ATS associates with Tef2 in <i>S. pombe</i> and <i>M. oryzae</i>.

<p>(<b>A</b>) Loss of SpTef2-function simulates ATS effect in <i>S. pombe</i>. Cell wall staining of the wild-type or <i>tef2</i>Δ <i>S. pombe</i> cells using CFW. Red arrowheads depict defective septal/cell wall deposition. Scale bar equals 10 micron. (<b>B</b>) Effect of digoxin on subcellular localization of SpTef2-RFP or Swo1-GFP in <i>S. pombe</i> cells. The strains expressing the indicated fusion proteins were stained with CFW and analysed by epifluorescence microscopy. Arrowheads show distinct aggregates of SpTef2-RFP. Bar = 10 µm. (<b>C</b>) Effect of ATS on localization of RFP-Tef2 in <i>M. oryzae</i> vegetative hyphae (upper panels; Scale Bar = 5 µm) and conidia (middle and lower panels; Bar represents 10 µm) co-stained with DAPI to aid visualization of nuclei. Arrowheads denote aberrant perinuclear aggregates and/or patches of RFP-Tef2. BF, Bright Field.</p

Excess ATS or digoxin induces cell death in the host plants and reduces blast disease severity.

<p>(<b>A</b>) Barley leaf explants were treated with residual solvent or ATS for 72 h, stained with trypan blue and observed using bright field optics. Arrowhead and arrows show visible (inset) and localized cell death, respectively, in the inoculation zone. (<b>B</b>) Transmission electron micrographs of residual solvent-, ATS- or digoxin-treated rice leaf explant stained with CeCl₃ after 48 h of treatment. Arrowheads depict cerium perhydroxide granules and/or plasmolysis after ATS or digoxin treatment for 48 h. CW, cell wall; M, mitochondrion; and V, vacuole. Bars = 1 µm. (<b>C</b>) Transcript levels of Pathogenesis Related genes tested by real-time qRT-PCR in rice after 24 h of treatment. Data represent mean ± SEM of two independent experiments with three replicates each. Perox, peroxidase; Tub, tubulin. (<b>D</b>) Detached barley leaf pieces were inoculated with wild-type conidia in the absence or presence of 200 µM digoxin (DG). The disease symptoms were evaluated at 6 dpi. Arrowhead denotes disease lesion. The data represents observations from 3 independent experiments.</p

ATS shares structural and functional properties with digoxin.

<p>Molecular mass of ATS (<b>A</b>) or digoxin (<b>B</b>) identified by APCI method. Molecular masses shown are sodium adducts of ATS or digoxin (both, <i>m/z</i> 780). Insets depict the predominant peaks of ATS or digoxin with their respective retention times. (<b>C</b>) and (<b>D</b>) Tandem mass spectra of ATS and digoxin, respectively. The ionization products characteristic of the steroidal nucleus (<i>m/z</i> 390), mono- and bi-sugar (<i>m/z</i> 520 and 650, respectively) molecules are highlighted. (<b>E</b>) Wild-type <i>S. pombe</i> cells were treated with residual solvent, ATS, or digoxin for 6 h and stained with CFW. Arrowheads show aberrant septal/cell wall biogenesis. Bars = 5 µm. (<b>F</b>) Conidia from wild-type <i>M. oryzae</i> were germinated on agarose in the presence of residual solvent, ATS, or digoxin and stained with CFW after 4 h. Excess cell wall deposits are indicated with arrowheads. Bar = 10 µm.</p

SpTef2 function and the F-actin cytoskeleton in <i>S. pombe</i>.

<p>(<b>A</b>) Sensitivity of <i>tef2</i>Δ <i>S. pombe</i> cells towards Ca⁺² in the growth medium. Serial dilutions of the wild-type or <i>tef2</i>Δ cells were inoculated under indicated growth conditions. (<b>B</b>) Morphology and dynamics of GFP-labelled F-actin cytoskeleton in wild-type <i>S. pombe</i> treated with ATS, digoxin or Ca⁺². The <i>tef2</i>Δ strain was analyzed in parallel. Arrowheads show excess accumulation of F-actin patches and/or short, spooling cables at the cell end(s). The maximum projection images shown here represent the compressed z-stack sections. Bar equals 10 µm.</p

Working model for the role of ATS in <i>M. oryzae</i> pathogenesis.

<p>(<b>A</b>) Schematic representation of accumulation of ATS, in the wild type or <i>abc3</i>Δ appressoria, affecting host entry. (<b>B</b>) The figure illustrates a proposed crosstalk/mechanistic link between ATS accumulation and ion homeostasis, Tef2-function, and F-actin dynamics during <i>M. oryzae</i> pathogenesis.</p

Exogenous ATS or digoxin alters the F-actin cytoskeleton in <i>M. oryzae</i>.

<p>Morphology (<b>A</b>) and dynamics (<b>B</b>) of the F-actin patches in wild type <i>M. oryzae</i> expressing Abp1-RFP and treated with ATS, digoxin, or 0.1 M CaCl₂. Arrowheads depict developing appressoria. Bars = 10 µm.</p

<i>Des1</i> ablation results in the activation of pro-survival kinase Akt/PKB.

<p><b>A.</b> Phospho-Akt^Ser473 level is upregulated in <i>Des1−/−</i> cells. <b>B.</b> Activated Akt phosphorylates its substrates at Ser/Thr. These phospho-Ser/Thr proteins detected by Phospho Akt Substrate (PAS) antibody shows increased phosphorylation in <i>Des1 −/−</i> cells. <b>C.</b> Among the Akt substrates, phospho-GSK-3β^Ser9, the inactive form of GSK-3β, is elevated in <i>Des1 −/−</i> cells. p27Kip which is inhibited by Akt, is downregulated in <i>Des1 −/−</i> cells. <b>D.</b> Pro-apoptotic tumor suppressor p53 is phosphorylated at Ser15 residue upon genotoxic stress such as etoposide. In <i>Des1 −/−</i> cells, phospho-p53^Ser15 and total p53 levels are significantly lower after treating with etoposide. p53 upregulates its negative regulator MDM2 at the transcriptional level. MDM2 can be phosphorylated by Akt or ERK at Ser 166 residue which in turn degrades non-phosphorylated p53. Paradoxical to the status of phospho-Akt^Ser473, phospho-MDM2^Ser166 is consistently higher in <i>Des1 +/+</i> cells.</p

Comprehensive analysis of phospholipids and glycolipids in the opportunistic pathogen <i>Enterococcus faecalis</i>

<div><p><i>Enterococcus faecalis</i> is a Gram-positive, opportunistic, pathogenic bacterium that causes a significant number of antibiotic-resistant infections in hospitalized patients. The development of antibiotic resistance in hospital-associated pathogens is a formidable public health threat. In <i>E</i>. <i>faecalis</i> and other Gram-positive pathogens, correlations exist between lipid composition and antibiotic resistance. Resistance to the last-resort antibiotic daptomycin is accompanied by a decrease in phosphatidylglycerol (PG) levels, whereas multiple peptide resistance factor (MprF) converts anionic PG into cationic lysyl-PG via a trans-esterification reaction, providing resistance to cationic antimicrobial peptides. Unlike previous studies that relied on thin layer chromatography and spectrophotometry, we have performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) directly on lipids extracted from <i>E</i>. <i>faecalis</i>, and quantified the phospholipids through multiple reaction monitoring (MRM). In the daptomycin-sensitive <i>E</i>. <i>faecalis</i> strain OG1RF, we have identified 17 PGs, 8 lysyl-PGs (LPGs), 23 cardiolipins (CL), 3 glycerophospho-diglucosyl-diacylglycerols (GPDGDAG), 5 diglucosyl-diacylglycerols (DGDAG), 3 diacylglycerols (DAGs), and 4 triacylglycerols (TAGs). We have quantified PG and shown that PG levels vary during growth of <i>E</i>. <i>faecalis in vitro</i>. We also show that two daptomycin-resistant (DapR) strains of <i>E</i>. <i>faecalis</i> have substantially lower levels of PG and LPG levels. Since LPG levels in these strains are lower, daptomycin resistance is likely due to the reduction in PG. This lipidome map is the first comprehensive analysis of membrane phospholipids and glycolipids in the important human pathogen <i>E</i>. <i>faecalis</i>, for which antimicrobial resistance and altered lipid homeostasis have been intimately linked.</p></div

List of primers used.

<p>List of primers used.</p

Status of ceramide and dihydroceramide to confirm the acceptability of our in vitro model.

<p><b>A.</b> Structure of dihydroceramide and ceramide. <b>B.</b> Des 1 knock out MEFs do not express Des1 mRNA (upper panel) as well as Des1 protein (lower panel). <b>C.</b> Des1 deletion inhibits ceramide formation and causes accumulation of dihydroceramide (*<i>P</i><0.01, **<i>P</i><0.003). <b>D.</b> To confirm the efficiency of <i>Des1</i> ablation, downstream metabolites of ceramide and dihydroceramide were detected. Ceramide containing <i>Des1 +/+</i> cells contain sphingomyelin while the <i>Des1 −/−</i> contain mostly its dihydro-species, dihydrosphingomyelin (*<i>P</i><0.05, # not significant). <b>E.</b> A similar pattern was observed for levels of sphingosine and dihydrosphingosine (*<i>P</i><0.01, **<i>P</i><0.05). The data represent the average values of experiments which have been done at least in triplicates. For all panels, paired t-test has been used for statistical analysis.</p