The glyoxylate cycle is required for
fungal virulence
Glyoxylate cycle and TCA share common reactions, only the isozymes specialized for the glyoxylate cycle that are
induced
mutant strain from S.cerevisiae and C. albican that lack of ICL1.
Methods
Introduction
cytosolic isozyme of MDH2, is induced 15.6-fold
Expression of glyoxylate cycle enzymes were not
changed significantly
In contrast, the mitochondrial (MDH1) and peroxisomal (MDH3) forms are not induced
Only the glyoxylate cycle-specific CIT2 is induced
Candida albicans
normal component of the mammalian gastrointestinal flora
In response to conditioned media, oxidative stress, or contact with heat-killed macrophages
responsible for most fungal infections in immunosuppressed patients
Phagocytosis specifically upregulates the glyoxylate cycle and its accessory proteins
normally phagocytosed by macrophages and neutrophils
Nutrient deprivation is the primary `stress' that confronts these cells.
secrete cytokines and induce hyphal development
neutopenia patient (low neutrophils) tend to get candidiasis
C. albicans genes were cloned for;
isocitrate lyase (ICL1)
malate synthase (MLS1)
The only enzymes whose activity is both
specific and limited to the glyoxylate cycle
Northern analysis of RNA from both S. cerevisiae and C. albicans cells grown in the presence of macrophages
genome-wide expression profiles of the related yeast Saccharomyces cerevisiae
both organisms the ICL1 or MLS1 genes are significantly induced by macrophage contact
Both mutant of icl mutant strain fail to use acetate or ethanol.
To determine the event in fungus on ingestion by a mammalian macrophage
Mutant construction and analysis
Murine virulence assay
Yeast±macrophage co-culture and gene expression analysis
Live S.cerevisiae cells isolated from
the phagolysosome
the induction of the glyoxylate enzymes is a conserved response to phagocytosis in these two yeasts
gene for glyoxylate cycle ( metabolic pathway that permits the use of two-carbon compounds as carbon sources) induced
click to edit
C. albican
heterozugous strain (mutant icl1/ICL1) and homozygous mutant ((¢icl1/ ¢icl1 + ICL1 grow well as wild type on acetate.
In C. albicans
phagocytosis also upregulates the principal enzymes of the glyoxylate cycle
isocitrate lyase (ICL1)
malate synthase (MLS1)
Systematic studies of host-pathogen interactions
lack of genetic tools in C. albicans
non-pathogenic yeast S. cerevisiae used to uncover relevant genes
cultured mammalian macrophages readily ingest both S. cerevisiae and C. albicans cells (in vitro)
A population of S. cerevisiae highly enriched for phagocytosed cells was isolated and subjected to whole-genome microarray analysis using oligonucleotide-based arrays (Affymetrix)
the growth rate of the mutant icl1/ICL1 strain is not significantly different from the parents strain on rich (Dextrose ) media.
Three hours after initiating the co-culture
this strain is not more sensitive to variety of invivo stress.
most of the phagocytosed cells were alive (67% alive)
transcriptional profiling of these cells reveals the response of fungal cells to phagocytosis
tested virulence in Mouse Model of systemic candidiasis.
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
wild type (strain SC5314) succumb rapidly to infection
isocitrate lyase, ICL1
malate synthase, MLS1
two independently constructed ¢icl1/¢icl1 strains survived longer.
malate dehydrogenase, MDH2
- 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.
citrate synthase, CIT2 (strongly induced (4.9-fold, ranking 24th))
Infection with the heterozygote (¢icl1/ICL1)
resulted in an intermediate mortality.
Several genes functionally related to the glyoxylate cycle are induced
acetyl coenzyme A (acetyl-CoA) synthase (ACS1)
Isocitrate lyase is not only induced by macrophage phagocytosis but is also essential for full virulence in this fungal pathogen.
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
Genes encoding the glyoxylate cycle
own to be required for virulence in both a bacterium (M. tuberculosis) and a fungus (C. albicans) that can survive inside a macrophage
Construct Saccharomyces cerevisiae Dicl1 mutants in the EM93 background using a PCR-mediated protocol with a G418-resistance cassette13.
Construct mutants in both mating types and mate them to produce homozygous ¢icl1/¢icl1 knockout strain (MLY283a/a).
Create an Dicl1 disruption construct for C. albicans by inserting a hisG±URA3±hisG cassette14 at a BglII site in the ICL1 open reading frame.
Linearize the construction and transform into CAI4 (a Ura- derivative of strain SC5314; refs 14, 15), and select by uracil prototrophy.
Identify the accurate integrants by PCR and passage on 5-FOA medium.
Use second round transformation to generate two independents homozygous ¢icl1/¢icl1 strains.
Re-introduce the wild-type ICL gene on linearized plasmid pRC2312 by transformation to produce a complemented strain.
Do the transformation of C. albicans by using the same description.
Use standard media and grow the strains at 37°C unless otherwise indicated.
Inhibitors of the glyoxylate cycle pathway
block nutrient availability and prevent survival of these pathogens inside the macrophage
microbe that find inside of the macrophages to be glucpse deficient environment
Compounds that inhibit nutrient availability have been developed into effective herbicides
e.g glyphosate and imidizolinones
because their targets are enzymes produced by plants but not by animals
click to edit
As theenzymes of the glyoxylate cycle are also not found in mammals, they are prime targets for antibacterial and antifungal agents
glucose is require for the synthesis of many macromolecules necessasy for proliferation
glycoxylate cycle is the only route to synthesis glucose in this environment
Both Saccharomyces and Candida induce the glyoxylate
cycle on macrophage contact, yet only Candida is virulent.
The wild-type strain uses oleic acid as well as acetate,
whereas the ¢icl1/¢icl1 strain is unable to metabolize oleic acid.
Dilute overnight cultures of C. albicans strains into fresh YPD and grown for 3±4 h at 37°C.
Collect the cultures by centrifuge and wash with PBS.
Inject cells 6 x 10^5 into the tail vein of 18±20-week-old female BALB/c mice.
Monitor the mice for 3 weeks after injection and kill the moribund animals.