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The glyoxylate cycle is required for fungal virulence (Glyoxylate cycle…

- - - - the growth rate of the mutant icl1/ICL1 strain is not significantly different from the parents strain on rich (Dextrose ) media.
        
        this strain is not more sensitive to variety of invivo stress.
    - - wild type (strain SC5314) succumb rapidly to infection
      - two independently constructed ¢icl1/¢icl1 strains survived longer.
      - Infection with the heterozygote (¢icl1/ICL1)
        resulted in an intermediate mortality.
    - - glucose is require for the synthesis of many macromolecules necessasy for proliferation
        
        glycoxylate cycle is the only route to synthesis glucose in this environment
- - - - Scrap the macrophage and the yeast to remove it. Pool the cells by centrifugation for 1 min at 500g.
      - Wash the cell mixture twice with ice-cold water to lyse the mammalian cells.
      - Use Poly(A)ttract kit (Promega) to isolate the RNA that is made from the pooled cell pellets using hot acidic phenol and the poly(A) fraction.
      - Extract and alter the array data to remove any genes whose expression did not change at least twofolds.
      - Identify the Candida albicans homologues of ICL1 and MLS1 by searching the genome sequence data from the Stanford DNA Sequencing and Technology Center
      - Perform macrophage interactions using 106 J774A cells in 5 ml media with 2 107 S. cerevisiae (EM93) or C. albicans (SC5314) cells for northern analysis.
      - The control population was grown for 3 h in rich media (YPD), or in tissue culture media without (RPMI) or with (serum) 10% fetal bovine serum.
      - Amplify species-species probes by PCR and label with a random primer.
      - Culture Murine macrophage-like cell line J774A in RPM1 plus 10% of fetal bovine serum at 37°C in 95% air/5% CO2.
      - Plate cell in 50 ml media at 2 x 10^7 cells per 750 ml flask.
      - Grow yeast strain EM93 overnight in YPD media at 37°C. Then, dilute in fresh YPD for 3-4 hours.
      - Centrifuge the yeast cells. Wash the pellet and resuspend in PBS. Add the suspension to the J774A cultures at 4 x 10^8 cells per flask.
      - Incubate the co-culture for 2,5-3.0 hours at 37°C in normal air.
- - - - neutopenia patient (low neutrophils) tend to get candidiasis
    - - secrete cytokines and induce hyphal development
  - - - Live S.cerevisiae cells isolated from
        the phagolysosome
      - gene for glyoxylate cycle ( metabolic pathway that permits the use of two-carbon compounds as carbon sources) induced
      - In C. albicans
        
        phagocytosis also upregulates the principal enzymes of the glyoxylate cycle
        
        isocitrate lyase (ICL1)
        
        malate synthase (MLS1)
- - - - most of the phagocytosed cells were alive (67% alive)
      - transcriptional profiling of these cells reveals the response of fungal cells to phagocytosis
        
        11/15 highly induced S. cerevisiae genes after phagocytosis encode proteins related to the glyoxylate cycle
        
        3/5 glyoxylate cycle enzymes are on this list
        
        isocitrate lyase, ICL1
        
        malate synthase, MLS1
        
        malate dehydrogenase, MDH2
        
        citrate synthase, CIT2 (strongly induced (4.9-fold, ranking 24th))
        
        Several genes functionally related to the glyoxylate cycle are induced
        
        acetyl coenzyme A (acetyl-CoA) synthase (ACS1)
        
        YDR384c, a homologue of the Yarrowia lipolytica glyoxylate pathway regulator (GPR1; refs 7, 8)
        
        several transporters and acetyltransferases
        
        used to traffic intermediates of the glyoxylate cycle and fatty-acid degradation between organelles (CRC1, ACR1, YAT1 and YER024w)
        
        ructose-1,6-bisphosphatase (FBP1)
        
        central regulatory point in gluconeogenesis9Ðthe production of glucose is the principal function of the glyoxylate cycle
        
        Induction of the glyoxylate cycleindicatesthatnutrientacquisitionanduseistheprimaryfocus of yeast cells upon phagocytosis, presumably because the phagolysosome is poor in complex carbon compounds
- - - - e.g glyphosate and imidizolinones
      - because their targets are enzymes produced by plants but not by animals