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The glyoxylate cycle is required for fungal virulence (Mutant…
The glyoxylate cycle is required for
fungal virulence
Candida albican
normally phagocytosed
by macrophages and neutrophils
secrete cytokines and induce hyphal development in the fungus
phagocytosis also upregulates the principal enzymes
malate synthase (MLS1)
isocitrate lyase (ICL1)
Candida albicans mutants lacking ICL1 is less virulent
less virulent in mice than
the wild type.
isocitrate lyase is upregulated and needed in virulence of Mycobacterium tuberculosis
host-pathogen interactions systemic study:
hampered by the lack of genetic tools in
C. albican
S. cerevisiae is
used to uncover relevant genes
mammalian macrophages readily ingest both both
S. cerevisiae
and
C. albicans cells
.
S.cerevisae
highly enriched for phagocytosed cells
subjected to whole-genome microarray analysis
most of the phagocytosed cells were alive after 3hrs
averaging 67% alive, as assayed by methylene blue staining
transcriptional profiling of these cells are reveals by fungal cells to phagocytosis
Methods
Yeast-macrophage co-culture and gene expression analysis
Murine macrophage-like cell line J774A (ATCC number TIB 67) cultured in RPMI
plus 10% fetal bovine serum at 37 C in 95% air/5% CO2.
Cells plated in 50 ml media at 2 X 10'7 cells per 750 ml flask, 18h before co-culture
Yeast strain EM93 grown overnight in YPD media at 37 C, diluted in fresh YPD for 3-4 h
Yeast cells centrifuged, washed once, resuspended in PBS, added to the J774A cultures at 4 x 10'8 cells per flask
The co-culture was incubated for 2.5- 3.0 h at 37 C in normal air
Yeast cells not associated with adherent macrophages removed by washing three times with ice cold PBS
The macrophages & yeast removed by scraping, pooled by centrifugation for 1 min at 500 xg. Cell number and viability determined by microscopy.
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the virulence of these C. albicans strains in a mouse model of systemic candidiasis.
Mice injected with wild-type
C. albicans strain SC5314 succumb rapidly to the infection
mice injected with
two independently constructed ¢icl1/¢icl1 strains survived longer
At day 28
6 out of 10 of an independent homozygous
mutant (MLC8).
7 out of 10 of the animals injected with one strain (MLC7)
remained alive
Infection with the heterozygote (¢icl1/ICL1)
resulted in an intermediate mortality
microbes find the inside of a macrophage
glucosede deficient environment
Glucose is required for the synthesis of many
macromolecules necessary for proliferation, including ribose and deoxyribose
Mutant construction and analysis
Saccharomyces cerevisiae
Dicl1 mutants
constructed in the EM93 background
using a PCR-mediated protocol
with a G418-resistance cassette
Mutants were constructed in
both mating types and mated
produce a homozygous ¢icl1/¢icl1 knockout strain (MLY283a/a)
C. albicans
an Dicl1 disruption construct was created
by inserting a hisG-URA3-hisG cassette
at a BglII site in the ICL1 open reading frame
This construct was linearized
transformed into CAI4
and selected by uracil prototrophy
Accurate integrants
identified by PCR
and passaged on 5-FOA medium
A second round of transformation
used to generate two independent homozygous ¢icl1/¢icl1 strains
The wild-type ICL1 gene
re-introduced on linearized plasmid pRC2312
by transformation to produce a complemented strain (MLC10)
Standard media was used
strains were grown at 37 C
phagolysosome
rich in fatty acids or their breakdown products (primarily acetyl-CoA).
AcetylCoA can only be assimilated through the glyoxylate cycle
Assayed growth of C. albicans in media
containing oleic
acid
an unsaturated 18-carbon fatty acid
e sole carbon source
wild-type strain uses oleic acid as well as acetate
icl1/icl1 mutant strain is unable to metabolize oleic acid.
Both Saccharomyces and Candida induce the glyoxylate
cycle on macrophage contact, yet only Candida is virulent
Inhibitors of the glyoxylate cycle pathway
block nutrient availability
Compounds that inhibit nutrient availability have been developed into effective herbicides (for example, glyphosate
and imidizolinones)
their targets are enzymes produced by
plants but not by animals
prevent survival of these pathogens inside the
macrophage
Murine virulence assay
Overnight cultures of
C. albicans
strains
diluted into fresh YPD
and grown for 3-4 h at 37 C
. Cultures were collected by centrifugation
and washed with PBS
Cell were injected
into the tail vein of 18±20-week-old female BALB/c mice
ten mice per strain were used
Mice were monitored for three weeks after injection
and moribund animals were killed
animals care according to NIH guidelines