conservation of morphogenesis and virulence in ... · gpg1/2 morphology determinant pheromone ste20...
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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