Human salivary histatin 5 causes disordered volume regulation and cell cycle arrest in Candida albicans

Abstract

Human salivary histatin 5 (Hst 5) is a nonimmune salivary protein with antifungal activity against an important human pathogen, Candida albicans. The candidacidal activity of histatins appears to be a distinctive multistep mechanism involving depletion of the C. albicans intracellular ATP content as a result of nonlytic ATP efflux. Hst 5 caused a loss of cell viability concomitant with a decrease in cellular volume as determined both by a classical candidacidal assay with exogenous Hst 5 and by using a genetically engineered C. albicans strain expressing Hst 5. Preincubation of C. albicans cells with pharmacological inhibitors of anion transport provided complete or substantial protection from Hst 5-induced killing and volume reduction of cells. Moreover, intracellular expression of Hst 5 resulted in a reduction in the population mean cell volume that was accompanied by an increase in the percentage of unbudded cells and C. albicans cells in the G(1) phase. Following expression of Hst 5, the smallest cells sorted by fluorescence-activated cell sorting from the total population did not replicate and were exclusively in the G(1) phase. Cells with intracellularly expressed Hst 5 had greatly reduced G(1) cyclin transcript levels, indicating that they arrested in the G(1) phase before the onset of Start. Our data demonstrate that a key determinant in the mechanism of Hst 5 toxicity in C. albicans cells is the disruption of regulatory circuits for cell volume homeostasis that is closely coupled with loss of intracellular ATP. This novel process of fungicidal activity by a human salivary protein has highlighted potential interactions of Hst 5 with volume regulatory mechanisms and the process of yeast cell cycle control.

FIG. 1.

Time-dependent reduction in mean cell volume of C. albicans cells induced by exogenous addition of Hst 5. C. albicans DS1 cells (2 × 10⁶ to 5 × 10⁶ cells) were treated with Hst 5 in 10 mM sodium phosphate buffer (pH 7.4) at room temperature and incubated for different times. The mean cell volume (average for 20,000 cells) was measured with a Coulter Counter Multisizer II-Channelyzer at different times following addition of Hst 5 and compared with the initial value. The data was analyzed by using AccuComp software, and the changes in mean cell volume are expressed as relative cell volumes compared to the pretreatment values. The data are means ± standard deviations for three independent observations.

FIG. 2.

Hst 5-induced cell volume change is inhibited by pretreatment with DIDS. C. albicans DS1 cells (5 × 10⁶ cells) were preincubated with 1 mM DIDS for 10 min before addition of 62 μM Hst 5 in 10 mM phosphate buffer at room temperature (○). Cells were also treated only with Hst 5 (•). Control cells were preincubated with 1 mM DIDS alone (▵). The mean cell volume for each treatment and each time point was measured with a Coulter Counter Multisizer II-Channelyzer, and cell volume changes are expressed as relative cell volumes compared to pretreatment values (zero time). All of experiments were carried out at room temperature. The data are means ± standard deviations for three independent experiments.

FIG. 3.

Induction of Hst 5 expression in C. albicans DB9 cells causes reduction in mean cell size. C. albicans DB9 and DB13 cells were grown for 23 h in sucrose to induce expression of Hst 5 (DB9) and a nontoxic control protein, statherin (DB13). Cells were collected, washed, and placed into isotonic NaCl buffer, and the cell volume for each population was measured with a Coulter Counter Multisizer II-Channelyzer. Cell size is expressed as the cell diameter. The overlay profile shows the reduction in mean cell size of DB9 cells expressing Hst 5 (left arrow) compared with the mean cell size of DB13 cells expressing statherin (right arrow).

FIG. 4.

Induction of Hst 5 expression in C. albicans DB9 cells results in an increased percentage of G₁ cells. DB9 cells were assayed at different times following inoculation into cultures containing glucose (without Hst 5) (•) or sucrose (inducing Hst 5) (▪). Cells were stained with Sytox Green, and the DNA content for the total DB9 cell population under inducing (▪) and noninducing (•) conditions was measured and analyzed with the FACScan by using Modifit software (Verity Software, Inc.) to determine the percentage of G₁ cells. The viability of DB9 cells expressing Hst 5 was measured by a dilution plate assay at each time point (▵).

FIG. 5.

Small cells sorted following Hst 5 expression in C. albicans DB9 cells are solely in the G₁ phase with a 1N DNA content. C. albicans DB9 cells were grown in medium containing 2% sucrose (inducing medium) for 23 h, washed, and measured by using FACS. The cells were gated by size according to forward scatter (FSC) and side scatter (SSC) intensity (gates indicated in panel B) and sorted according to the smallest 30% (R1) (A) and largest 30% (R2) (C) of the cells. Collected cells from the R1 and R2 regions were analyzed for DNA content by flow cytometry following fixation and staining with Sytox Green. Analysis of the G₁ and G₂-M phase contents (1N and 2N DNA contents, respectively) was done by using Modifit software. Data from one experiment are shown, and similar results were observed in repeated experiments.

FIG. 6.

Hst 5 expression results in reduced Cln1 and Cln2 transcript levels. Analysis of the RT-PCR products in total RNA samples obtained from C. albicans strain DB9 (Hst 5-expressing) (A) and DB13 (statherin-expressing) (B) cells was done to examine the levels of specific G₁ cyclin transcripts. Lanes 1, 3, 5, and 7, RNA samples obtained from cultures grown on YNB-glucose (noninducing conditions); lanes 2, 4, 6, and 8, RNA samples obtained from cultures grown on YNB-sucrose (inducing conditions). The designations of the C. albicans genes examined are indicated at the top. Lane M contained molecular mass markers.