Adaptation of Cryptococcus neoformans to the mammalian...
Transcript of 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
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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)
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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, …
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Interactions with phagocytic cells: Survival and dissemination
Crossing the Blood-Brain Barrier: On the road to meningoencephalitis
Cryptococcal pathogenesis
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Some C. neoformans strains are aneuploids
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13a
Two clinical isolates of C. neoformans are disomic for chromosome 13
Strain CBS7779 (VNI): AIDS patient – Argentina
Strain WM626 (VNII): AIDS patient - Australia
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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
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White strains are disomic at chromosome 13
White disomic
Black • monosomic
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Disomy at chromosome 13 correlates with increased susceptibility to fluconazole and brefeldin A, and slower growth
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Disomy influences gene expression – especially for genes on chromosome 13
White vs. Black
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Disomy at chromosome 13 is correlated with attenuated virulence
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BRAIN LUNG
Disomic and monosomic strains achieve similar fungal burdens
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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
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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?
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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
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Mixed infections are common in AIDS patients
Disomy influences resistance to azole drugs
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Cryptococcal giant cells have increased ploidy
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Giant cell formation during pulmonary disease
Okagaki, L. H. et al. 2010. PLoS Pathogens Zaragoza, O. et al. 2010. PLoS Pathogens
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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
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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)
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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
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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?
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Transferrin
Other trxn factors: HapX, Rim101
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C. neoformans grows to a high cell density on heme
Heme may be an additional iron source during infection
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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?
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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
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Secretion of capsule polysaccharide, laccase, and other enzymes
Cig1?
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Growth in LIM+10uM hemin
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A cig1 mutant shows delayed growth on heme
Deletion of CIG1 alone does not influence virulence
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*Experiment terminated on Day 60
Deletion of CIG1 further attenuates the virulence of a cfo1Δ (high affinity uptake) mutant
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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)
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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
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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