Clinostat Rotation Affects Metabolite Transportation and Increases Organic Acid Production by Aspergillus carbonarius, as Revealed by Differential Metabolomic Analysis.

Contamination by fungi may pose a threat to the long-term operation of the International Space Station because fungi produce organic acids that corrode equipment and mycotoxins that harm human health. Microgravity is an unavoidable and special condition in the space station. However, the influence of microgravity on fungal metabolism has not been well studied. Clinostat rotation is widely used to simulate the microgravity condition in studies carried out on Earth. Here, we used metabolomics differential analysis to study the influence of clinostat rotation on the accumulation of organic acids and related biosynthetic pathways in ochratoxin A (OTA)-producing Aspergillus carbonarius As a result, clinostat rotation did not affect fungal cell growth or colony appearance but significantly increased the accumulation of organic acids, particularly isocitric acid, citric acid, and oxalic acid, and OTA both inside cells and in the medium, as well as resulted in a much higher level of accumulation of some products inside than outside cells, indicating that the transport of these metabolites from the cell to the medium was inhibited. This finding corresponded to the change in the fatty acid composition of cell membranes and the reduced thickness of the cell walls and cell membranes. Amino acid and energy metabolic pathways, particularly the tricarboxylic acid cycle, were influenced the most during clinostat rotation compared to the effects of normal gravity on these pathways.IMPORTANCE Fungi are ubiquitous in nature and have the ability to corrode various materials by producing metabolites. Research on how the space station environment, especially microgravity, affects fungal metabolism is helpful to understand the role of fungi in the space station. This work provides insights into the mechanisms involved in the metabolism of the corrosive fungus Aspergillus carbonarius under simulated microgravity conditions. Our findings have significance not only for preventing material corrosion but also for ensuring food safety, especially in the space environment.

Conversion of DON to 3-epi-DON in vitro and toxicity reduction of DON in vivo by Lactobacillus rhamnosus.

Deoxynivalenol (DON) is a mycotoxin that contaminates grains and feed. Degradation and toxicity reduction of DON by probiotics benefit human and animal health. Lactobacillus rhamnosus is an FDA-approved probiotic that can be used in children. After screening seven L. rhamnosus strains isolated from human milk, SHA113 showed the highest DON degradation rate of 60% under the optimal conditions of 37 °C, pH 6.0, OD600 = 1.5, 5 mg L-1 DON, and 48 h. When dead and live SHA113 cells were used separately, only the live cells reduced the DON concentration and transformed it into 3-epi-DON. Mice feeding experiments showed that pretreatment with SHA113 for 48 h reduced the toxicity of DON to the immunological system and organs. Directly feeding SHA113 cells could also slightly reduce the DON toxicity. Both in vitro and in vivo experiments indicated that L. rhamnosus has potential to reduce DON toxicity.

Capability of iturin from Bacillus subtilis to inhibit Candida albicans in vitro and in vivo.

Candida albicans is a fungal pathogen that is difficult to cure clinically. The current clinic C. albicans-inhibiting drugs are very harmful to humans. This study revealed the potential of iturin fractions from Bacillus subtilis to inhibit C. albicans in free status (MIC = 32 µg/mL) and natural biofilm in vitro. The inhibition mechanism was identified as an apoptosis pathway via the decrease of mitochondrial membrane potential, the increase of the reactive oxygen species (ROS) accumulation, and the induction of nuclear condensation. For in vivo experiments, the C. albicans infection model was constructed via intraperitoneal injection of 1 × 108C. albicans cells into mice. One day after the infection, iturin was used to treat infected mice at different concentrations alone and in combination with amphotericin B (AmB) by intraperitoneal injection. The treatment with AmB alone could cause the death of infected mice, whereas treatment with 15 mg/kg iturin per day alone led to the survival of all infected mice throughout the study. After continuously treated for 6 days, all mice were sacrificed and analyzed. As results, the combination of 15 mg/kg iturin and AmB at a ratio of 2:1 had the most efficient effect to remove the fungal burden in the kidney and cure the infected mice by reversing the symptoms caused by C. albicans infection, such as the loss of body weight, change of immunology cells in blood and cytokines in serum, and damage of organ structure and functions. Overall, iturin had potential in the development of efficient and safe drugs to cure C. albicans infection.

Effect of cell culture models on the evaluation of anticancer activity and mechanism analysis of the potential bioactive compound, iturin A, produced by Bacillus subtilis.

Two-dimensional (2D) cell culture is widely used to evaluate the potential of food compounds in anticancer activity in vitro. However, 3D culture is rarely used. In this study, we compared the obtained anticancer activity and mechanisms of iturin A, a multiple functional compound produced by Bacillus subtilis, in 2D and 3D cultures of HepG2 cells. 3D culture resulted in a much higher 50% inhibitory concentration (55.26 µM) compared to 2D culture (11.91 µM). Reactive oxygen species accumulation, autophagy, apoptosis characterized by cytochrome c release, high apoptotic protein expression and caspase activation were detected in both 2D and 3D cultures. Induction of paraptosis was also detected in 2D culture and the cytoplasmic vacuoles occurred in large numbers. Compared with 2D culture, 3D culture can simulate the microenvironment in vivo and provide more accurate data. Therefore, 3D culture was recommended for the evaluation of anticancer activity of food compounds towards solid tumors.

Iturin A-like lipopeptides from Bacillus subtilis trigger apoptosis, paraptosis, and autophagy in Caco-2 cells.

This study revealed that iturin A-like lipopeptides produced by Bacillus subtillis induced both paraptosis and apoptosis in heterogeneous human epithelial colorectal adenocarcinoma (Caco-2) cells. Autophagy was simultaneously induced in Caco-2 cells treated with iturin A-like lipopeptides at the early stage and inhibited at the later stage. A western blot analysis showed that the lipopeptides induced apoptosis in Caco-2 cells via a mitochondrial-dependent pathway, as indicated by upregulated expression of the apoptotic genes bax and bad and downregulated expression of the antiapoptotic gene bcl-2. The induction of paraptosis in Caco-2 cells was indicated by the occurrence of many cytoplasmic vacuoles accompanied by endoplasmic reticulum (ER) dilatation and mitochondrial swelling and dysfunction. ER stress also occurred with significant increases in reactive oxygen species and Ca2+ levels in cells. Autophagy was detected by a transmission electron microscopy analysis and by upregulated expression of LC3-II and downregulated expression of LC3-I. The inhibition of autophagy at the later stage was shown by upregulated expression of p62. This study revealed the capability of iturin A-like B. subtilis lipopeptides to simultaneously execute antitumor potential via multiple pathways.

Potential of iturins as functional agents: safe, probiotic, and cytotoxic to cancer cells.

The iturin produced by Bacillus subtilis was previously found to exhibit significant antifungal activities, showing potential for application to food to protect it from spoilage and decay. However, its safe level must be evaluated before it can be used in foods consumed by mammals. Therefore, we carried out acute toxicity (7 days) and subacute toxicity (28 days) evaluations by intragastric administration of the iturin at 5000 mg per kg (bw) and 2000 mg per kg (bw) to Kunming mice. At the end of the subacute toxicity evaluation, the blood parameters of the mice were evaluated, and the visceral organs and intestinal flora were examined. No acute toxicity was found for 5000 mg per kg (bw) iturin and no significant negative effects were found for 2000 mg per kg (bw) iturin on blood parameters or organ functions. A more important observation was that the application of the iturin at the concentration of 2000 mg per kg (bw) showed great enhancement of the abundance of probiotics in the intestinal microflora. The iturin also showed inhibitory effects on cancer cells HepG2, Caco-2, MCF-7, A549, and BIU-87, at the level of 30 µM. The results indicate that the B. subtilis iturin has the potential to be developed as a functional food additive with antitumor potential and the capability to enhance the abundance of probiotics in the intestinal microflora. This study indicates a new possible source of functional food additives.

Potential of Bacillus subtilis lipopeptides in anti-cancer I: induction of apoptosis and paraptosis and inhibition of autophagy in K562 cells.

The lipopeptide iturin from Bacillus subtilis has been found to have a potential inhibitory effect on breast cancer, alveolar adenocarcinoma, renal carcinoma, and colon adenocarcinoma. In this study, the potential of B. subtilis lipopeptides (a mixture of iturin homologues, concentration of 42.75%) to inhibit chronic myelogenous leukemia was evaluated using K562 myelogenous leukemia cells. The results showed that the lipopeptides could completely inhibit the growth of K562 at 100 µM, with an IC50 value of 65.76 µM. The lipopeptides inhibited the profile of K562 via three pathways: (1) induction of paraptosis indicated by the occurrence of cytoplasmic vacuoles, and swelling of the mitochondria and endoplasmic reticulum (ER) without membrane blebbing in the presence of a caspase inhibitor; (2) inhibition of autophagy progress illustrated by the upregulated expression of LCII and P62; and (3) induction of apoptosis by causing ROS burst, and induction of the intrinsic pathway indicated by the upregulated expression of cytochrome c (Cyto-c), bax, and bad, together with downregulated expression of Bcl-2. The ROS-dependent apoptosis and caspase-independent paraptosis were verified using the ROS inhibitor and caspase inhibitor, respectively. The extrinsic apoptosis pathway was not involved in the lipopeptide's effects on K562. Overall, the B. subtilis lipopeptides (consisting of a majority of iturin) exhibited promising potential in inhibiting chronic myelogenous leukemia in vitro via simultaneously causing paraptosis, apoptosis, and inhibition of autophagy.

Fungal Spores Promote the Glycerol Production of Saccharomyces cerevisiae by Upregulating the Oxidative Balance Pathway.

Fungal contamination is prevalent in grape berries and unavoidable during the winemaking process. In botrytized wine, Botrytis cinerea contamination of grape berries beneficially promotes the wine flavor, which is desirable especially with high glycerol content. To investigate the underlying mechanism, Aspergillus carbonarius and B. cinerea spores were separately cocultured with two different Saccharomyces cerevisiae strains in both grape juice and synthetic nutrient media. The results showed that both A. carbonarius and B. cinerea promoted glycerol accumulation and the consumption of sugars in the coculture systems but could not synthesize glycerol by themselves. The metabolites produced by fungal spores triggered these reactions. Reverse transcription-polymerase chain reaction analysis showed that the presence of A. carbonarius spores regulated the expression of GPP1 and GPD2, indicating that the reaction was triggered by regulating the oxidative balance pathway. The study revealed the beneficial impact of fungal contamination on wine quality by influencing yeast metabolism.