Xiaorong Lin Texas A&M University

Conservation of morphogenesis and virulence in Cryptococcus neoformans

Cryptococcus infection

Yeast cells cross the blood-brain barrier

Lymphatic and hematogenous dissemination

Survival in macrophages

Pulmonary infection

Fungal cells Ramify in neurophil and parenchyma

Cryptococcal meningitis

Lin and Heitman, Annual Review Microbiology 2006

Conserved association of morphogenesis and virulence in human pathogenic fungi

Lo et al, Cell 1997 Liu, Curr Opin Microbiol 2001 Nemecek, Wüthrich, and Klein, Science 2006 Klein and Tebbets, Curr Opin Microbiol 2007 Lin, Infection, Genetics & evolution 2009

Paracoccidioides brasiliensis

Sporothrix schenckii Candida albicans

Hortaea werneckii Wangiella dermatitidis

Pneumocystis carinii

Rhizopus arrhizus / R. oryzae Cryptococcus neoformans

Zygomycota

Ascomycota

Taphrinomycotina Saccharomycotina

Sordariomycetes

Blastomyces dermatitidis Histoplasma capsulatum

Coccidioides immitis

Aspergillus fumigatus Penicillium marneffei

Onygenales

Eurotiales

Pezizomycotina Dothideomycetes

Eurotiomycetes

Eurotiomycetidae

Basidiomycota

Chaetothyriomycetidae

Dimorphic Transition

Hyphal form

Yeast form

Correlation between morphological transition and virulence in Cryptococcus

Virulent (1) Avirulent as inocula in animal models (2) Immunization against lethal yeast infection (3) Regain yeast growth, then regain virulence

Shadomy and Utz, Mycologia 1966 Neilson et al., IAI 1978 Fromtling et al., Mycopathologia 1979 Neilson et al., Mycopathologia 1981 Zimmer et al., Mycopathologia1983 Lin, Infection, Genetics & evolution 2009

Yeast Filament (hyphae/pseudohyphae)

The molecular link between morphogenesis and virulence in Cryptococcus remains unknown

Morphological transition in Cryptococcus has historically been associated with mating

Kwon-Chung, Mycologia 1975, 1976 Wickes, et al. PNAS 1996 Lin et al., Nature 2005 Lin et al., PLoS Genet 2006 Lin et al., PLoS Genet 2007

Bisexual mating (αxa)

Unisexual mating

Gpg1/2

Signaling pathways controlling mating

Pheromone

Ste20

Ste11

Ste7

Cpk1

Ste3 GPCR Receptor Cpr2

Ligand

Gpa 2/3 Gpb1 β G Proteins

PAK1

MAPK components

Sxi2a Sxi1α

Cell identity complex

Pheromone sensing pathways

Mat2

Ras1

Ste12

X X ?

Mating

H2O High temperature CO2

Host conditions differ drastically from mating-inducing conditions

Components specifically regulating mating do NOT control morphogenesis under host-relevant conditions.

Host physiological environment

Gpg1/2

Morphology determinant

Pheromone

Ste20

Ste11

Ste7

Cpk1

Ste3 GPCR Receptor Cpr2

Ligand

Gpa 2/3 Gpb1 β G Proteins

PAK1

MAPK components

Sxi2a Sxi1α

Cell identity complex

Pheromone sensing pathways

Mat2

Ras1

Ste12

Znf2 Zinc finger transcription factor

Lin et al., PLoS Genetics 2010

?

Pheromone (MFα)

Znf2

Ste12 Mat2

Znf2: a terminal regulator of morphogenesis but not important for pheromone signaling

Overexpression of ZNF2 confers filamentation in these mutants, but it does NOT restore defects in pheromone signaling.

Overexpression of MAT2 or STE12 does NOT restore filamentous growth in znf2∆ mutant.

Strains in JEC21 background

Znf2 can drive filamentation under mating-inducing and host-relevant conditions

Cu2+ inhibits ZNF2 expression.

BCS, a copper chelator, induces ZNF2 expression.

ZNF2

Copper chelator BCS

Copper ion

PCTR4-2

Inducible expression of ZNF2

Time-course analysis of Znf2-mediated morphological transition.

Znf2 determines the morphological transition

Inducer (BCS)

Inhibitor (Cu2+)

Znf2 mediates virulence

Inhalation model of cryptococcosis

Mechanisms of Znf2-mediated virulence

Znf2

Virulence

1.

2. Other properties normally associated with morphogenesis

Znf2 quantitatively controls cellular and colony morphology

Cell adhesins (flocculins) cause wrinkled colony morphology across kingdoms

Schembri et al., J. Bacteriol 2004 Zara et al., AEM 2005

FLO11

flo11∆

S. cerevisiae E. coli

Ag43 (flu) +

Ag43(flu) ̶

Sedimentation test of WT and ZNF2OE strain in YPD medium.

Znf2 mediates cell flocculation

CFL1: Cell Flocculin 1 CMF1: Cellular Morphology Factor 1

(Log2 ZNF2OE/WT)

133 fold CMF1

WT

znf2∆

CMF1

CFL1

ACT1

(znf2∆/WT)

Znf2 regulates many extracellular proteins

Cfl1 regulates cell-cell adhesion, but it is not critical for filamentation

CFL1 also regulates cell-substrate adhesion and agar invasion

•  Cfl1 is the first Cryptococcus adhesin discovered. •  Cfl1 is a novel adhesin:

–  short (<400 aa), no GPI anchor, no repeats, shares no homology with known adhesins in sequence or domain.

Znf2 regulates additional adhesins besides Cfl1

Znf2 downstream factor Cmf1 regulates cell morphology, but not adhesion

Host relevant condition

WT PGPD1-CMF1

Mating inducing condition

WT

PGPD1-CMF1

Both the adhesin Cfl1 and the morphogenesis factor Cmf1 mediate virulence

WT

PGPD1-CFL1 WT

PGPD1-CMF1

Summary

•  Znf2 is the first molecular link between morphological transition and virulence in Cryptococcus.

–  Activate Znf2 decreases Cryptococcus virulence potential.

•  Znf2 regulates a family of novel adhesins and morphogenesis factors.

Acknowledgments

Collaborators •  Dr. Paul de Figueiredo

•  Dr. Qingming Qin •  Dr. Matt Sachs

•  Dr. Cheng Wu •  Dr. Shuping Zhang •  Dr. Floyd Wormley

•  Dr. Karen Wozniak

Lin lab

Linqi Wang Bing Zhai (postdoc) (graduate student)

Srijana Upadhyay Rachana Gyawali (research associate) (graduate student)

Funding •  NIAID (R21) •  AHA •  NHARP