New Insights into the Pathogenesis of Cryptococcosis ... Speaker... · 3 Cryptococcus neoformans Environmental stresses-varying temperatures-solar radiation-desiccation/osmotic stress-nutrient

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New Insights into the Pathogenesis of

Cryptococcosis: Focus on the Organism

Andrew Alspaugh, MD

Duke University School of Medicine

Departments of Medicine and

Molecular Genetics/Microbiology

No disclosures

>100,000Validly described species

of fungi

150primary human

fungal pathogens

Fungi yet to be discovered

Candida,Aspergillus,

Crypto, Blasto, Histo,Cocci, Dermatophytes

The Fungi

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9,700

162,500

3,300 4,400

43,200

0

20,000

40,000

60,000

80,000

100,000

120,000

140,000

160,000

180,000

North/SouthAmericas and

Carribean

Sub-SaharanAfrica

North Africaand Middle

East

Europe Asia andPacific

Esti

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Cas

es

-Rajasingham, R. et al. Global burden of disease of HIV-associated cryptococcalmeningitis: an updated analysis. Lancet Infect Dis 17, 873-881, 2017.

Cryptococcus neoformans – epidemiology of infection

Cryptococcus neoformans

Environmental stresses-varying temperatures-solar radiation-desiccation/osmotic stress-nutrient deprivation-natural predators

-adaptation to the environment

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Environmental stresses-varying temperatures-solar radiation-desiccation/osmotic stress-nutrient deprivation-natural predators

Host-induced stresses-host physiological temperature-intra- and extra-cellular growth-reactive oxygen species-reactive nitrogen species (NO)-nutrient deprivation (Fe)-host immune cells/Ab’s

-adaptation to the environment

-adaptation to the infected host


MelaninCapsule

Phenotypic plasticity

Phenotypic plasticity – ability to rapidly alter phenotypes in response to changing environmental conditions

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Phenotypic plasticity Genomic plasticity Titan cell formation

-Altamirano et al. mSphere

2017;2:e00205-17

-Okagaki, PLoS Pathogens 2010

-Zaragoza, PLoS Pathogens, 2010

Cryptococcus neoformansPhenotypic plasticity• Epigenetic regulation by histone modifications

-Brandao et al., Virulence 2015; Scientific Reports 2018

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Histone de-acetylases (HDACs) regulate capsule expression





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Phenotypic plasticity• Epigenetic regulation by histone modifications

-Brandao et al., Scientific Reports 2018

Cryptococcus neoformansPhenotypic plasticity• Epigenetic regulation by histone modifications• Regulated expression of “exo-GXM”

-Steven Denham and Jessica Brown, Infect Immun 2018

Un

-in

du

ced

Ind

uce

d

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Un

-in

du

ced

Ind

uce

d



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Cryptococcus neoformansPhenotypic plasticity• Epigenetic regulation by histone modifications• Regulated expression of “exo-GXM”• Active remodeling of cell wall in vivo

WT rim101Δ

WG

A –

exp

ose

d c

hit

inTE

M

O'Meara et al. PLoS Path. 2010, O’Meara et al. mBio 2013, O’Meara et al MCB 2013, Ost mBio 2017

WT

rim101Δ

Cryptococcus neoformansPhenotypic plasticity• Epigenetic regulation by histone modifications• Regulated expression of “exo-GXM”• Active remodeling of cell wall in vivo

WT rim101Δ

WG

A –

exp

ose

d c

hit

inTE

M

O'Meara et al. PLoS Path. 2010, O’Meara et al. mBio 2013, O’Meara et al MCB 2013, Ost mBio 2017

WT rim101Δ

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-Ost, et al., mBio, 2017

Altered chitin/cell wall exposure controls immune recognition

Altered chitin/cell wall exposure controls immune recognition

-Specht, et al., mBio, 2017

-Uphadya, et al., mBio, 2016

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Vaccination of CBA/J mice with 107 CFU of live cda1Δ2Δ3Δ cells conferred robust protective

immunity to subsequent infection with wild-type KN99α C. neoformans cells.

Rajendra Upadhya et al. mBio 2016;

doi:10.1128/mBio.00547-16

Cryptococcus neoformans-Genomic plasticity – changes to the genome that allow adaptation to new stresses

Phenotypic plasticity Genomic plasticity


2017;2:e00205-17

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Cryptococcus neoformans-Genomic plasticity

FLC heteroresistance mediated by alterations

of ploidy and ERG11 azole target gene

Edward Sionov et al. Antimicrob. Agents Chemother.

2009;53:2804-2815

Edward Sionov et al. Antimicrob. Agents Chemother.

2013;57:5127-5130


FLC heteroresistance mediated by alterations

of ploidy and ERG11 azole target gene

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FLC treatment results in an increase in

ploidy in a significant fraction of cells.

Sophie Altamirano et al. mSphere 2017;2:e00205-17

A delay or complete block in cell

separation during FLC treatment may

lead to an increase in DNA content in

unseparated cells.



Analysis of centromere dynamics

in FLC-treated cells.

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C. neoformans is remarkably tolerant of ploidy changes

-potentially reversible-important for azole resistance

Cryptococcus neoformans-mechanisms of adaptation

Phenotypic plasticity Genomic plasticity Titan cell formation


2017;2:e00205-17

-Okagaki, PLoS Pathogens 2010

-Zaragoza, PLoS Pathogens, 2010

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Experimental mouse lung infection with C. neoformans• Intact lung architecture• Small foci of inflammatory cell infiltration• Numerous fungal cells

Experimental mouse lung infection with C. neoformans• Intact lung architecture• Small foci of inflammatory cell infiltration• Numerous fungal cells

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Titan cell formation – reversible morphological transition

-only observed in vivo-large cells (>15 um)-large capsules-resistant to phagocytosis-resistant to ROS/RNS-polylpoid-generate haploid progeny


Titan cell formation

-only observed in vivo-large cells (>15 um)-large capsules-resistant to phagocytosis-resistant to ROS/RNS-polylpoid-generate haploid progeny

-in vitro cultivation

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Titan cell formation-only observed in vivo-large cells (>15 um)-large capsules-resistant to phagocytosis-resistant to ROS/RNS-polylpoid-generate haploid progeny

-in vitro cultivation-role of lung resident bacteria(peptidoglycan subunit muramyl dipeptide)

20 um

5 um

Cryptococcus neoformans – an adaptable pathogenPhenotypic plasticity Genomic plasticity Titan cell formation

Rapid phenotypic changes:-Histone modification, epigenetic modifications-Regulation of surface and secreted capsule-Active remodeling of cell wall/immune evasion

Genomic changes:-Polyploidy-Aneuploidy

?reversible-RNAi/Transposons

Morphogenetic changes:-Resistant to host stresses-Direct immune response-?Persister cell

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Alspaugh LabConnie NicholsKaila PianaltoHannah BrownCalla TelzrowSandra Breeding

Elizabeth Ballou-Univ Birmingham, UKTeresa O’Meara – Univ TorontoKyla Selvig Ost – Univ UtahFabiana Brandao – Univ BrasiliaShannon Esher – Tulane Univ.Diana NortonJessie NarlochNaureen HudaFabiana BrandaoMaria KohlbrennerDayton GroganHannah Shepard

Duke Mycology UnitJohn PerfectFred Dietrich/Josh GranekJoe HeitmanLorena Beese

Aaron Mitchell, Carnegie-Mellon

Jim Kronstad, UBCFloyd Wormley, UTSAMichael Price, Liberty Univ.Lukasz Kozubowski – Clemson Univ

Aberdeen Fungal GroupCarol MunroJeanette WagenerLouise Walker

Funding:NIH/NIAIDAHABurroughs Wellcome Fund

Acknowledgements

Documents

New Insights into the Pathogenesis of Cryptococcosis ... Speaker... · 3 Cryptococcus neoformans Environmental stresses-varying temperatures-solar radiation-desiccation/osmotic stress-nutrient