Transcriptional profiling of the early stages of germination in by real-time RT-PCR

Toyoda, Mika; Cho, Tamaki; Kaminishi, Hidenori; Sudoh, Masayuki; Chibana, Hiroji

doi:10.1016/j.femsyr.2004.08.004

Cited by 22 publications

(9 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2A). This pattern is consistent with the single-time-point result of Toyoda et al (45), who showed that TUP1 mRNA levels increased slightly at 180 min after induction of filamentation. In the presence of farnesol, we found that TUP1 mRNA consistently increased 2.5-fold Ϯ 0.6-fold (n ϭ 4) from 20 to 60 min.…”

Section: Resultssupporting

confidence: 92%

Candida albicans Tup1 Is Involved in Farnesol-Mediated Inhibition of Filamentous-Growth Induction

et al. 2008

View full text Add to dashboard Cite

Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. Its ability to regulate morphogenesis is strongly correlated with virulence. Tup1, a transcriptional repressor, and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion. Based on this overlap in function, we tested the hypothesis that the cellular response to farnesol involves, in part, the activation of Tup1. Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. The tup1/tup1 and nrg1/nrg1 mutants, but not the rfg1/rfg1 mutant, failed to respond to farnesol. Treatment of C. albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. Importantly, this increase corresponds with the commitment point, beyond which added farnesol no longer blocks germ tube formation, and it correlates with a strong decrease in the expression of two Tup1-regulated hypha-specific genes, HWP1 and RBT1. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Farnesol did not affect EFG1 (a transcription regulator of filament development), NRG1, or RFG1 mRNA levels, demonstrating specific gene regulation in response to farnesol. Furthermore, the tup1/tup1 and nrg1/nrg1 mutants produced 17-and 19-fold more farnesol, respectively, than the parental strain. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Our data are consistent with the role of Tup1 as a crucial component of the response to farnesol in C. albicans.

show abstract

Section: Resultssupporting

confidence: 92%

Candida albicans Tup1 Is Involved in Farnesol-Mediated Inhibition of Filamentous-Growth Induction

et al. 2008

View full text Add to dashboard Cite

show abstract

“…A total of 1 g of RNA was treated with amplification grade DNase I (Invitrogen, Carlsbad, CA) and used for cDNA synthesis with a cMaster RT Kit (Eppendorf AG, Hamburg, Germany) as per the manufacturer's instructions. The following primer sets were used in conjunction with SYBR Green PCR Master Mix (Applied Biosystems, Forest City, CA) and twin.tec real-time 96-well PCR plates (Eppendorf AG, Hamburg, Germany) in an ABI 7300 Real Time PCR System (Applied Biosystems, Forest City, CA): for NRG1, CAC CTC ACT TGC AAC CCC and GCC CTG GAG ATG GTC TGA (29); ACT1, ATG TGT AAA GCC GGT TTT GCC G and CCA TAT CGT CCC AGT TGG AAA C (29); HWP1, TCA GCC TGA TGA CAA TCC TC and GCT GGA GTT GTT GGC TTT TC; ALS3, CAA CTT GGG TTA TTG AAA CAA AAA CA and AGA AAC AGA AAC CCA AGA ACA ACC T (13); EFG1, GCC TCG AGC ACT TCC ACT GT and TTT TTT CAT CTT CCC ACA TGG TAG T; and TUP1, GCT TCA GGT AAC CCA TTG TTG AT and CTT CGG TTC CCT TTG AGT TTA GG. Parameters for primer design were set according to the recommendations of Applied Biosystems.…”

Section: Methodsmentioning

confidence: 99%

Characteristics of Candida albicans Biofilms Grown in a Synthetic Urine Medium

et al. 2009

View full text Add to dashboard Cite

Urinary tract infections (UTIs) are the most common type of nosocomial infection, and Candida albicans is the most frequent organism causing fungal UTIs. Presence of an indwelling urinary catheter represents a significant risk factor for UTIs. Furthermore, these infections are frequently associated with the formation of biofilms on the surface of these catheters. Here, we describe the characterization of C. albicans biofilms formed in vitro using synthetic urine (SU) medium and the frequently used RPMI medium and compare the results. Biofilms of C. albicans strain SC5314 were formed in 96-well microtiter plates and on silicon elastomer pieces using both SU and RPMI media. Biofilm formation was monitored by microscopy and a colorimetric XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. As in biofilms grown in RPMI medium, time course studies revealed that biofilm formation using SU medium occurred after an initial adherence phase, followed by growth, proliferation, and maturation. However, microscopy techniques revealed that the architectural complexity of biofilms formed in SU medium was lower than that observed for those formed using RPMI medium. In particular, the level of filamentation of cells within the biofilms formed in SU medium was diminished compared to those in the biofilms grown in RPMI medium. This observation was also corroborated by expression profiling of five filamentation-associated genes using quantitative real-time reverse transcriptase PCR. Sessile C. albicans cells were resistant to fluconazole and amphotericin B, irrespective of the medium used to form the biofilms. However, caspofungin exhibited potent in vitro activity at therapeutic levels against C. albicans biofilms grown in both SU and RPMI media.Candida albicans is a nosocomial pathogen that has a predilection for the urinary tract, where it can cause infections with a broad spectrum of disease severity. It is thereby known to be the most frequent organism causing fungal urinary tract infections (UTIs) (26). Presence of an indwelling urethral catheter represents a significant risk factor for C. albicans UTI as these infections are frequently associated with the formation of biofilms on the surface of these catheters (8,19). C. albicans biofilms are highly organized communities of yeast, hyphae, and pseudohyphae attached to the surface of biomaterials and enclosed in a matrix of polysaccharides and are highly resistant to most major classes of antifungal drugs (21).C. albicans biofilms have been developed on several different model systems in vitro (4,14,24,30). Several experimental variables have been incorporated into these systems in order to mimic conditions in patients, such as flow to simulate blood flow, coating of substrate surfaces with various host blood and salivary proteins, variation in the nutrient medium employed, and use of common device materials (14,20). In the present study we grew C. albicans biofilms in synthetic urine (SU) medium-an in vitro model mimicking an in vivo...

show abstract

“…However, at such a low concentration of farnesol, a high percentage of the cells finally formed germ tubes data not shown , but we needed a higher concentration of farnesol to completely block germination. Strain JCM 9061 germinated in GlcNAc medium at a lower inoculum than strain CAI4 11 . Even at an inoculum of 5 10 5 cells/ml, strain JCM 9061 cells budded instead.…”

Section: Resultsmentioning

confidence: 98%

Transcriptional Changes in Candida albicans Genes by Both Farnesol and High Cell Density at an Early Stage of Morphogenesis in N-acetyl-D-glucosamine Medium

Cho

Aoyama

Toyoda

et al. 2007

Nippon Ishinkin Gakkai Zasshi

View full text Add to dashboard Cite

Transcriptional profiling of the early stages of germination in by real-time RT-PCR

Cited by 22 publications

References 28 publications

Candida albicans Tup1 Is Involved in Farnesol-Mediated Inhibition of Filamentous-Growth Induction

Candida albicans Tup1 Is Involved in Farnesol-Mediated Inhibition of Filamentous-Growth Induction

Characteristics of Candida albicans Biofilms Grown in a Synthetic Urine Medium

Transcriptional Changes in Candida albicans Genes by Both Farnesol and High Cell Density at an Early Stage of Morphogenesis in N-acetyl-D-glucosamine Medium

Contact Info

Product

Resources

About