Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata

Abstract

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.

Keywords: AMP resistance; Candida glabrata; CgTpo4; Galleria mellonella; polyamine resistance; virulence.

Figure 1

CgTPO4 expression is required for C. glabrata virulence against the G. mellonella infection model. The survival of larvae injected with approximately 5 × 10⁶ CFU/larvae of wild-type KUE100 C. glabrata, harboring the pGREG576 cloning vector ( ), or the pGREG576_MTI_CgTPO4 expression plasmid ( ), and of C. glabrata deletion mutant Δcgtpo4, harboring the pGREG576 cloning vector ( ) or the pGREG576_MTI_CgTPO4 expression plasmid ( ), with PBS for control ( ) are displayed as Kaplan–Meier survival curves. The displayed results are the average of at least three independent experiments, with standard deviation represented by the grey lines. ** p < 0.01, *** p < 0.001.

Figure 2

CgTpo4 is a determinant of histatin-5 resistance. Relative concentration of viable cells of wild-type C. glabrata KUE100, harboring the pGREG576 cloning vector (black), or the pGREG576_MTI_CgTPO4 expression plasmid (dark grey) and the derived ∆cgtpo4 deletion mutant, harboring the cloning vector pGREG576 (grey), or the expression plasmid pGREG576_MTI_CgTPO4 (light grey), in the presence of 35 µM of histatin-5. The average percentage of survival, obtained from at least three independent experiments, is indicated by the black lines, with standard deviation being represented by error bars. * p < 0.05, ** p < 0.01, *** p < 0.001, and ***** p < 0.00001.

Figure 3

Expression of genes encoding antimicrobial peptide (AMPs) is activated in G. mellonella, during infection by C. glabrata. Comparison of the variation of the gallerimycin (A), galliomycin (B), inducible metalloproteinase inhibitor (IMPI) (C), lysozyme (D) and cecropin (E) transcript levels in G. mellonella, after 1, 4, 8, 12, and 24 h of infection by KUE100 C. glabrata wild-type cells (black bars) and derived Δcgtpo4 mutant cells (grey bars). The presented transcript levels were obtained by quantitative RT-PCR and are normalized to the GmACT1 mRNA levels, relative to the values registered in G. mellonella larvae, in the same time points, treated with PBS buffer as a control. The indicated values are averages of at least three independent experiments. Error bars represent the corresponding standard deviations. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

CgTPO4 confers resistance to polyamines in C. glabrata. Comparison of the susceptibility to the polyamines, spermidine and putrescine, at the indicated concentrations, of the C. glabrata KUE100 wild-type strain harboring the pGREG576 cloning vector (KUE100 + vv) or the pGREG576_MTI_CgTPO4 vector (KUE100 + CgTPO4) and the derived KUE100_Δcgtpo4 mutant cells harboring the pGREG576 cloning vector (Δcgtpo4 + vv) or the pGREG576_MTI_CgTPO4 vector (Δcgtpo4 + CgTPO4), in YPD agar plates, by spot assays. The inocula were prepared as described in the Materials and Methods section. Cell suspensions used to prepare the spots were 1:5 (b) and 1:25 (c) dilutions of the cell suspension used in (a). The displayed images are representative of at least three independent experiments.

Figure 5

CgTpo4 is localized in the plasma membrane of yeast cells. Fluorescence of exponential-phase L5U1 C. glabrata cells or BY4741 S. cerevisiae cells, harboring the pGREG576_MTI_CgTPO4 or pGREG576_CgTPO4 plasmids, after 5 h of copper- or galactose-induced recombinant protein production, respectively. Results indicate that the CgTpo4_GFP fusion protein localizes to the plasma membrane in both S. cerevisiae and C. glabrata cells.

Figure 6

CgTPO4 expression leads to decreased spermidine accumulation in C. glabrata. Time course accumulation ratio of [³H]-Spermidine in nonadapted cells of KUE100 C. glabrata wild-type (■) or KUE100_Δcgtpo4 (♦) strains, during cultivation in YPD liquid medium in the presence of 8.5 mM unlabeled spermidine. The accumulation ratio values are averages of at least three independent experiments. Error bars represent the corresponding standard deviations. *** p < 0.001.