A controllable gene-expression system for the pathogenic fungus Candida glabrata.
A system for controlling gene expression was established in the pathogenic fungus Candida glabrata to elucidate the physiological functions of genes. To control the expression of the gene of interest, the C. glabrata cells were first transformed with the plasmid carrying the tetracycline repressor-transactivator fusion tetR::GAL4, then with the DNA fragment containing the controllable cassette, the tetracycline operator chimeric promoter (tetO::ScHOP1). The peptide elongation factor 3 (CgTEF3) and DNA topoisomerase II (CgTOP2) genes from C. glabrata were cloned and their expression assessed using this system. When the promoter of CgTEF3 or CgTOP2 was replaced with tetO::ScHOP1, doxycycline almost completely repressed the expression of both mRNAs, and impaired growth. Repression of the TOP2 or TEF3 gene by doxycycline also hampered the survival of C. glabrata cells in mice; in mouse kidneys the number of C. glabrata cells, in which the TOP2 or TEF3 promoter was replaced with the tetO::ScHOP1 controllable cassette, did not increase when the mice were given doxycycline. Thus, it appears that the gene repression mediated by doxycycline occurred not only in culture media but also in animals; therefore, this system can be used to elucidate the function of the gene in fungal infections and pathogenesis.
Nakayama
Hironobu
H
Department of Mycology, Nippon Roche Research Center,200 Kajiwara, Kamakura, Kanagawa 247,Japan.
Izuta
Miho
M
Department of Mycology, Nippon Roche Research Center,200 Kajiwara, Kamakura, Kanagawa 247,Japan.
Nagahashi
Shigehisa
S
Department of Mycology, Nippon Roche Research Center,200 Kajiwara, Kamakura, Kanagawa 247,Japan.
Sihta
Emi Y
EY
Department of Mycology, Nippon Roche Research Center,200 Kajiwara, Kamakura, Kanagawa 247,Japan.
Sato
Yasuko
Y
Department of Mycology, Nippon Roche Research Center,200 Kajiwara, Kamakura, Kanagawa 247,Japan.
Yamazaki
Toshikazu
T
Department of Mycology, Nippon Roche Research Center,200 Kajiwara, Kamakura, Kanagawa 247,Japan.
Arisawa
Mikio
M
Department of Mycology, Nippon Roche Research Center,200 Kajiwara, Kamakura, Kanagawa 247,Japan.
Kitada
Kunio
K
Department of Mycology, Nippon Roche Research Center,200 Kajiwara, Kamakura, Kanagawa 247,Japan.
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002175:Candida / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D002177:Candidiasis / Q000209:etiology / Q000382:microbiology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D000091262:TEA Domain Transcription Factors; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics; D014170:Transformation, Genetic; D014774:Virulence / Q000235:genetics
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009124:Muscle Proteins; D000091262:TEA Domain Transcription Factors; C100498:TEFR1 protein, isoform b, mouse; C101204:Tead4 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D004318:Doxycycline