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10/2/2018
1
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
10/2/2018
2
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
mat
ed
Yea
rly
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
10/2/2018
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Cryptococcus neoformans
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
Cryptococcus neoformans
MelaninCapsule
Phenotypic plasticity
Phenotypic plasticity – ability to rapidly alter phenotypes in response to changing environmental conditions
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Cryptococcus neoformans
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
10/2/2018
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Cryptococcus neoformansPhenotypic plasticity• Epigenetic regulation by histone modifications
Histone de-acetylases (HDACs) regulate capsule expression
-Brandao et al., Virulence 2015; Scientific Reports 2018
Cryptococcus neoformansPhenotypic plasticity• Epigenetic regulation by histone modifications
Histone de-acetylases (HDACs) regulate capsule expression
-Brandao et al., Virulence 2015; Scientific Reports 2018
10/2/2018
6
Cryptococcus neoformans
Histone de-acetylases (HDACs) regulate capsule expression
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
10/2/2018
7
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
Cryptococcus neoformansPhenotypic plasticity• Epigenetic regulation by histone modifications• Regulated expression of “exo-GXM”
-Steven Denham and Jessica Brown, Infect Immun 2018
10/2/2018
8
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
-Altamirano et al. mSphere
2017;2:e00205-17
10/2/2018
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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
Cryptococcus neoformans-Genomic plasticity
FLC heteroresistance mediated by alterations
of ploidy and ERG11 azole target gene
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Cryptococcus neoformans-Genomic plasticity
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.
Sophie Altamirano et al. mSphere 2017;2:e00205-17
Cryptococcus neoformans-Genomic plasticity
Analysis of centromere dynamics
in FLC-treated cells.
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Sophie Altamirano et al. mSphere 2017;2:e00205-17
Cryptococcus neoformans-Genomic plasticity
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
-Altamirano et al. mSphere
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
10/2/2018
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Cryptococcus neoformans
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
Cryptococcus neoformans
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
10/2/2018
16
Cryptococcus neoformans
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