Expression plasmids for use in Candida glabrata

Abstract

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata.

Keywords: Candida glabrata; MET3; expression vector; inducible; macrophage.

Figure 1

Schematic diagrams of the pCU and pCN series of C. glabrata vectors. Naming convention was chosen to indicate the plasmids have a CEN/ARS and either URA3 (pCU; A) or nourseothricin resistance (NAT^R) (pCN; B) cassettes for selection in C. glabrata. Both backbones carry an Ap^R gene and pMB1 (ColEI family) origin (not shown) for growth and selection in E. coli. The restriction sites in the multiple cloning site (MCS) between the promoter and terminator are listed. *The XbaI site is not present in the MCS of pCU-LYS21 or pCN-LYS21. Refer to Table 4 to determine which MCS restriction sites are unique in each plasmid, because this depends on the particular backbone and promoter combination. XXX# is a placeholder to designate the name of the promoter present in a particular vector. (C) The lengths of the promoters cloned into the pCU and pCN vectors using SacI-XbaI restriction digestion for all constructs except LYS21, which used SacI-SpeI, thereby eliminating the XbaI site in the MCS, are depicted.

Figure 2

Fluorescence of exponential phase C. glabrata strains carrying pCU series plasmids. Flow cytometry was used to measure fluorescence levels in C. glabrata strains carrying pCU series plasmids containing EGD2 (A), HHT2 (B), PDC1 (C), ACO2 (D), and LYS21 (E) promoters. The strains were diluted from a saturated overnight culture and grown to OD₆₀₀ of approximately 0.3 before fluorescence was measured. Each panel depicts histograms of fluorescence for two C. glabrata strains containing the empty vectors (red and blue lines) and two C. glabrata strains containing GFP reporter vectors (green and orange lines) for a given promoter. The median fluorescence value for each strain population is shown.

Figure 3

Fluorescence of stationary phase C. glabrata strains carrying pCU series plasmids. Flow cytometry was used to measure fluorescence levels in C. glabrata strains carrying pCU series plasmids containing EGD2 (A), HHT2 (B), PDC1 (C), ACO2 (D), and LYS21 (E) promoters. Fluorescence of saturated overnight cultures was measured. Each panel depicts histograms of fluorescence for two C. glabrata strains containing the empty vectors (red and blue lines) and two C. glabrata strains containing GFP reporter vectors (green and orange lines) for a given promoter. The median fluorescence value for each strain population is shown.

Figure 4

Quantitative reverse-transcription PCR (qRT-PCR) results of GFP expression during phagocytosis. qRT-PCR assessment of relative expression of GFP (normalized to TUB1) in C. glabrata grown in media or after phagocytosis by J774A.1 macrophage-like cells. The data are averages of two biological replicates (qPCR performed in triplicate) for each strain; error bars indicate the SD between the averages. The numbers above the bars indicate the average fold-change in GFP expression (normalized to TUB1) in C. glabrata that have been phagocytosed by J774A.1 (macrophage) vs. growth in tissue culture (TC) media (media). Data are shown for one C. glabrata strain carrying pCU-ACO2-GFP and two independent strains carrying pCU-LYS21-GFP.

Figure 5

Expression from pCU-LYS21-GFP is increased in phagocytosed C. glabrata. C. glabrata strains carrying pCU-LYS21-GFP were grown in media only (left panels) or used to infect J774A.1 macrophage-like cells (right panels). After 2 hr, the cells were fixed and GFP expression was monitored by bright-field differential image contrast (DIC) and fluorescence microscopy. Merge panels were created by combining the bright-field and pseudo-colored GFP images using ImageJ. Scale bar is 10 μm.

Figure 6

Fluorescence of C. glabrata strains carrying pCU-MET3 plasmids. Each panel depicts histograms of fluorescence for two C. glabrata strains containing the empty vectors (red and blue lines) and two C. glabrata strains containing GFP reporter vectors (green and orange lines). The median fluorescence value for each strain population is shown. “OFF” indicates the strains were grown in media containing 2 mM Met and Cys, which represses the MET3 promoter. “ON” indicates the strains were grown in media lacking Met and Cys, which induces expression from the MET3 promoter.