Unraveling Fungal Radiation Resistance Regulatory

Networks through the Genome-Wide Transcriptome and

Genetic Analyses of Cryptococcus neoformans
Maulana Malik Nashrulloh
Ahmad Nuruddin Khoiri
Advisor : Dr. Yaowaluck M. Roshorm

BIT 612
FINAL PRESENTATION
Outlines

1. Introduction
2. Objectives
3. Methods + Results
4. Conclusion
5. References
Introduction
Azzam et al., 2012
Cryptococcus neoformans
 Naturally free living and
found in plants and animals

 Can be opportunistic
pathogen, and also causing
fatal meningoencephalitis

 Radiotrophic fungi, that is,

able to use the pigment
melanin to convert gamma
radiation into chemical
energy for growth
(Dadachova et al., 2007)
Objective
 To understand the genome wide radiation resistance networks and
mechanism in the radioactive stress management in Cryptococcus
neoformans
Comparison of gamma radiation tolerance in several fungi

Fungi
Exposed Serially
Cultured in spotted Incubated Survived
by diluted
YPD broth on YPD in 30 C, 3 Colony
radiation (100 -
medium plate d counted
for 1 h 104)
(30 C, 24 h)

 C. neoformans H99 was

most tolerant to gamma
radiation compared to
other fungi.
Melanin production by Laccases in C. neoformans
C neoformans Exposed by 1 and 3 kGy
RNA
Cultured in YPD gamma radiation (0’, 30’, 60, qRT-PCR
extraction
medium 120’)

 Laccases (LAC1 and

LAC2) expressed
during initial radiation,
but gradually
decreased over the
time post radiation.

 Laccases play a role in melanin production. While melanin physically shields the cell
and helps quenching ROS (Dadachova & Casadevall, 2008), but it does not play a
role in mitigating other effects such as DNA damage repairing.
Remodeling of transcriptome profiles in response to gamma radiation
of C. neoformans

 Microarray

Wild Type Radiated by 3 kGy, 1 h

Sample taken at 30’, 60’, 120’

Remodeling of transcriptome profiles in response to gamma radiation
of C. neoformans (Cont’d)

 Totals of 209, 129, and 142 genes (P < 0.01)

are significantly exhibited more than 2-fold
induction at different recovery time points.
Among these genes, 56 genes (P < 0.01) are
upregulated by more than 2-fold at all of the
time points.

 Totals of 34, 299, and 424 genes (P < 0.01)

are downregulated by more than 2-fold at
different recovery time points. The
expression level of 14 genes (P < 0.01) are
decreased more than 2-fold at all time points.
Remodeling of transcriptome profiles in response to gamma radiation
of C. neoformans (Cont’d)
 At 30 min, genes involved in DNA
replication and repair, signal
transduction, and posttranslational
modification and chaperone functions
were induced.
 Cells immediately activated defense
systems to counteract the effects of
gamma radiation.

 A number of genes involved in amino

acid metabolism and transport, RNA
processing and modification, and
translation were significantly
downregulated at 60 and 120 min.
 Cell decreases basic cellular function to
avoid toxic effect from resulting the
production of abnormal molecules.
Enzymes that play critical roles in the survival of C. neoformans
upon exposure to gamma radiation
Not exposed by
gamma radiation
C neoformans
RNA
Cultured in YPD qRT-PCR
extraction
medium
Exposed by
gamma radiation
(30’)
 Under radiation, RAD51, RDH54, RAD54,
and PSO2 are highly expressed

 RAD51 is recombinase enzymes, that

worked in repairing broken DNA.

 RAD54 and RDH54 are ATPase that work in

the chromosomal remodelling.

 PSO2 is a nuclease that work in excising

Inter Cross Linking (ICL), required for repair
DNA topology.
Enzymes that play critical roles in the survival of C. neoformans
upon exposure to gamma radiation (Cont’d)

WT and KO-ed Five level 10-fold

Radiated under Incubated at 30 °C
C. neoformans culture in YPD
1 and 2.5 kGy 3d
culture medium

 In rad51∆, rad54∆, rdh54∆, the cells

cannot survive in the radiation, while
WT and pso2∆ cell can survive.

104 103 102 101 100

Effects of Gene Knockout on genotoxic agent resistance in wild type and mutants

WT and KO-ed Five level 10-fold Exposure with

Incubated at 30 °C
C. neoformans culture in YPD Genotoxic
3d
culture medium Agents

104 103 102 101 100

 In rad51∆, rad54∆, rdh54∆, the cells cannot survived in the genotoxic

agents exposure, while WT and pso2∆ cell can survived.
Oxidative stress defense genes required for the survival of C. neoformans

C neoformans
Exposed by 3 kGy gamma RNA
Cultured in YPD qRT-PCR
radiation (0’, 30’, 60’) extraction
medium

 A number of oxidative stress

defense genes were highly
upregulated in response to
gamma radiation.

 Srx1 gene was required for

the gamma radiation
resistance that occurred in a
peroxiredoxin-independent
manner.
Oxidative stress defense genes required for the survival of C.
neoformans (Cont’d)
WT and KO-ed Five level 10- Exposure with 3
Incubated at 30
C. neoformans fold culture in kGy gamma
°C 3 d
culture YPD medium radiation

 srx1∆ cannot survive under

radiation

 srx1 encode Sulfiredoxin, an

antioxidant enzyme. It is
required for restoring
Peroxiredoxins (a group of
Peroxidases), normalized the
enzymes when these
enzymes are inhibited by
over-oxidation.
Protein quality control systems for gamma radiation resistance in C.
neoformans

 the ire1∆ mutant exhibited growth

defects when exposed to gamma
radiation, suggesting that Ire1 was
involved in radiation resistance in an
Hxl1-independent manner.

 The expression levels of KAR2,

LHS1, PDI1, and SCJ1 were all
increased in response to gamma
radiation.

 UPR pathway played a role in the

gamma radiation resistance in C.
neoformans.
Unfolded protein response (UPR) pathway
 Protein quality control systems for gamma radiation resistance in C.
neoformans (Cont’d)
C. neoformans Exposed by 3 kGy
RNA
Cultured in YPD gamma radiation (0’, qRT-PCR
extraction
medium 30’, 60’, 120’)

 The expression levels of UBC6,

UBC62, and UBC7 genes were
increased and then decreased. In
contrast, expression of UBC1
increased after radiation
exposure.
 UPR and ERAD pathways
contribute to radiation resistance
by regulating the expression
levels of genes of molecular
chaperones and protein
Endoplasmic reticulum-associated degradation (ERAD) pathway
degradation.
The role of ergosterol homeostasis in gamma radiation resistance of
C. neoformans
C. neoformans Exposed by 3 kGy
RNA
Cultured in YPD gamma radiation (0’, qRT-PCR
extraction
medium 30’, 60’, 120’)

 ERG1, ERG3, ERG5, and ERG11

were downregulated.
 Fluconazole treatment increased
gamma radiation resistance in
wild-type cells, and the effect was
observed more clearly with high
doses of radiation, suggesting that
decreased ergosterol content
contributed to gamma radiation
resistance.
The role of ergosterol homeostasis in gamma radiation resistance of
C. neoformans (Cont’d)
WT and KO-ed Five level 10-
Radiated under Incubated at 30
C. neoformans fold culture in
2 and 3 kGy °C 3 d
culture YPD medium

 Sre1 is the major positive regulator of

expression of ergosterol biosynthesis
gene.
 homeostasis of ergosterol
biosynthesis was critical for gamma
radiation resistance in C.
neoformans.
Discovery of novel gamma radiation resistance genes in C. neoformans
qRT-PCR
WT and KO-ed Five level 10-
Radiated under
C. neoformans fold culture in
1 and 3 kGy
culture YPD medium
Incubated at 30 °C 3 d

 RIG1, RIG2, and RIG3 are novel

gamma radiation resistance genes
in C. neoformans.
 The expression pattern of RIG1,
RIG2, and RIG3 were increased
during radiation exposure.
 RIG1, RIG2, and RIG3 were mainly
involved in gamma radiation
resistance, as well as DNA damage
response.
Discovery of novel gamma radiation resistance genes in C. neoformans
(Cont’d)
Fungi in YPD culture
RNA
were treated with qRT-PCR
isolated
MMS or HU

 The expression of RIG1, RIG2, and RIG3

was gradually increased in response to either
HU or MMS treatment.
Discovery of novel gamma radiation resistance genes in C. neoformans
(Cont’d)
WT and KO-ed Five level 10-fold Exposure with
Incubated at 30 °C
C. neoformans culture in YPD Genotoxic
3d
culture medium Agents

 Rig1∆ and Rig2∆ mutants showed severe sensitivity to all of the DNA-
damaging stresses that was tested. While, Rig3∆ mutant exhibited increased
susceptibility to MMS and 4-NQO.
 Radiation-induced genes, such as RIG1, RIG2, and RIG3, contributed to
gamma radiation resistance, as well as DNA damage stress responses, in C.
neoformans.
Identification and characterization of a novel bZIPTF, Bdr1, for
radiation resistance in C. neoformans
qRT-PCR
Microarray Unknown
data transcript Phylogenetic
CD Analysis
Analysis

0.1

 Expression of BDR1 was increased at 30 min post-radiation recovery and

then decreased.
Identification and characterization of a novel bZIPTF, Bdr1, for
radiation resistance in C. neoformans (Cont’d)
Incubated at 30 °C
3d
Exposure with 1
WT and KO-ed Five level 10-fold
and 1.5 kGy; and
C. neoformans culture in YPD
Genotoxic
culture medium
Agents
RNA
qRT-PCR
isolated

 Deletion of BDR1 caused severe growth defects upon genotoxic agents exposure.
Identification of downstream targets and upstream regulators of Bdr1 for radiation
resistance in C. neoformans

 Bdr1 was enriched in the nucleus under

basal conditions, indicating that Bdr1 was
likely to be a TF in C. neoformans.
 Radiation-responsive induction of
expression of RAD51, RDH54, RAD54,
PSO2, RIG1, RIG2, and RIG3 was
considerably reduced in the bdr1∆ mutant
compared to the wild type, suggesting that
Bdr1 played critical roles in the regulating
expression of DNA repair genes.
Identification of downstream targets and upstream regulators of Bdr1 for radiation
resistance in C. neoformans (Cont’d)

 The rad53∆ mutant showed severe

growth defects after gamma radiation,
as well as following exposure to
genotoxic stress inducers, such as
UV-C, 4-NQO, and bleomycin.
 The increased BDR1 gene
expression in the wild-type strain
post-radiation was significantly
decreased by deletion of RAD53,
indicating that Rad53 was an
upstream regulator of Bdr1 in C.
neoformans.
 Conclusion
Free Radical
Radiation Chaperone
Melanin DNA Neutralization
Resistance and
Production Repair (Antioxidant
Proteins Proteasome
Enzyme)
Bdr1
Upregulation Rad53

Gamma radiation

Downregulation

Ergosterol
Protein translation Metabolic process
synthesis
References
Jung KW, Yang DH, Kim MK, Seo HS, Lim S, Bahn YS. 2016. Unraveling Fungal Radiation Resistance
Regulatory Networks through the Genome-Wide Transcriptome and Genetic Analyses of Cryptococcus
neoformans. mBio. 7(6): e01483-16. **

Azzam EI, Jay-Gerin JP, Pain D. 2012. Ionizing radiation-induced metabolic oxidative stress and
prolonged cell injury. Cancer Letter. 327:48–60.

Dadachova E, Bryan RA, Huang X, Moadel T, Schweitzer AD, Aisen P, Nosanchuk JD, Casadevall A.
2007. Ionizing Radiation Changes the Electronic Properties of Melanin and Enhances the Growth of
Melanized Fungi. PLoS ONE, 2(5), e457.

Dadachova E., & Casadevall A. 2008. Ionizing Radiation: how fungi cope, adapt, and exploit with the help
of melanin. Current Opinion in Microbiology, 11(6), 525–531.