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

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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

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

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OD6

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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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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