A controllable gene-expression system for the pathogenic fungus Candida glabrata

Abstract

A system for controlling gene expression was established in the pathogenic fungus Candida glabrata to elucidate the physiological functions of genes. T o Control the expression O f the gene O f interest, the c. glabrata Cells Were first transformed with the plasmid carrying the tetracycline repressortransactivator fusion tetR:: G A U , then with the DNA fragment containing the controllable cassette, the tetracycline operator chimeric promoter ( t e t 0 : :ScHOP7). 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 CgT€F3 or CgTOP2 was replaced with tet0: :ScHOP7, doxycycline almost completely repressed the expression of both mRNAs, and impaired growth. Repression of the TOP2 or T€F3 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 T€F3 promoter was replaced with the tetO::ScHOP7 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.

The inositol regulon controls viability in Candida glabrata

“…The CgTRP1 gene was disrupted in Cg33 using a plasmid from Karl Kuchler's lab to create strain EBCg046. EBCg046 was used to create the tetO : : HOPI1 : : CgOPI1 strains EBCg048 and EBCg049 by first integrating plasmid pINTG4 carrying the tetR : : GAL4AD repressor-activator into its genome as described by Nakayama et al (1998). Then the tetO : : HOP1 : : CgOPI1 construct was PCR-amplified from plasmid pEB48 (Table 3) and was used to replace the CgOPI1 locus on the chromosome.…”

“…The pINT4 vector was a gift from Hironobu Nakayama (Nakayama et al, 1998) and was integrated into the EBCg046 genome after cutting it with EcoRV (Nakayama et al, 1998). Plasmid pEB48 was generated by subcloning an~500 bp 59 NCR of the CgOPI1 ORF into p97CGH upstream (59) of the HIS3 tetO-HOP1 cassette using primers EBO76 and EBO77.…”

“…To determine if CgOpi1p could repress CgINO1, CgOPI1 was placed under the control of a doxycycline-repressible promoter (Nakayama et al, 1998). C. glabrata strain BG14 (Cgura3D) was modified by disrupting both the CgHIS3 gene (making it a histidine auxotroph) and the CgTRP1 gene (making it a tryptophan auxotroph).…”

Target validation and ligand development for a pathogenic fungal profilin, using a knock‐down strain of pathogenic yeast Candida glabrata and structure‐based ligand design

“…In this study, two strains were constructed from a parent strain, ACG4 (Table 3), with the Tet-OFF promoter system described in previous studies (Nakayama et al, 1998;Ueno et al, 2007). In this system, the promoter of the target gene is replaced with the Tet-OFF promoter, which can be repressed by adding tetracycline or tetracycline derivatives, such as doxycycline, to the medium.…”

“…In this strain, the endogenous promoter of the profilin gene was replaced with the tetracycline-regulatable (Tet-OFF) promoter (Nakayama et al, 1998) (Figure 2A, B).…”