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The glyoxylate cycle is required for fungal virulence (Methods (Yeast…
The glyoxylate cycle is required for
fungal virulence
Methods
Mutant construction and analysis
Construct
Saccharomyces cerevisiae icl1
mutants in the EM93 background using a PCR-mediated protocol with a G418-resistance cassette
Construct mutants in both mating types and mated to produce a homozygous
icl1/icl1
knockout strain (MLY283a/
a
)
For
C. albicans
,
hisG-URA3-hisG
cassette is inserted at a
Bgl
II site in the ICL1 open reading frame to construct an
icl1
disruption
Linearize the construct and then transform it into CAI4 (a Ura- derivative of strain SC5314) and then selected by uracil prototrophy.
Identify the accurate integrants by PCR and passage it on 5-FOA medium
Generate two independent homozygous
icl1/icl1
(MLC7 and MLC8) strains using the second round of transformation
Re-introduced the wild-type
ICL1
gene on linearized plasmid pRC2312 by transformation to produce a complemented strain (MLC10).
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Murine virulence assay
Dilute O/N
C. albicans
culture into fresh YPD
Incubate to grow the culture for 3-4H at 37C
Centrifuge the culture and then wash it with PBS to collect the culture
Inject the tail vein of 18±20-week-old female BALB/c mice with 6x10^5 cells collected from the culture
After injection, monitor the mice for 3 weeks
Notes:
Use 10 mice per stain
Kill moribund animals
Care for animals according to NIH guidelines
Yeast-macrophage co-culture and gene expression analysis
Culture Murine macrophage-like cell line J774A (ATCC number TIB-67) in RPMI plus 10% fetal bovine serum at 37C in 95% air/ 5% CO2
Plate cells in 50 ml media at 2x10^7 cells per 750 ml flask about 18H before a co-culture experiment
Incubate to grow yeast strain EM93 (MAT
a
/
a
prototroph) overnight in YPD media at 37C, and then dilute it in fresh YPD for 3-4H
Centrifuge yeast cell to pellet
Wash 1 time with PBS buffer
Add yeast pellet to J774A culture at 4x10^8 cells per flask
Incubate co-culture for 2.5-.3.0H at 37C in normal air
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Prepare the RNA from pooled cell pellets using hot acidic phenol
Isolate the poly(A) fraction using the Poly(A)ttract kit (Promega)
Label the poly(A) RNA (2 mg per sample) and hybridize it to the Ye6100 oligo nucleotide array set (Affymetrix)
Extract and alter the array data to remove any genes whose expression did not change at least twofold (or 100 units) in the experiment
Identify
Candida albicans
homologues of
ICL1
and
MLS1
by searching currently available
C. albicans
genome sequence data from the Stanford DNA Sequencing and Technology Center
Accession numbers in Genbank of gene sequences for
C. albicans
ICL1
- AF222905,
MLS1
- AF222907
For northern analysis, perform the macrophage interactions using 106 J774A cells in 5 ml media with 2x10^7
S. cerevisiae
(EM93) or
C. albicans
(SC5314) cells.
Grow the control populations for 3 h in rich media (YPD), or in tissue culture media without (RPMI) or with (serum) 10% fetal bovine serum
Amplify the species-species probes by PCR and labelled with a random primer.
Abstract
Candida albicans
Normal mammalian gastrointestinal flora, but cause infections in immunocompromised patients
Candida phagocytosed by macrophage and neutrophils, cytokine sreleased will induce hyphal development
Phagocytosis
upregulates the principles the principal enzymes of the glyoxylate cycle, isocutrate lyase (ICL1) & malate synthase (MLS1)
Mutant
lacks ICL1
less virulent
Glyoxylate cycle has a wide range of significance in microbil pathogenesis
Saccharomyces cerevisiae isolated from phagolysosome are induced for genes of the glyoxylate cycle
Glyoxylate cycle
a metabolic pathway that permits the use of two-carbon compounds as carbon source
In vitro, cultured mammalian
macrophages readily ingest both S. cerevisiae and C. albicans cells
population of S. cerevisiae highly enriched for phagocytosed cells was isolated and subjected to whole-genome microarray analysis using oligonucleotide-based arrays (Affymetrix)
3 hours later, 67% phagocytosed cells were alive
Glyoxylate cycle enzyme
isocitrate lyase (ICL1)
malate synthase (MLS1)
malate dehydrogenase (MDH2)
citrate synthase (CIT2)
Acetyl coenzyme A synthase (ACS1)
YDR384c (homologue of Y. lipolytica glyoxylate pathway regulator; GPR1)
transporters and acetyltransferase (CRC1, ACR1, YAT1 and YER024w)
fructose-1,6-bisphosphatase (FBP1); a central regulatory
point in gluconeogenesis