sirolimus and deoxynivalenol

We investigated the effects of rapamycin (RAPA) and chloroquine (CQ) in supporting growth performance and the intestinal mucosal barrier in response to deoxynivalenol (DON) in piglets. A total of 32 healthy weaned piglets (bodyweight 7.10 ± 0.58 kg) were divided into four groups and treated daily with RAPA (1 mg/kg BW), CQ (10 mg/kg BW), or a control volume of normal saline (two groups) until the end of the experiment. After feeding a basal diet for seven days, three groups were then switched to mildewed feed containing 1 mg kg/DON for a further seven days. In contrast to the control group, DON-treated piglets showed decreased average daily gain (ADG) and daily feed intake (ADFI), as well as negatively affected intestinal morphology as indicated by villus height, crypt depth, and tight junction protein expression. A group treated with RAPA and DON showed increased intestinal autophagy, aggravated inflammatory responses, and damage to the intestinal mucosa and permeability, leading to reduced growth performance. Meanwhile, a group treated with CQ and DON showed indices comparable to the non-DON control group, with alleviated inflammatory cytokines and healthy intestinal morphology and structure. They also showed better growth performance compared to DON treatment alone. These findings have important implications for mediating autophagy against DON

Topics: Amine Oxidase (Copper-Containing); Animals; Antioxidants; Autophagy; Cadherins; Chloroquine; Cytokines; Diet; Inflammation; Integrins; Intestinal Mucosa; Lactic Acid; Occludin; Proliferating Cell Nuclear Antigen; RNA, Messenger; Sirolimus; Swine; Trichothecenes; Zonula Occludens-1 Protein

Autophagy is involved in the modulation of nutrition, immunity, and disease in humans and animals. To understand the impact of autophagy modulation on a rainbow trout gill cell line, RTgill-W1, treatments including reduced nutrition (2% fetal bovine serum compared with 10% control), rapamycin, 3-methyladenine, deoxynivalenol, and chloroquine were tested. Western blot and immunofluorescence were used to detect microtubule-associated protein 1A/1B-light chain protein and quantitative polymerase chain reaction was used to detect the expression of 10 autophagy-related genes. At 3-d post-treatment, reduced nutrition significantly (p < 0.05) increased autophagy while deoxynivalenol significantly (p < 0.01) suppressed it compared to controls. These phenomena were confirmed by using immunofluorescence to detect the number of autophagosomes in RTgill-W1. Chloroquine is critical to allow observation of microtubule-associated protein 1A/1B-light chain protein in this model. The commonly used autophagy-modulating chemicals rapamycin and 3-methyladenine either activated or suppressed microtubule-associated protein 1A/1B-light chain protein, respectively, as expected from the literature, but did not act in a consistently significant manner. Expression of five of the 10 Atg genes, including lc3, gabarap, atg4, atg7, and atg12, were altered in a similar pattern to microtubule-associated protein 1A/1B-light chain protein. The consistent trend of autophagy-related gene upregulation including becn1, lc3, gabarap, and atg9 following treatment with 3-methyladenine and chloroquine is suggestive of a novel feedback regulation in the autophagy machinery.

Topics: Adenine; Animals; Autophagosomes; Autophagy; Cell Line; Cell Survival; Chloroquine; Fish Proteins; Gene Expression Regulation; Gills; Microtubule-Associated Proteins; Nutrients; Oncorhynchus mykiss; Pharmaceutical Preparations; Serum; Sirolimus; Time Factors; Trichothecenes

Lipid droplets (LDs) control lipid metabolism in eukaryotic cells in general. However, the biogenesis regulation and biological functions of LDs are largely unknown in pathogenic fungi. Rapamycin treatment results in a significant increase of LD biogenesis in Fusarium graminearum. Molecular mechanisms of the target of rapamycin (TOR) pathway in regulating LD biogenesis and the functions of LD in virulence of F. graminearum were investigated in depth by combining genetic, cytological and phenotypic strategies. TOR in Fusarium graminearum (FgTOR) inhibition by rapamycin induces LD biogenesis through the FgPpg1/Sit4 signaling branch. FgPpg1 promotes phosphorylation of protein phosphatase FgNem1 by the protein kinase FgCak1. The phosphorylated FgNem1 dephosphorylates the phosphatidate phosphatase FgPah1. Dephosphorylated FgPah1 is active and stimulates LD biogenesis. Moreover, deletion of FgNem1/Spo7 or FgPah1 leads to serious defects in vegetative growth, sexual development and virulence. The results of this study provide novel insights into the regulatory mechanism and biological functions of the LDs in the devastating pathogenic fungus F. graminearum.

Topics: Fungal Proteins; Fusarium; Lipid Droplets; Phosphorylation; Protein Binding; Signal Transduction; Sirolimus; Trichothecenes; Virulence

The target of rapamycin (TOR) signaling pathway plays critical roles in controlling cell growth in a variety of eukaryotes. However, the contribution of this pathway in regulating virulence of plant pathogenic fungi is unknown. We identified and characterized nine genes encoding components of the TOR pathway in Fusarium graminearum. Biological, genetic and biochemical functions of each component were investigated. The FgFkbp12-rapamycin complex binds to the FgTor kinase. The type 2A phosphatases FgPp2A, FgSit4 and FgPpg1 were found to interact with FgTap42, a downstream component of FgTor. Among these, we determined that FgPp2A is likely to be essential for F. graminearum survival, and FgSit4 and FgPpg1 play important roles in cell wall integrity by positively regulating the phosphorylation of FgMgv1, a key MAP kinase in the cell wall integrity pathway. In addition, the FgPpg1 interacting protein, FgTip41, is involved in regulating mycelial growth and virulence. Notably, FgTip41 does not interact with FgTap42 but with FgPpg1, suggesting the existence of FgTap42:FgPpg1:FgTip41 heterotrimer in F. graminearum, a complex not observed in the yeast model. Collectively, we defined a genetic regulatory framework that elucidates how the TOR pathway regulates virulence and vegetative development in F. graminearum.

Topics: Drug Resistance, Fungal; Fusarium; Gene Expression Regulation, Fungal; Genes, Fungal; Genetic Complementation Test; Saccharomyces cerevisiae; Sequence Deletion; Signal Transduction; Sirolimus; Tacrolimus Binding Protein 1A; TOR Serine-Threonine Kinases; Trichothecenes; Two-Hybrid System Techniques; Virulence