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The glyoxylate cycle is required for fungal virulence (8 (acetyl-coA only…
The glyoxylate cycle is required for
fungal virulence
4
Isoenzyme specialized for glyoxylate cycle
-cytosolic isoenzyme of MDH2 induced 15,6-fold.
Mitochondrial MDH1 and peroxisomal MDH3 not induced
Only glyoxylate cycle-sp CIT2 induced (citrate synthase isoforms)
glyoxylate cycle enzymes were not changed significantly in response to conditioned media, oxidative stress, or contact with heat-killed macrophages (control array)
phagocytosis sp. upregulates cycle and accesory protein
response precedence any conventional stress response (nutrient deprive is primary)
1
Candida albicans
normal component of mammalian gastrointestinal flora
responsible for most fungal infection in immunosuppressed patients
normally phagocytosed by macrophages and neutrophils (which secrete cytokines and induce hyphal development)
phagocytosis also upregulates the principal enzymes of the glyoxylate cycle, isocitrate
lyase (ICL1) and malate synthase (MLS1)
C. albicans
mutants lacking
ICL1
less virulent in mice than the wild type
conjuction with reports that isocitrate lyase is both upregulated and required for virulence of
M. tuberculosis
(wide ranging significance of the glyoxylate cycle in microbial pathogenesis)
Neutrophenic patients
deficient in these immune cells
susceptible to systematic candidiasis
use genome-wide expression profiles of the related yeast
Saccharomyces cerevisisae
to obtain a signature of the events take place in fungus ingestion by mammalian macrophage
S.cerevisisae cells isolated from phagosome are induced for genes of the glycoxylate cycle (metabolic pathway that use 2 C compounds as C source)
3
Eleven of highly induced S.cerevisiae genes
(after phagocytosis)
encode protein related to glyoxylate cycle
two carbon compound assimilated into TCA cycle.
Three of five glyoxylate cycle enzyme strongly induced
isocitrate lyase , ICL1
malate synthase, MLS1
malate dehydrogenase, MDH2
fourth- citrate synthase, CIT2
Genes induced (cycle)
acetyl-coA synthase, ACS1
YDR384C (homologue
Yarrowia lipolytica
to GPR1)
transporter & acetyltransferase (traffic intermediate of glyoxylate cycle and fatty acid degradation
FBP1- central regulator point of gluconeogenesis
8
microbe find inside of macrophage become glucose-deficient environment
gluscose is required for the synthesis of many macromolecules that require for proliferation
phagolysosome rich in fatty acid or their products.
acetyl-coA only can be assimilated through glyoxylate cycle
gyloxylate cycle is the only route to synthesis of glucose
the wild-type strain use oleic acid as well as acetate
icl1 mutant unable to metabolize oleic acid
saccharomyces and candida induce glyoxylate cycle on macrophage contact yet only candida is virulent.
2
Systematic studies of host-pathogen interactions have been
hampered by the lack of genetic tools in
C. albicans
related but non-pathogenic yeast
S. cerevisiae
is often
used to uncover relevant genes
In vitro
, cultured mammalian macrophages readily ingest both:
S. cerevisiae
cells
C. albicans
cells
population of
S.cerevisisae
enriched for phagocytosed cells was isolated
subjected to whole-genome microarray analysis using oligonucleotide-based arrays (Affymetrix).
3 hours after initiating the co-culture, most phagocytes cells alive (average 67% alive by methylene blue staining)
reveals to response of fungal cells to phagocytosis
6
mutant strains of both s.cerevisiae and c.albicans that constructed is lacke of ICL1
icl1 mutant strains fail to use acetate or ethanol as expected/
both heterozygous strain and homozygous mutant in c.albicans has been re-introduced grow
the growth rate of c.albicans not significantly different from the parent strain on media and this strain not sensitive to variety of vivo stresses
5
c.albicans genes is cloned for isocitrate lyasae(ICL1) and malate synthase (MLS1)
the activity of both enzyme are specific and limited to glyoxylate cycle.
both genes sahe same homology with protein from fungi, plant and bacteria
RNA from c.cerevisiae and c.albicans cells grow in the presence of macrophage
genes significantly induced by ,acrophage contact
induction of glyoxylate enzyme is a response to phagocytosis in the yeast.
7
tested the virulence of
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 deltaicl1/deltaicl1 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 (deltaicl1/ICL1)
resulted in an intermediate mortality (median of 8 days).
isocitrate lyase not only induced by macrophage phagocytosis but
is also essential for full virulence in this fungal pathogen
9
Genes encoded need for virulence (
M.tuberculosis
&
C.albicans
)
Cycle inhibitor should block nutrient availability, prevent survival of pathogen in macrophage
Herbicides- inhibit nutrient availability (glyposate & imidizolinones)- target enzyme produced by plant not animals
Cycle's enzyme not found in mammal (prime target antibacterial & antifungal agents)