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The glyoxylate cycle is required for fungal virulence (Induction of…
The glyoxylate cycle is required for fungal virulence
ABSTRACT
Candida albicans
, a normal component of the mammalian gastrointestinal - responsible for most fungal infections in immunosuppressed patients
Phagocytosed
by macrophages and neutrophils, which secrete cytokines and induce hyphal development in this fungus
Use genome-wide expression profile of the related yeast
S. cerevisae
- obtain signature event that take place in the fungus
on ingestion by a mammalian macrophage
Method
Live
S. cerevisiae
cells isolated from the phagolysosome
Conclusion
Isocitrate lyase
is both upregulated and required for the
virulence of *Mycobacterium tuberculosis - significance of the glyoxylate cycle in microbial pathogenesis.
C. albicans
mutant - lacks of
IL1
- less virulent in mice than wild type
Test virulence of
C. albicans
strains in a mouse
model of systemic candidiasis.
RESULT : Mice injected with wild-type
C. albicans
strain SC5314 succumb rapidly to the infection (median survival 3 days)
RESULT : mice injected with
two independently constructed delta icl1/delta icl1 strains survived longer.
Day 28 : 7/10 animals injected with strain (MLC7) remain alive as did 6/10 of independent homozygous mutant (MCL8)
Infection with the heterozygote (delta icl1/ICL1)
resulted in an intermediate mortality (median 8 days)
Indicate that isocitrate lyase not only induced by macrophage phagocytosis but also essential for full virulence in fungal pathogen
From genome array and virulence studies suggest
that microbes inside of a macrophage to be a glucose-decient environment
Glucose - required for the synthesis of many
macromolecules necessary for proliferation, including ribose and deoxyribose
Phagolysosome rich in fatty acids or their breakdown products (primarily acetyl-CoA)
AcetylCoA only can be assimilated through the glyoxylate cycle
bypasses the catabolic steps of the TCA cycle; thus glyoxylate
cycle the only route to the synthesis of glucose
Growth of C. albicans in media containing oleic
acid (unsaturated 18-carbon fatty acid) as the sole carbon source
wild-type strain uses oleic acid and acetate,
whereas the deltaicl1/deltaicl1 strain is unable to metabolize oleic acid
Although the glyoxylate pathway is necessary for virulence, it is clearly not enough.
Both
Saccharomyces
and
Candida
induce the glyoxylate cycle on macrophage contact, Thus only
Candida
virulent
Genes encoding the glyoxylate cycle required for virulence in both bacterium (
M. tuberculosis
) and a
fungus (
C. albicans
) that can survive inside a macrophage
Inhibitors of glyoxylate cycle pathway block nutrient availability and prevent survival of pathogens inside the
macrophage
Compound inhibit nutrient availability developed into effective herbicides (ex: glyphosate & imidizolinones) - they target enzyme produced by plant not animals
As enzyme of glyoxylate cycle not found in mammals become prime targets for antibacterial & antifungal agents
Host-pathogen interactions
Lack of genetic tools in
C. albicans
Non-pathogenic yeast
S. cerevisiae
: Often
used to uncover relevant genes
In vitro
Cultured mammalian macrophages readily ingest both
S. cerevisiae
and
C. albicans
cells.
Affymetrix
:
S. cerevisae
that highly enriched for phygocytosed cells isolated
Transcriptional profiling of these cells reveals the response of
fungal cells to phagocytosis.
Most highly induced
S. cerevisiae
genes after phagocytosis encode proteins related to the
glyoxylate cycle
- two-carbon compounds are assimilated into
TCA cycle
Strongly induced
ICL1
; malate synthase
MLS1
; and malate dehydrogenase, MDH2
CIT2
: citrate synthase
Other genes related to glyoxylate cycle that induced are
acetyl coenzyme A (acetyl-CoA) synthase (
ACS1
);
YDR384c
: a homologue of the
Yarrowia lipolytica
glyoxylate pathway regulator (
GPR1
)
several transporters and acetyltransferases, which are used to trafficintermediates of the glyoxylate cycle and fatty-acid degradation between organelles (
CRC1, ACR1, YAT1
and
YER024w
); and
fructose-1,6-bisphosphatase (
FBP1
).
Central regulatory point in gluconeogenesis - production of glucose
Induction of the glyoxylate cycle
indicates that nutrient acquisition and use is the primary focus of yeast cells upon phagocytosis, presumably because the phagolysosome is poor in complex carbon compounds
Induction of specialized isozymes
Isozymes specialized for glyoxylate cycle are only induced
Cytosolic isozymes of MDH2 is induced 15.6-fold
Mitochondrial (MDH1) and peroximal (MDH3) forms are not induced
Out of three citrate synthase isoforms only the glyoxylate cycle-specific CIT2 is induced
Nothern analysis of RNA from both S.cerevisiae and C. albicans cells grown in the presence of macrophages shows that in both organisms the ICLI or MLSI genes are significantly induced by macrophage contact when compared with cells grown in media alone
Thus, induction of glyoxylate enzymes is a conserved response to phagocytosis in these two yeast
Methods
Mutant construction and analysis
Saccharomyces cerevisiae
Dicl1 mutants constructed in the EM93 background using a
PCR-mediated protocol with a G418-resistance cassette13
Mutants constructed in both mating types and mated to produce a homozygous ¢icl1/¢icl1 knockout strain (MLY283a/a)
C. albicans
, an Dicl1 disruption construct was created by inserting a his G±URA3±hisG cassette14 at a BglII site in the ICL1 open reading frame
linearized & transformed into CAI4 (aUra-derivative strain of SC5314; refs 14, 15) selected by uracil prototrophy
Accurate integrants were identifed by PCR and passaged
on 5-FOA medium.
2nd round of transformation was done to generate 2 independent homozygous¢icl1/¢icl1 strains (MLC7 & MLC8)
2nd round of transformation was done to generate 2 independent homozygous¢icl1/¢icl1 strains (MLC7 & MLC8)
wild-type ICL1 gene was re-introduced on
linearized plasmid pRC2312 (ref. 16) by transformation & produce complement strain MLC10
transformation of
C.albicans
was done where standard media was used n strains were grown at 37^C
Murine virulence assay
Overnight cultures of C.
albicans
strains were diluted into fresh YPD and grown for 3-4 h at 37 degree celcius
Cultures were collected by centrifugation and washed with PBS
Cells (6 x 10^5) were injected into the tail vein of 18-20-week-old female BALB/c mice
(10 mices for each strain were used)
Mice were monitored for three weeks after injection and moribund animals
were killed
Yeast-macrophage co-culture and gene expression analysis
Murine macrophage-like cell line J774A was cultured in RPMI plus 10% fetal bovine serum at 37 8C in 95% air/5% CO2
About 18 h before a co-culture
experiment, cells were plated in 50 ml media at 2x10^7 cells per 750 ml flask
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
The co-culture was incubated for 2.5-3.0 h at 37 degree celcius in normal air
Yeast cells not associated with the adherent macrophages were removed by washing three times with ice-cold PBS
The macrophages and associated yeast were removed by scraping, and pooled by centrifugation for 1min at 500g
Cell number and viability are determined (using
methylene blue) by microscopy
The cell mixture was washed twice with ice-cold water to
lyse the mammalian cells
The resulting cell pellet consisting mostly of yeast cells was
frozen at -80 degree celcius
RNA was made from the pooled cell pellets using hot acidic phenol and the poly(A) fraction was isolated using the Poly(A)ttract kit (Promega)
Poly(A) RNA (2 mg per sample) was labelled in duplicate as described12 and hybridized to the Ye6100 oligooligonucleotide array set (Affymetrix)
Array data were extracted and altered to remove any
genes whose expression did not change at least twofold (or 100 units) in the experiment
Candida albicans
homologues of ICL1 and MLS1 were identified by searching currently available C.
albicans
genome sequence data from the Stanford DNA Sequencing and Technology Center
For northern analysis, macrophage interactions were performed as described above using 106 J774A cells in 5ml media with 2x10^7 S.
cerevisiae
(EM93) or C.
albicans
(SC5314) cells
Control populations were grown for 3 h in rich media (YPD),
or in tissue culture media without (RPMI) or with (serum) 10% fetal bovine serum
Species-specific probes were amplified by PCR and labelled with a random primer