 26 M−1 s−1), dihydrolipoamide (k = 53 M−1 s−1), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the kcat values ranging from 0.03 s−1 to 10 s−1 at 25°C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced flavin rather than by the active-site dithiol Cys58/Cys63. The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H2O2 at the expense of O2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cell-pathological and clinical contexts

<i>B. cereus</i> spore germination at low spore concentrations.

<p>(A) <i>B. cereus</i> spores were diluted in 10 or 4000 ml (OD₅₈₀ of 1 or 0.0025, respectively) of germination buffer supplemented with 0.2 mM inosine and 0 or 40 µM alanine. Thirty min post-inosine exposure, spores were collected by centrifugation, and pellets were treated with malachite green to stain resting spores and safranin-O to stain germinated cells. Samples were placed under a microscope and a field selected at random. (B) <i>B. cereus</i> spores were diluted in germination buffer (OD₅₈₀ of 1 to 0.0025) in the presence of 0.2 mM inosine. Stained samples were placed under a light microscope and the amount of resting spores and germinated cells were counted on three different fields selected at random. The percentage of germinated spores was plotted against the initial spore optical density.</p

Enhanced germination rate of B. cereus spores germinated in conditioned supernatants.

<p>Wild-type <i>B. cereus</i> spores were germinated with a 0.2 mM inosine solution (•) or in conditioned supernatants containing 0.2 mM inosine (▪). <i>B. cereus</i> spores were also germinated with 0.2 mM inosine and 20 µM alanine (X).</p

7-AMC adducts detected in <i>wt B. cereus</i> conditioned supernatants.

<p>Wild type <i>B. cereus</i> 569 spores were resuspended in 200 µl TMB buffer to OD₅₈₀ = 1. Spores were treated with 0.2 mM inosine and supernatants were collected 30 min post-inosine addition. Collected supernatants were labeled with 7-AMC. 7-AMC adducts sere separated by RP-HPLC and identified by mass spectrometry. Concentrations were calculated by fluorescence spectroscopy.</p

<i>B. cereus</i> spore germination in the presence of Ca-DPA.

<p>(A) Wild-type <i>B. cereus</i> spores were germinated in the presence of 0.2 mM inosine (□). <i>B. cereus</i> spores were also germinated in the presence of conditioned supernatants containing 0.2 mM inosine (+). Spores were also germinated with 0.2 mM inosine supplemented with 0.18 mM Ca-DPA (▪).</p