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The glyoxylate cycle is required for fungal virulence (Yeast±macrophage…
The glyoxylate cycle is required for
fungal virulence
Candida albicans
mammalian gastrointestinal flora that can induce fungal infection in immunocomprimised patients.
albicans, phagocytosis upregulates principal enzymes (isocitrate lyase (ICL1) and malate synthase (MLS1) of the glyoxylate cycle.
mutants that lack ICL1 are less virulant compared to the wild types.
ICL1 is required for both upregulation anf virulance of ofMycobacterium tuberculosis
Once phagocytosed the Candida albicans will secrete cytokins and will induce the fungal hypha development
genome-wide expression profiles related yeast Saccharomyces cerevisiae (less virulant)
obtain signature of the events thattakes place in fungus on ingestion by a mamanlian macrophage.
Live S. cerevisiae cells isolated from the phagolysosome
In vitro, cultured mammalian
macrophages readily ingest both S. cerevisiae and C. albicans cells.
population of S. cerevisiae highly enriched for phagocytosed cells are isolated and subjected to whole-genome microarray analysis with oligonucleotide-based assays (Affymetrix)
host±pathogen interactions is hampered as the lacking of genetic tool in the Candida albicans.
Glyoxylate cycle
Metabolic pathway that permits the use of two-carbon compounds as carbon sources
Enzymes
Two principle enzymes
Isocitrate lyase (
ICL1
)
Malate synthase (
MLS1
)
Other related enzymes
Malate dehydrogenase (
MDH2
)
cytosolic isozym
Citrate synthase (
CIT2
)
citrate synthase isoforms
: glyoxylate cycle-specific
Genes related to glyoxylate cycle
Acetyk coenzyme A (actetyl-CoA) synthase (
ACS1
)
YDR384c
homologue
Yarrowia lipolytica
glyoxylate pathway regulator (
GPR1
)
Transporter and acetyltransferases
: function to traffic intermediates of glyoxylate cycle and fatty-acid degradation between organelles
CRC1
ACR1
YAT1
YER024w
Fructose-1,6-bisphosphatase (
FBP1
)
central regulatory point in gluconeogensis: production of glucose
Mutant strains of S. cerevisiae and C. albicans
Both icl1 mutant strains fail to use acetate or ethanol
C. albicans, both the heterozygous strain (¢icl1/ICL1) and a homozygous mutant in which ICL1 has been re-introduced (¢icl1/¢icl1 + ICL1) grow as well as a wild-type strain on acetate media
The growth rate of the C. albicans ¢icl1/¢icl1 strain is not significantly different from the parent strain on rich (YP-Dextrose) media.
nor is this strain any more sensitive to a variety of in vivo stresses, including salt, heat shock, ethanol (assayed on glucose media), or oxidative stress (Fig. 3c, d). ¢icl1/ ¢icl1 strains form filaments (on solid medium) and germ tubes (in liquid medium) in response to serum or neutral pH
Mice injected with wild-type C. albicans strain SC5314 succumb rapidly to the infection (median survival of 3 days; Fig. 4)
, mice injected with two independently constructed ¢icl1/¢icl1 strains survived longer.
At day 28, 7 out of 10 of the animals injected with one strain (MLC7) remained alive as did 6 out of 10 of an independent homozygous mutant (MLC8).
Infection with the heterozygote (¢icl1/ICL1) resulted in an intermediate mortality (median of 8 days).
isocitrate lyase is not only induced by macrophage phagocytosis but is also essential for full virulence in this fungal pathogen.
Microbes find the inside of a macrophage to be a glucose deficient environment. Glucose is required for the synthesis of many macromolecules necessary for proliferation, including ribose and deoxyribose
the phagolysosome is rich in fatty acids or their breakdown products (primarily acetyl-CoA). Acetyl- CoA can only be assimilated through the glyoxylate cycle, which bypasses the catabolic steps of the TCA cycle; thus the glyoxylate cycle is the only route to the synthesis of glucose in this environment.
The wild-type strain uses oleic acid as well as acetate, whereas the ¢icl1/¢icl1 strain is unable to metabolize oleic acid. The glyoxylate pathway is necessary for virulence but it is clearly not sufficient
Both Saccharomyces and Candida induce the glyoxylate cycle on macrophage contact, yet only Candida is virulent.
Genes encoding the glyoxylate cycle have now been shown to be required for virulence in both a bacterium (M. tuberculosis) and a fungus (C. albicans) that can survive inside a macrophage.
Inhibitors of the glyoxylate cycle pathway should block nutrient availability and prevent survival of these pathogens inside the macrophage.
Yeast±macrophage co-culture and gene expression analysis
1) Murine macrophage-like cell line J774A (ATCC number TIB-67) was cultured in RPMI
plus 10% fetal bovine serum at 37 8C in 95% air/5% CO2
2) 18 h before a co-culture
experiment, cells were plated in 50 ml media at 2 ´ 107 cells per 750 ml ¯ask.
3)
Yeast strain EM93 (MATa/a prototroph11) was grown overnight in YPD media at 37 8C,
diluted infresh YPD for 3±4 h
4) Yeast cells were pelleted by centrifugation, washed once, resuspended in PBS, and added to the J774A cultures at
4 x 10^8 cells per flask
5) co-culture was incubated for 2.5±3.0 h at 37 8C in normal air
Yeast cells associated with the adherent macrophages were removed by washing three times with icecold PBS
macrophages and associated yeast were removed by scraping, and pooled by centrifugation for 1min at 500g. (calculate the number of viable cells
