人體病原真菌隱球菌生殖,菌絲生長及致病分子機制之探討(I)

Fungal infection has drawn lots of
attention due to dramatically increased
number of immunocompromised patients
caused by HIV infection, organ and tissue
transplantation, and cancer chemotherapy in
the past two decades. Cryptococcus
neoformans, a human pathogenic
basidiomycetous yeast, causes the
life-threatening meningoencephalitis mainly
in such individuals with compromised
immune functions. Studies of the
pathogenesis in C. neoformans have revealed
several important virulence factors such as
capsule, melanin, and interestingly mating
type locus. Environmental and clinical
prevalence of MATα strains and virulence
2
studies of the congenic pair of C. neoformans
serotype D strains have suggested that MAT
α locus might involve in regulating the
virulence in C. neoforman. Following these
observations, MAT α locus has been
identified and characterized. Three copies of
pheromone precursor genes were identified in
the MATα locus. Characterization of three
pheromone genes deletion mutant strains
have suggested an autocrine signaling loop
may function and contribute to the virulence
of the MATá cells (Shen et al., 2002).
The purpose of this proposal is to
characterize the components in the
pheromone response pathway of C.
neoforman serotype D strain and further
address how mating type locus regulates the
virulence and autocrine signaling loop
functions in C. neoforman.
C. neoformans serotype D strain GPB1
gene were identified and disrupted in the
MATa strain. MATa gpb1 mutants were also
identified in the progeny of cross between the
MATαgpb1 mutant and MATa strain. The
MATαgpb1 and MATagpb1mutant strains
were mating impaired but not sterile when
coincubated with the wild-type strain of
opposite mating type on V8 mating medium.
Haploid fruiting was reduced, but not
completely abolished, in the MATαgpb1
mutant strains, similar to the mfα 1,2,3
pheromoneless mutant.
To further address how pheromones act
in the autocrine signaling loop, we have
identified STE6 homologue, a pheromone
transporter, in the C. neoformans genome
project at Stanford Genome Technology
Center and begun to dissect its function. By
disrupting the STE6, we found that ste6
mutants in either MATá or MATa background
showed partially impaired mating function,
although slight differences were noticed.
However, when ste6 MATá and MATa
mutants cross with each other, the mating
process was nearly completely abolished. Our
data indicates that the STE6 functions
bilaterally and is required but not essential
for mating in C. neoformans.
Currently we are constructing the gpb1
and ste6 reconstituted strains, virulence test
will be conducted when verified those strains.
We are also conducting epistasis and
functional analysis on these two genes, and
hoping to claify their role in virulence and
other physiological processes.
To identify novel targets in the
downstream of the signaling pathway, we
have optimized growth conditions and RNA
extraction procedures. We will start the
subtractive PCR screen shortly.