The glyoxylate cycle is required for fungal virulence (Results (Genome…
The glyoxylate cycle is required for
: metabolic pathway where permits use of two carbon compound as carbon sources
11 of 15 most highly induced S. cerevisiae genes after phagocytosis encode proteins related to the glyoxylate cycle.
enzymes related to glyoxylate pathway that was iduced:
Isocitrate lyase , ICL1
Malate synthase, MLS1
Malate dehydrogenase, MDH2
Acetyl-CoA synthase, ASC1
Citrate synthase, CIT2
glyoxylate and TCA share common rxn - however in glyoxylate only isoenzyme
induced, MDH1,MDH3 not
- phagocytosis glyoxylate cycle, isocitrate lyases (ICL1), malate synthase (MLS 1)
Yeast-macrophage co-culture and gene expression analysis
1) Murine macrophage-like cell line (J774A) -> cultured in RPMI + 10% fetal bovine serum (37C in 95% air/5% CO2)
2)18 h before a co-culture:
cells were plated in 50 ml media->at 2x10^7 cells per 750 ml flask
3) Yeast was grown overnight -> in YPD media at 37C -> diluted in YPD (3-4hrs)
4) Yeast cell undergo centrifugation -> wash -> pellet resuspended in PBS -> added to J774A culture (4x10^8 cells per flask)
5) co-culture was incubated for 3hrs at 37C in normal air
6) Yeast cell that is not associated with macrophage -> removed by washed three times by ice cold PBS.
7) macrophage+ associated yeast remove by scraping -> centrifuge (1min, 500xg) -> cell mixture washed 2x w cold water -> frozen at -80C
Mutant construction and analysis
Saccharomyces cerevisiae icl1
mutants -> constructed in the EM93 -> using a PCR-mediated w G418-resistance cassette.
2) Mutants were constructed -> in both mating types and mated
For C. albicans
disruption construct -> created by inserting a
2) transformed into CAI4 -> selected by uracil prototrophy
3) identified by PCR
4) second round of transformation -> generate two
independent homozygous mutant
gene was re-introduced -> by transformation
6) Standard media was used -> strains grow at 37C
1) RNA -> made from the pooled cell pellets ->using hot acidic phenol
2) Poly(A) fraction -> isolated using the Poly(A)ttract kit (Promega)
3) Poly(A) RNA > hybridized to the Ye6100 oligonucleotide array set (Affymetrix)
4) Array data -> extracted and filtered (to remove any
genes whose expression did not change)
Murine virulence assay
1) Overnight cultures of C. albicans strains -> diluted into fresh YPD-> grow for 4 hrs, 37C
2) undergo centrifuge-> washed with PBS
3) cell were injected into mice
4) Mice monitored for 3 weeks -> moribund animals
isocitrate lyases (ICL1) & ,alate synthase (MLS1) - specific and limited to the glyoxylate cycle
Genome arrays and virulence studies suggested that the inside of macrophages are a glucose deficient environment
Glucose are required for synthesis of ribose and deoxyribose important in cell proliferation.
Phagolysosome is rich in fatty acids and acetyl-coA which will be assimilated through glycoxylate cycle as the only route for glucose synthesis because it bypasses the TCA cycle.
(wild type) can grow in media containing only oleic acids as sole carbon source but
Although glycoxylate cycle is important for virulence but by blocking it only by blocking nutrient availability is not sufficient for cancelling pathogenicity and its survival inside a macrophage.
Compounds that inhibit nutrient availability such as glyphosate and imidizolinones targeting plant enzymes were developed into herbicides. The enzymes of a glycoxylate cycle were not found in mammals hence effective as antibacterial and antifungal agents.
icl 1 mutant strains-fail to use acetate or ethanol
experiment of mice: infection with heterozygote resulted in an intermediate mortality
isocitrate lyase- not only induced by macrophage phagocytosis and essential for full virulence in the fungal pathogen
glucose- required for synthesis of many macromolecules for proleferation includes ribose and deoxyribose