In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans

Abstract

Environmental or chemically induced stresses often trigger physiological responses that regulate intracellular pH. As such, the capacity to detect pH changes in real time and within live cells is of fundamental importance to essentially all aspects of biology. In this respect, pHluorin, a pH-sensitive variant of green fluorescent protein, has provided an invaluable tool to detect such responses. Here, we report the adaptation of pHluorin2 (PHL2), a substantially brighter variant of pHluorin, for use with the human fungal pathogen Candida albicans. As well as a cytoplasmic PHL2 indicator, we describe a version that specifically localizes within the fungal vacuole, an acidified subcellular compartment with important functions in nutrient storage and pH homeostasis. In addition, by means of a glycophosphatidylinositol-anchored PHL2-fusion protein, we generated a cell surface pH sensor. We demonstrated the utility of these tools in several applications, including accurate intracellular and extracellular pH measurements in individual cells via flow cytometry and in cell populations via a convenient plate reader-based protocol. The PHL2 tools can also be used for endpoint as well as time course experiments and to conduct chemical screens to identify drugs that alter normal pH homeostasis. These tools enable observation of the highly dynamic intracellular pH shifts that occur throughout the fungal growth cycle, as well as in response to various chemical treatments. IMPORTANCECandida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays.

Keywords: Candida albicans; chemical screening; pH dynamics; vacuoles.

FIG 1

C. albicans-optimized PHL2 and CPP-PHL2 probes localize to the cytoplasm and vacuole, respectively. (A) The P_TEF1-PHL2 construct was introduced into wild-type strain CAI4, and PHL2 fluorescence was observed by confocal microscopy. (B) The P_ACT1-CPP-PHL2 expression construct was introduced into CAI4 (WT) (left panels) or the VATPase-deficient vph1Δ/Δ mutant (right panels). Cells were grown in minimal medium and preincubated with FM4-64 to label the vacuolar membrane. Images were then acquired by confocal microscopy using differential interference contrast optics and 488-nm and 546-nm lasers for PHL2 and FM4-64, respectively. (C) CAI4 cells expressing PHL2 or CPP-PHL2 were resuspended in precalibrated buffers (containing 10 mM sodium azide) at the indicated pH. Fluorescence emission intensity was then measured at a fixed wavelength of 509 nm (9-nm bandwidth), following excitation at 395 (I₃₉₅) and 470 nm (I₄₇₀) (9-nm bandwidth), by using a monochromator-based plate reader. The I₃₉₅/I₄₇₀ ratio was calculated and plotted against the buffer’s pH to generate a calibration curve. (D) CAI4 (WT) and vph1Δ/Δ strains expressing PHL2 (cytoplasmic) or CPP-PHL2 (vacuolar) were grown in unbuffered minimal medium, and the I₃₉₅/I₄₇₀ ratios of intact cells were determined as described for panel C, except without permeabilization. Cytoplasmic and vacuolar pHs were then estimated for each strain by using a third-degree polynomial regression equation based on each strain’s calibration curve. Means and standard deviations were calculated from four biological replicates for each strain. *, P < 0.0001.

FIG 2

PHL2-Pga59p fusion provides a cell surface pH sensor for C. albicans. (A) CAI4 cells expressing the PHL2-Pga59p fusion protein were grown in minimal medium at pH 7. Bright-field (right panel) and fluorescent images (enhanced GFP filter at 485 nm [20-nm bandwidth]) (left panel) were acquired at 60× magnification using an epifluorescence microscope. (B) CAI4 cells expressing PHL2-Pga59p were grown overnight in minimal medium buffered at pH 6.2, 6.4, and 6.8 (open symbols) and at pH 5.7 and transferred to nonlytic medium prepared at the indicated pHs. Fluorescence intensity was then measured at a fixed wavelength of 509 nm (9-nm bandwidth), following excitation at 395 (I₃₉₅) and 470 nm (I₄₇₀) (9-nm bandwidth) using a monochromator-based plate reader. The I₃₉₅/I₄₇₀ ratio was calculated and plotted against the buffer’s pH to generate a calibration curve.

FIG 3

Intracellular pH varies with growth phase in C. albicans. CAI4 (WT) (left panel) and vph1Δ/Δ mutant (right panel) expressing PHL2 (full circles), or CPP-PHL2 (open squares) were grown in minimal medium in 96-well plates at 35°C for 24 h within a BioTek Cytation 5 plate reader. I395, I470, and OD600nm were measured every 30 min as described in Fig. 1. Background fluorescence at either wavelength was measured at the same intervals from a strain carrying the empty vector (minus PHL2 control), and subtracted from the total fluorescence intensity to yield PHL2-derived fluorescence at either wavelength. I395/I470 ration was then calculated for each time point and plotted against time.

FIG 4

Cytoplasmic and vacuolar pHs vary among clinical isolates of C. albicans. Cytoplasmic PHL2 and CPP-PHL2 expression constructs were introduced into CAI4 (filled circles), vph1Δ/Δ mutant strain (open triangles), the clinical isolate DR17 (open circles), and SC5314 (gray squares). (A) To generate calibration curves, cultures of each strain expressing CPPPHL2 were resuspended in precalibrated buffers at the indicated pH. The I₃₉₅ and I₄₇₀ was then measured using a monochromator-based plate reader as described for previous figures. The I₃₉₅/I₄₇₀ ratio was calculated and plotted against each buffer’s pH to generate the calibration curve. Strains expressing PHL2 (B) or CPP-PHL2 (C) were grown in minimal medium in a 96-well plate, and the I₃₉₅/I₄₇₀ ratios of intact cells as well as the OD₆₀₀ were determined every 30 min as described for Fig. 3. For each strain, a third-degree polynomial regression equation based on the respective calibration curve was generated to calculate the cytoplasmic (B) and vacuolar (C) pH.

FIG 5

Diflunisal and Benzbromarone differentially affect cytoplasmic and vacuolar pHs in C. albicans. PHL2- and CPP-PHL2-expressing strains of C. albicans (CAI4 strain background) were used to screen the NCC compound library in time course assays as described for Fig. 3, with each compound at a final concentration of 10 μM. No-drug control wells contained an equivalent amount of the DMSO vehicle alone. Growth (the OD₆₀₀) and fluorescence intensity was measured every 30 min, and I₃₉₅/I₄₇₀ ratios were calculated for each time point after background fluorescence (measured from a strain carrying the empty expression vector) was subtracted. Data for two compounds in the collection, diflunisal (A) and Benzbromarone (B), are shown. In a separate follow-up experiment (bottom graphs in both panels A and B), endpoint measurements were taken from the PHL2- and CPP-PHL2-expressing strains grown for 24 h at 30°C in minimal medium plus 50 mM diflunisal (labeled 50 mM in panel D) or Benzbromarone (labeled 50 mM in panel B). Growth (the OD₆₀₀) and fluorescence intensity were measured as described above, and cytoplasmic as well as vacuolar pHs were estimated from the respective I₃₉₅/I₄₇₀ ratios by using a third-degree polynomial regression equation based on each strain’s respective calibration curve (Fig. 1). Means and standard deviations were calculated from three biological repeats for each strain. *, P < 0.001.

FIG 6

Fluconazole dysregulates cytoplasmic and vacuolar pH homeostasis in C. albicans. CAI4 cells expressing PHL2 or CPP-PHL2 were grown in minimal medium at 35°C for 24 h in the presence of 10 mM fluconazole (open circles) or an equivalent amount of DMSO solvent (full circles). Fluorescence intensity from cytoplasmic PHL2 (A) or vacuolar CPP-PHL2 (B) and the OD₆₀₀ (C) were measured every 30 min, and I₃₉₅/I₄₇₀ ratios were calculated for each time point, as described for Fig. 3.