Conservation of morphogenesis and virulence in Cryptococcus neoformans. Xiaorong Lin Texas A&M University

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1 Conservation of morphogenesis and virulence in Cryptococcus neoformans Xiaorong Lin Texas A&M University

2 Cryptococcus infection Lymphatic and hematogenous dissemination Pulmonary infection Cryptococcal meningitis Fungal cells Survival in macrophages Ramify in neurophil and parenchyma Yeast cells cross the blood-brain barrier Lin and Heitman, Annual Review Microbiology 2006

3 Conserved association of morphogenesis and virulence in human pathogenic fungi Hyphal form Dimorphic Transition Ascomycota Basidiomycota Zygomycota Yeast form Pezizomycotina Saccharomycotina Taphrinomycotina Eurotiomycetes Dothideomycetes Sordariomycetes Coccidioides immitis Onygenales Paracoccidioides brasiliensis Blastomyces dermatitidis Eurotiomycetidae Histoplasma capsulatum Eurotiales Chaetothyriomycetidae Aspergillus fumigatus Penicillium marneffei Wangiella dermatitidis Hortaea werneckii Sporothrix schenckii Candida albicans Pneumocystis carinii Cryptococcus neoformans Rhizopus arrhizus / R. oryzae 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

4 Correlation between morphological transition and virulence in Cryptococcus Yeast Virulent Filament (hyphae/pseudohyphae) (1) Avirulent as inocula in animal models (2) Immunization against lethal yeast infection (3) Regain yeast growth, then regain virulence The molecular link between morphogenesis and virulence in Cryptococcus remains unknown 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

5 Morphological transition in Cryptococcus has historically been associated with mating Bisexual mating (αxa) Unisexual 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

6 Signaling pathways controlling mating Pheromone Ligand Cpr2 Ste3 GPCR Receptor Pheromone sensing pathways Gpg1/2 Gpb1 β Gpa 2/3 G Proteins Ste20 PAK1 Ras1? Ste12 Ste11 Ste7 Cpk1 X Mat2 MAPK components X Sxi2a Sxi1α Cell identity complex

7 Host conditions differ drastically from mating-inducing conditions Host physiological environment H 2 O High temperature CO 2 Mating Components specifically regulating mating do NOT control morphogenesis under host-relevant conditions.

8 Morphology determinant Pheromone Ligand Cpr2 Ste3 GPCR Receptor Pheromone sensing pathways Gpg1/2 Gpb1 β Gpa 2/3 G Proteins Ste20 PAK1 Ras1 Ste11 Ste7 MAPK components? Ste12 Cpk1 Mat2 Sxi2a Sxi1α Cell identity complex Znf2 Zinc finger transcription factor Lin et al., PLoS Genetics 2010

9 Znf2: a terminal regulator of morphogenesis but not important for pheromone signaling Pheromone (MFα) Ste12 Mat2 Znf2 Strains in JEC21 background 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.

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

11 Inducible expression of ZNF2 Copper ion P CTR4-2 ZNF2 Copper chelator BCS Cu 2+ inhibits ZNF2 expression. BCS, a copper chelator, induces ZNF2 expression.

12 Znf2 determines the morphological transition Inducer (BCS) Inhibitor (Cu 2+ ) Time-course analysis of Znf2-mediated morphological transition.

13 Znf2 mediates virulence Inhalation model of cryptococcosis

14 Mechanisms of Znf2-mediated virulence Znf Other properties normally associated with morphogenesis Virulence

15 Znf2 quantitatively controls cellular and colony morphology

16 Cell adhesins (flocculins) cause wrinkled colony morphology across kingdoms E. coli S. cerevisiae Ag43(flu) flo11 Ag43 (flu) + FLO11 Schembri et al., J. Bacteriol 2004 Zara et al., AEM 2005

17 Znf2 mediates cell flocculation Sedimentation test of WT and ZNF2 OE strain in YPD medium.

18 Znf2 regulates many extracellular proteins CMF1 133 fold (znf2 /WT) WT znf2 CFL1 CMF1 ACT1 (Log 2 ZNF2 OE /WT) CFL1: Cell Flocculin 1 CMF1: Cellular Morphology Factor 1

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

20 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.

21 Znf2 regulates additional adhesins besides Cfl1

22 Znf2 downstream factor Cmf1 regulates cell morphology, but not adhesion Host relevant condition WT P GPD1 -CMF1 WT Mating inducing condition P GPD1 -CMF1

23 Both the adhesin Cfl1 and the morphogenesis factor Cmf1 mediate virulence WT P GPD1 -CFL1 WT P GPD1 -CMF1

24 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.

25 Acknowledgments Lin lab Linqi Wang (postdoc) Bing Zhai (graduate student) Collaborators Dr. Paul de Figueiredo Dr. Qingming Qin Dr. Matt Sachs Dr. Cheng Wu Dr. Shuping Zhang Dr. Floyd Wormley Dr. Karen Wozniak Srijana Upadhyay (research associate) Rachana Gyawali (graduate student) Funding NIAID (R21) AHA NHARP

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