title: Fatty acid synthesis is essential for survival of Cryptococcus neoformans and a potential fungicidal target.
abstract: Fatty acid synthase in the yeast Cryptococcus neoformans is composed of two subunits encoded by FAS1 and FAS2 genes. We inserted a copper-regulated promoter (P(CTR4-2)) to regulate FAS1 and FAS2 expression in Cryptococcus neoformans (strains P(CTR4-2)/FAS1 and P(CTR4-2)/FAS2, respectively). Both mutants showed growth rates similar to those of the wild type in a low-copper medium in which FAS1 and FAS2 were expressed, but even in the presence of exogenous fatty acids, strains were suppressed in growth under high-copper conditions. The treatment of C. neoformans with fluconazole was shown to have an increased inhibitory activity and even became fungicidal when either FAS1 or FAS2 expression was suppressed. Furthermore, a subinhibitory dose of fluconazole showed anticryptococcal activity in vitro in the presence of cerulenin, a fatty acid synthase inhibitor. In a murine model of pulmonary cryptococcosis, a tissue census of yeast cells in P(CTR4-2)/FAS2 strain at day 7 of infection was significantly lower than that in mice treated with tetrathiomolybdate, a copper chelator (P < 0.05), and a yeast census of P(CTR4-2)/FAS1 strain at day 14 of infection in the brain was lower in the presence of more copper. In fact, no positive cultures from the brain were detected in mice (with or without tetrathiomolybdate treatment) infected with the P(CTR4-2)/FAS2 strain, which implies that this mutant did not reach the brain in mice. We conclude that both FAS1 and FAS2 in C. neoformans are essential for in vitro and in vivo growth in conditions with and without exogenous fatty acids and that FAS1 and FAS2 can potentially be fungicidal targets for C. neoformans with a potential for synergistic behavior with azoles.
authors: Department of Medicine, Division of Infectious Diseases, Duke University Medical Center, Durham, NC 27710, USA.
authors: Methee
authors: M
authors: Chayakulkeeree
authors: Thomas H
authors: TH
authors: Rude
authors: Dena L
authors: DL
authors: Toffaletti
authors: John R
authors: JR
authors: Perfect
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001393:Azoles / Q000494:pharmacology; D001921:Brain / Q000382:microbiology; D002569:Cerulenin / Q000494:pharmacology; D015169:Colony Count, Microbial; D003300:Copper / Q000502:physiology; D003453:Cryptococcosis / Q000188:drug therapy / Q000382:microbiology; D003455:Cryptococcus neoformans / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors* / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics; D008168:Lung / Q000382:microbiology; D051379:Mice; D008826:Microbial Sensitivity Tests; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D004271:DNA, Fungal; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D007527:Isoenzymes; D002569:Cerulenin; D003300:Copper; D064429:Fatty Acid Synthases
references: Annu Rev Nutr. 2001;21:97-119
references: 11375431
references: Laryngoscope. 2001 Apr;111(4 Pt 1):696-701
references: 11359142
references: Microbiology. 2002 Aug;148(Pt 8):2607-15
references: 12177355
references: Infect Immun. 2002 Oct;70(10):5684-94
references: 12228298
references: J Nat Prod. 2002 Dec;65(12):1909-14
references: 12502337
references: Eukaryot Cell. 2003 Dec;2(6):1336-49
references: 14665467
references: Yeast. 2004 Aug;21(11):919-26
references: 15334556
references: Microbiol Mol Biol Rev. 2004 Sep;68(3):501-17, table of contents
references: 15353567
references: Eukaryot Cell. 2004 Oct;3(5):1249-60
references: 15470254
references: Mol Gen Genet. 1986 Jun;203(3):479-86
references: 3528750
references: J Biol Chem. 1988 Sep 5;263(25):12315-25
references: 2900835
references: J Biol Chem. 1989 Oct 5;264(28):16537-44
references: 2674136
references: Eur J Biochem. 1992 Feb 1;203(3):607-14
references: 1735446
references: J Bacteriol. 1993 Mar;175(5):1405-11
references: 8444802
references: J Biol Chem. 1993 May 25;268(15):10946-52
references: 8098706
references: Microbiology. 1996 Sep;142 ( Pt 9):2509-14
references: 8828218
references: Infect Immun. 1997 Feb;65(2):829-32
references: 9009352
references: Invest Ophthalmol Vis Sci. 2005 Jan;46(1):299-303
references: 15623788
references: Eukaryot Cell. 2005 Feb;4(2):487-9
references: 15701811
references: Nat Rev Microbiol. 2005 Oct;3(10):753-64
references: 16132036
references: FEMS Yeast Res. 2006 Jun;6(4):469-79
references: 16696643
references: Infect Dis Clin North Am. 2006 Sep;20(3):507-44, v-vi
references: 16984867
references: J Clin Invest. 2007 Mar;117(3):794-802
references: 17290306
references: J Biol Chem. 2001 Oct 5;276(40):37051-9
references: 11477098