title: Calcineurin plays key roles in the dimorphic transition and virulence of the human pathogenic zygomycete Mucor circinelloides.
abstract: Many pathogenic fungi are dimorphic and switch between yeast and filamentous states. This switch alters host-microbe interactions and is critical for pathogenicity. However, in zygomycetes, whether dimorphism contributes to virulence is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits hyphal growth in aerobic conditions but switches to multi-budded yeast growth under anaerobic/high CO₂ conditions. We found that in the presence of the calcineurin inhibitor FK506, Mucor exhibits exclusively multi-budded yeast growth. We also found that M. circinelloides encodes three calcineurin catalytic A subunits (CnaA, CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Mutations in the latch region of CnbR and in the FKBP12-FK506 binding domain of CnaA result in hyphal growth of Mucor in the presence of FK506. Disruption of the cnbR gene encoding the sole calcineurin B subunit necessary for calcineurin activity yielded mutants locked in permanent yeast phase growth. These findings reveal that the calcineurin pathway plays key roles in the dimorphic transition from yeast to hyphae. The cnbR yeast-locked mutants are less virulent than the wild-type strain in a heterologous host system, providing evidence that hyphae or the yeast-hyphal transition are linked to virulence. Protein kinase A activity (PKA) is elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, suggesting a novel connection between PKA and calcineurin. cnaA mutants lacking the CnaA catalytic subunit are hypersensitive to calcineurin inhibitors, display a hyphal polarity defect, and produce a mixture of yeast and hyphae in aerobic culture. The cnaA mutants also produce spores that are larger than wild-type, and spore size is correlated with virulence potential. Our results demonstrate that the calcineurin pathway orchestrates the yeast-hyphal and spore size dimorphic transitions that contribute to virulence of this common zygomycete fungal pathogen.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Soo Chan
authors.initials: SC
authors.lastname: Lee
authors.forename: Alicia
authors.initials: A
authors.lastname: Li
authors.forename: Silvia
authors.initials: S
authors.lastname: Calo
authors.forename: Joseph
authors.initials: J
authors.lastname: Heitman
mesh_terms: D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D025301:Hyphae / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity; D007166:Immunosuppressive Agents / Q000494:pharmacology; D009089:Mucor / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D009091:Mucormycosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017434:Protein Structure, Tertiary; D016559:Tacrolimus / Q000494:pharmacology; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D007166:Immunosuppressive Agents; D037521:Virulence Factors; D019703:Calcineurin; D016559:Tacrolimus
references.citation: J Gen Microbiol. 1974 May;82(1):1-13
references.pmid: 4851999
references.citation: Curr Opin Microbiol. 2007 Aug;10(4):314-9
references.pmid: 17719267
references.citation: Clin Microbiol Rev. 2000 Apr;13(2):236-301
references.pmid: 10756000
references.citation: Microbiology (Reading). 2004 Jan;150(Pt 1):143-150
references.pmid: 14702407
references.citation: Eukaryot Cell. 2012 Nov;11(11):1391-8
references.pmid: 23002105
references.citation: PLoS Pathog. 2010 Jun 17;6(6):e1000945
references.pmid: 20585557
references.citation: Eur J Clin Microbiol Infect Dis. 2006 Apr;25(4):215-29
references.pmid: 16568297
references.citation: EMBO J. 1997 May 15;16(10):2576-89
references.pmid: 9184205
references.citation: Eukaryot Cell. 2009 Jul;8(7):933-44
references.pmid: 19411621
references.citation: G3 (Bethesda). 2012 Jun;2(6):675-91
references.pmid: 22690377
references.citation: Microbiol Mol Biol Rev. 2011 Jun;75(2):213-67
references.pmid: 21646428
references.citation: Clin Infect Dis. 2002 Apr 1;34(7):909-17
references.pmid: 11880955
references.citation: PLoS Pathog. 2012;8(10):e1002936
references.pmid: 23055925
references.citation: Mol Gen Genet. 1997 Sep;256(2):104-14
references.pmid: 9349701
references.citation: N Engl J Med. 2012 Dec 6;367(23):2214-25
references.pmid: 23215557
references.citation: J Antimicrob Chemother. 2011 Oct;66(10):2312-4
references.pmid: 21795260
references.citation: J Bacteriol. 1982 Nov;152(2):880-7
references.pmid: 7130131
references.citation: Cell. 1998 Jan 9;92(1):39-49
references.pmid: 9489698
references.citation: J Bacteriol. 1980 Apr;142(1):196-201
references.pmid: 7372569
references.citation: J Microbiol Methods. 2011 Mar;84(3):442-6
references.pmid: 21256886
references.citation: Methods Enzymol. 1983;99:3-6
references.pmid: 6316096
references.citation: J Clin Invest. 2010 Jun;120(6):1914-24
references.pmid: 20484814
references.citation: Mol Plant Microbe Interact. 2009 Oct;22(10):1293-301
references.pmid: 19737102
references.citation: Eukaryot Cell. 2011 Jun;10(6):803-19
references.pmid: 21531874
references.citation: J Antimicrob Chemother. 2010 Apr;65(4):701-3
references.pmid: 20130026
references.citation: J Bacteriol. 1973 Nov;116(2):972-80
references.pmid: 4745438
references.citation: Antimicrob Agents Chemother. 2009 Aug;53(8):3549-51
references.pmid: 19451295
references.citation: Eur J Biochem. 1989 Feb 1;179(2):429-34
references.pmid: 2537210
references.citation: Microbiol Rev. 1991 Jun;55(2):234-58
references.pmid: 1886520
references.citation: Fungal Genet Biol. 1999 Dec;28(3):171-89
references.pmid: 10669583
references.citation: Microbiology (Reading). 2000 Aug;146 ( Pt 8):1949-1958
references.pmid: 10931899
references.citation: Ann N Y Acad Sci. 2007 Sep;1111:225-35
references.pmid: 17513466
references.citation: Sci Transl Med. 2012 Dec 19;4(165):165rv13
references.pmid: 23253612
references.citation: J Bacteriol. 1962 Oct;84:841-58
references.pmid: 13969720
references.citation: PLoS Pathog. 2011 Jun;7(6):e1002086
references.pmid: 21698218
references.citation: Cell. 2002 Apr;109 Suppl:S67-79
references.pmid: 11983154
references.citation: Mol Biol Cell. 1993 Dec;4(12):1225-38
references.pmid: 8167406
references.citation: J Bacteriol. 1971 Jul;107(1):114-20
references.pmid: 4327506
references.citation: Arch Biochem Biophys. 2000 Sep 1;381(1):74-82
references.pmid: 11019822
references.citation: Infect Dis Clin North Am. 2006 Sep;20(3):581-607, vi
references.pmid: 16984870
references.citation: Protoplasma. 2003 Sep;222(1-2):23-30
references.pmid: 14513308
references.citation: Infect Immun. 2009 Aug;77(8):3491-500
references.pmid: 19451235
references.citation: Eukaryot Cell. 2004 Oct;3(5):1147-53
references.pmid: 15470242
references.citation: Cell Microbiol. 2009 Mar;11(3):370-80
references.pmid: 19170685
references.citation: Eukaryot Cell. 2003 Oct;2(5):1053-60
references.pmid: 14555488
references.citation: Cell. 1997 Sep 5;90(5):939-49
references.pmid: 9298905
references.citation: BMC Bioinformatics. 2007 Nov 22;8:460
references.pmid: 18034891
references.citation: Mol Microbiol. 2001 Feb;39(4):835-49
references.pmid: 11251806
references.citation: Microbiology (Reading). 2002 Aug;148(Pt 8):2607-2615
references.pmid: 12177355
references.citation: EMBO J. 1995 Jun 15;14(12):2772-83
references.pmid: 7540976
references.citation: EMBO J. 2002 Feb 15;21(4):546-59
references.pmid: 11847103
references.citation: J Bacteriol. 1973 Sep;115(3):1198-204
references.pmid: 4353871
references.citation: Genetics. 2000 May;155(1):57-67
references.pmid: 10790384
references.citation: PLoS One. 2012;7(7):e41761
references.pmid: 22844520
references.citation: J Bacteriol. 1962 Oct;84:829-40
references.pmid: 13969719
references.citation: Protoplasma. 2006 Sep;228(4):189-99
references.pmid: 16983486
references.citation: Curr Opin Microbiol. 2010 Aug;13(4):437-42
references.pmid: 20570552
references.citation: J Infect Dis. 2009 Sep 15;200(6):1002-11
references.pmid: 19659439
references.citation: Clin Infect Dis. 2012 Jun;54(11):1629-36
references.pmid: 22431807
references.citation: Syst Biol. 2003 Oct;52(5):696-704
references.pmid: 14530136
references.citation: Mycopathologia. 1982 May 22;78(2):107-22
references.pmid: 7099241
references.citation: EMBO J. 2004 Apr 21;23(8):1845-56
references.pmid: 15071502
references.citation: Curr Opin Microbiol. 2006 Dec;9(6):572-8
references.pmid: 17045514
references.citation: Cell. 1994 Nov 4;79(3):437-47
references.pmid: 7525078
references.citation: FEMS Yeast Res. 2002 May;2(2):203-13
references.pmid: 12702308
references.citation: FEMS Microbiol Lett. 2008 Jul;284(2):127-34
references.pmid: 18479435
references.citation: Fungal Genet Biol. 2010 Feb;47(2):107-16
references.pmid: 19755168
references.citation: Nature. 1989 Oct 26;341(6244):755-7
references.pmid: 2477714
references.citation: Eukaryot Cell. 2008 Oct;7(10):1856-64
references.pmid: 18776037
references.citation: J Infect Dis. 2005 Apr 15;191(8):1350-60
references.pmid: 15776383
references.citation: Eukaryot Cell. 2012 Aug;11(8):989-1002
references.pmid: 22635921
references.citation: Proc Natl Acad Sci U S A. 2009 Jan 13;106(2):351-2
references.pmid: 19129500
references.citation: Cell. 1998 Apr 17;93(2):215-28
references.pmid: 9568714
references.citation: Infect Genet Evol. 2009 Jul;9(4):401-16
references.pmid: 19460306
references.citation: Clin Infect Dis. 2005 Sep 1;41(5):634-53
references.pmid: 16080086
references.citation: PLoS One. 2012;7(8):e44192
references.pmid: 22952924
references.citation: Antonie Van Leeuwenhoek. 2007 Apr;91(3):237-51
references.pmid: 17080289
references.citation: Fungal Genet Biol. 2007 Jun;44(6):504-16
references.pmid: 17074518
references.citation: Eukaryot Cell. 2012 Mar;11(3):270-81
references.pmid: 22210828
references.citation: Bacteriol Rev. 1963 Sep;27:293-304
references.pmid: 14063856
references.citation: PLoS Pathog. 2010 Jun 17;6(6):e1000953
references.pmid: 20585559
references.citation: J Infect Dis. 2013 Mar 1;207(5):834-41
references.pmid: 23242544
references.citation: Biochim Biophys Acta. 1962 Nov 5;64:548-51
references.pmid: 13969721
references.citation: Cell. 1991 Aug 23;66(4):807-15
references.pmid: 1715244
references.citation: Eukaryot Cell. 2006 Jul;5(7):1091-103
references.pmid: 16835453
references.citation: Clin Microbiol Rev. 2005 Jul;18(3):556-69
references.pmid: 16020690
references.citation: Nature. 1992 Dec 17;360(6405):682-4
references.pmid: 1281518
references.citation: Infect Immun. 2009 Oct;77(10):4345-55
references.pmid: 19620339
references.citation: Eur J Biochem. 1994 Jun 1;222(2):501-6
references.pmid: 8020487