adaptation of cryptococcus neoformans to the mammalian...
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Adaptation of Cryptococcus neoformans to the mammalian host environment
www.kronstadlab.msl.ubc.ca
Microbiology & Immunology UBC - Vancouver
Outline 1. Genomic adaptation (variation in chromosomal copy number)
- disomy and virulence
- disomy in isolates from HIV/AIDS patients
2. Metabolic adaptation (host – pathogen competition for iron)
- Heme utilization (Heme oxygenase, VPS41, Endocytosis, Cig1)
Cryptococcosis and virulence traits Immunocompromised people, e.g., AIDS patients ~One million cases per year, ~600,000 deaths
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Pulmonary infection
Meningoencephalitis
Spores or yeast
(Park et al. 2009. AIDS 23: 525-30)
Growth at 370C Survival in macrophages
Capsule
Melanin
C. neoformans is an environmental fungus: Pigeon excreta, Soil, Trees, …
Interactions with phagocytic cells: Survival and dissemination
Crossing the Blood-Brain Barrier: On the road to meningoencephalitis
Cryptococcal pathogenesis
Some C. neoformans strains are aneuploids
2
3
4
5 6
7 8 9 10 11 12
13b
14
1
13a
Two clinical isolates of C. neoformans are disomic for chromosome 13
Strain CBS7779 (VNI): AIDS patient – Argentina
Strain WM626 (VNII): AIDS patient - Australia
Is there a correlation between disomy for chr13 and melanin production?
Strain CBS7779 (poor melanin formation) White to beige colonies
Isolate melanin+ variants Black colonies (1/103)
Copy number (chr 13) 1.00 1.07 1.09 0.95 1.84 2.01 1.97
Quantitative PCR
Strain H99 Black 1 Black 2 Black 3 White 1 White 2 White 3
~ 1
~ 2
Non-melanized sector
White strains are disomic at chromosome 13
White disomic
Black • monosomic
Disomy at chromosome 13 correlates with increased susceptibility to fluconazole and brefeldin A, and slower growth
Disomy influences gene expression – especially for genes on chromosome 13
White vs. Black
Disomy at chromosome 13 is correlated with attenuated virulence
BRAIN LUNG
Disomic and monosomic strains achieve similar fungal burdens
Black White Control
Emphysema/Airway damage Fibrosis Uninfected
Day 14
Infections with disomic and monosomic strains result in different lung pathology
Similar inflammation for both black and white infections
Black White Control
Disomy is correlated with melanin production, gene expression and virulence
So how common is it -
- in environmental and clinical isolates?
- in fresh isolates from HIV/AIDS patients?
Chromosome copy number differences are seen in clinical and environmental strains, and in fresh isolates from AIDS patients
2/13 HIV/AIDS pa8ents
2/19 clinical and environmental isolates
Mixed infections are common in AIDS patients
Disomy influences resistance to azole drugs
Cryptococcal giant cells have increased ploidy
Giant cell formation during pulmonary disease
Okagaki, L. H. et al. 2010. PLoS Pathogens Zaragoza, O. et al. 2010. PLoS Pathogens
Disomy is associated with reduced melanin production, changes in gene expression, reduced virulence, and pulmonary fibrosis.
Fresh isolates from the CSF of HIV/AIDS patients show variation in chromosomal copy number.
Summary for genomic adaptation
Genome plasticity may influence the ability of Cryptococcus to: - withstand the immune response - establish latency - disseminate to the CNS - resist antifungal drugs
Outline 1. Genomic adaptation (variation in chromosomal copy number)
- disomy and virulence
- disomy in isolates from HIV/AIDS patients
2. Metabolic adaptation (host – pathogen competition for iron)
- Heme utilization (Heme oxygenase, VPS41, Endocytosis, Cig1)
Capsule size: Iron, Serum, CO2,Tissue (lung>brain),cAMP
Low Iron
High Iron
+ DOPA - DOPA
Melanin
Dissemination to CNS >
Iron influences capsule size
Iron overload exacerbates cryptococcal meningoencephalitis Barluzzi et al. 2002. J. Neuroimmunol. 132: 140-146
Iron transport, regulation and homeostasis in C. neoformans
CIR1: Jung et al. 2006. SIT1: Tangen et al. 2007. CFT1, CFO1: Jung et al. 2008; 2009. CIG1: Lian et al., 2005; Cadieux et al. In prep.
Jacobson and Petro, 1987; Jacobson and Var8varian, 1992 Var8varian et al. 1995 Nyhus et al. 1997, 2002; Nyhus and Jacobson, 1999 Jacobson and Hong, 1997; Jacobson et al. 1998, 2005
Physiology and gene8cs of iron acquisi8on in C. neoformans Molecular gene8cs of iron acquisi8on in C. neoformans
3HAA
C[2 Cfo2
Heme?
?
Transferrin
Other trxn factors: HapX, Rim101
C. neoformans grows to a high cell density on heme
Heme may be an additional iron source during infection
1. Intracellular heme processing HMX1 - heme oxygenase Heme + NADPH + H+ + 3 O2 → biliverdin + Fe2+ + CO + NADP+ + H2O Role in virulence, no growth defect on heme. 2. Heme trafficking and processing (T-DNA mutagenesis and candidate genes) VPS41 - vacuolar protein sorting and endocytosis (HOPS complex) Role in virulence, growth defect on heme and inorganic iron. 3. Extracellular heme capture CIG1 - exported mannoprotein Role in virulence, growth defect on heme
What functions are required for heme utilization?
3. Extracellular mannoproteins may facilitate heme capture
SAGE TAG LIM LIM+Fe Fold diff. Annotation CATGCAAGTAATTT 547 52 10.5 cytokine inducing glycoprotein
The most abundant transcript in cells from low iron medium (LIM) encodes the mannoprotein Cig1
Fe + -
Red = anti-capsule antibody
Green = anti-HA (Cig1) antibody
rRNA
CIG1
Secretion of capsule polysaccharide, laccase, and other enzymes
Cig1?
Growth in LIM+10uM hemin
0
0.5
1
1.5
2
2.5
3
3.5
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90Time (h)
OD6
00
H99cig1cig1CIG1
A cig1 mutant shows delayed growth on heme
Deletion of CIG1 alone does not influence virulence
*Experiment terminated on Day 60
Deletion of CIG1 further attenuates the virulence of a cfo1Δ (high affinity uptake) mutant
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0 10 20 30 40 50 60 70
Num
ber o
f mic
e
Days post infection
Survival of female A/J mice inoculated with Cryptococcus neoformans
Group 1 (cig1cfo1:CIG1)
Group 2 (cfo1)
Group 3 (H99)
Group 4 (cig1)
Group 6 (cig1:CIG1)
Group 7 (cig1cfo1)
Summary: An emerging pathway for heme utilization
Cft1/Cfo1
CAPSULE
Fe+2
Fe+3
Fe+3
Fe+3 melanin
Cig1
CAPSULE - CELL WALL
MELANIN DEPOSITION - CELL WALL
Heme
Hmx1
Vps41 Vacuole
endocytosis facilitator
recycling?
processing/ storage
Heme Heme
Heme
Vps41
Transferrin
Siderophores
Siderophores
Transferrin
Sit1
Joyce Wang
Sanjay Saikia
Funding: NIH (NIAID), Canadian Institutes of Health Research, Burroughs Wellcome Fund
Guanggan Hu Iris Liu
John Perfect Tom Mitchell Ana Litvintseva June Kwon-‐Chung Louis de Repen8gny
Collaborators
Won Hee Jung
Brigitte Cadieux
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